InteractiveFly: GeneBrief

moleskin: Biological Overview | References


Gene name - moleskin

Synonyms -

Cytological map position - 66B6-66B6

Function - signaling

Keywords - importin, nucleo-cytoplasmic transport , ras/MAP kinase pathway, TGFβ/Dpp pathway

Symbol - msk

FlyBase ID: FBgn0026252

Genetic map position - 3L: 8,109,278..8,114,681 [+]

Classification - Importin-beta

Cellular location - nuclear and cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Li, Q., Zhang, X., Hu, W., Liang, X., Zhang, F., Wang, L., Liu, Z. J. and Zhong, Y. (2016). Importin-7 mediates memory consolidation through regulation of nuclear translocation of training-activated MAPK in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26929354
Summary:
Translocation of signaling molecules, MAPK in particular, from the cytosol to nucleus represents a universal key element in initiating the gene program that determines memory consolidation. Translocation mechanisms and their behavioral impact, however, remain to be determined. This study reports that a highly conserved nuclear transporter, Drosophila importin-7 (DIM-7), regulates import of training-activated MAPK for consolidation of long-term memory (LTM). Silencing DIM-7 functions results in impaired LTM, whereas overexpression of DIM-7 enhances LTM. This DIM-7-dependent regulation of LTM is confined to a consolidation time window and in mushroom body neurons. Image data show that bidirectional alteration in DIM-7 expression results in proportional changes in the intensity of training-activated MAPK accumulated within the nuclei of mushroom body neurons during LTM consolidation. Such DIM-7-regulated nuclear accumulation of activated MAPK is observed only in the training specified for LTM induction and determines the amplitude, but not the time course, of memory consolidation.
Vishal, K., Brooks, D. S., Bawa, S., Gameros, S., Stetsiv, M. and Geisbrecht, E. R. (2017). Adult muscle formation requires Drosophila Moleskin for proliferation of wing disc-associated muscle precursors. Genetics [Epub ahead of print]. PubMed ID: 28249984
Summary:
Adult muscle precursor (AMP) cells located in the notum of the larval wing disc undergo rapid amplification and eventual fusion to generate the Drosophila melanogaster indirect flight muscles (IFMs). This study finds that loss of Moleskin (Msk) function in these wing disc-associated myoblasts reduces the overall AMP pool size, resulting in the absence of IFM formation. This myoblast loss is due to a decrease in the AMP proliferative capacity and is independent of cell death. In contrast, disruption of Msk during pupal myoblast proliferation does not alter the AMP number, suggesting that Msk is specifically required for larval AMP proliferation. It has been previously shown that Wingless (Wg) signaling maintains expression of the Vestigal (Vg) transcription factor in proliferating myoblasts. However, other factors that influence Wg-mediated myoblast proliferation are largely unknown. This study examined the interactions between Msk and the Wg pathway in regulation of the AMP pool size. A myoblast-specific reduction of Msk was shown to result in the absence of Vg expression and a complete loss of the Wg pathway readout β-catenin/Armadillo (Arm). Moreover, msk RNAi knockdown abolishes expression of the Wg target Ladybird (Lbe) in leg disc myoblasts. Collectively, these results provide strong evidence that Msk acts through the Wg signaling pathway to control myoblast pool size and muscle formation by regulating Arm stability or nuclear transport.

BIOLOGICAL OVERVIEW

The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants (see Myospheroid), either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation is not significantly affected, and ERK accumulates in a perinuclear ring. Tyrosine phosphorylation of DIM-7 is reduced in cells expressing integrin mutants, indicating a mechanistic link between these components. DIM-7 and integrins localize to the same actin-containing peripheral regions in spreading cells, but DIM-7 is not concentrated in paxillin-positive focal contacts or stable focal adhesions. The Corkscrew (SHP-2) tyrosine phosphatase binds DIM-7, and Corkscrew is required for the cortical localization of DIM-7. These data suggest a model in which ERK phosphorylation must be spatially coupled to integrin-mediated DIM-7 activation to make a complex that can be imported efficiently. Moreover, dpERK nuclear import can be restored in DIM-7-deficient cells by Xenopus Importin-7, demonstrating that ERK import is an evolutionarily conserved function of this protein (James, 2007).

The integrin cell surface receptors regulate numerous cellular processes, including growth, differentiation, apoptosis and migration. Integrins are heterodimers made up of α and β subunits, each with short cytoplasmic tails and large extracellular domains. Integrins function as adhesion molecules and frequently form a physical connection between the extracellular matrix (ECM) and the actin cytoskeleton (James, 2007).

In addition to their function in cell adhesion, integrins are critical to many of the signaling pathways of cells. Of particular relevance to these studies, numerous examples have been documented in which integrins regulate the activity of mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK), or in turn are regulated by these enzymes. Integrins may directly mediate ERK activation, or in other cases, they may function to modulate the activities of growth factor receptors on ERK signaling (James, 2007).

ERK-induced gene expression requires the transport of ERK into the nucleus (Pouysségur, 2002). In the absence of stimulation, ERK is maintained in the cytoplasm through an interaction with its upstream activator mitogen-activated protein kinase kinase (MEK) (Fukuda, 1997). MEK phosphorylates both tyrosine and threonine residues in the activation loop of ERK (Pouysségur, 2002). After phosphorylation by MEK, diphospho-ERK (dpERK) probably dimerizes and enters the nucleus via an active transport mechanism (Görlich, 1998; Khokhlatchev, 1998; Adachi, 1999). The subcellular localization of dpERK after activation offers an additional level of regulation of ERK signaling (Pouysségur, 2002; Kumar, 2003; Smith, 2004; Marenda, 2006; Vrailas, 2006; James, 2007 and references therein).

In general, cells in suspension respond weakly to growth factor stimulation compared with cells adhering to the ECM, and regulation of ERK nuclear import is one potential step where integrin and receptor tyrosine kinase (RTK) signals may be integrated. For example, after activation by MEK in NIH 3T3 cells maintained in suspension the majority of ERK remains in the cytoplasm, and ERK activation of the transcription factor Elk-1 is reduced compared with adherent cells (Aplin, 2001). A β4 integrin signaling domain has been shown to affect the nuclear translocation of MAP kinases and NF-kappaB although the large cytoplasmic domain of β4 is not homologous to that of other integrin β subunits (Nikolopoulos, 2005). The nuclear localization of other transcriptional regulators also has been shown to be altered by integrin function in mammalian cells, including the c-Abl tyrosine kinase in mouse fibroblasts and the transcriptional coactivator JAB1 in a variety of cell types. Additional connections between integrins and nuclear import are suggested by studies on proteins that are typically considered to be downstream of integrins. For example, integrin-linked kinase (ILK) has been shown to regulate the nuclear import of a c-Jun coactivator protein (James, 2007).

A potential link between integrins and nuclear import has been further suggested by studies of wing development in Drosophila. In Drosophila, integrins are required to maintain the attachment of the dorsal and ventral wing epithelia during adult morphogenesis, and this process depends on the differential expression αPS1 and αPS2 integrin subunits on the dorsal and ventral cells, respectively. Loss of integrin function leads to wing blisters, where the two surfaces separate after eclosion of the adult from the pupal case. Surprisingly, wing blisters can also occur when an α subunit is inappropriately expressed on the wrong side of the wing, and experiments with various mutants have demonstrated that this is a gain-of-function phenotype. That is, the activity of an integrin in the wrong place during a specific morphogenetic event causes a subsequent loss of epithelial attachment. A genetic screen for dominant suppressors of this gain-of-function wing blister phenotype (Baker, 2002) identified null mutations in a gene named moleskin (msk) (James, 2007).

The moleskin gene encodes Drosophila Importin-7 (DIM-7), which is a close homologue of vertebrate Importin-7 (Lorenzen, 2001), also known as Ran Binding Protein-7 (RanBP-7). Importin-7 is a member of the importin β superfamily of nuclear importers, which can bind directly to the nuclear pore complex (Görlich, 1997; Jäkel, 1999). Vertebrate Importin-7 has been shown to mediate nuclear import of ribosomal proteins, histone H1, the HIV-1 reverse transcription complex and the glucocorticoid receptor (Jäkel, 1999; Fassati, 2003; Freedman, 2004). In Drosophila, DIM-7 is tyrosine phosphorylated in response to growth factor stimulation of RTKs, and it physically binds Drosophila ERK (Lorenzen, 2001). Additionally, DIM-7 binds the tyrosine phosphatase Corkscrew (CSW), the Drosophila homologue of SHP-2 (Perkins, 1996; Lorenzen, 2001). Corkscrew is generally required for ERK signaling via RTKs, and in vertebrate cells SHP-2 has been associated with integrin signaling and regulation of integrin activity, although the molecular bases of these interactions remain unclear (James, 2007).

Until recently, it has not been clear how dpERK gains entry to the nucleus after activation. In addition to regulated nuclear import, the cellular localization of phosphorylated ERK dimers can be influenced by release from cytoplasmic anchors and regulated nuclear retention or export (Pouysségur, 2002), and in at least one case it has been suggested that ERK2 may not require any additional import proteins. Genetic experiments with Drosophila embryos demonstrate that DIM-7 is largely responsible for the nuclear import of activated ERK in this system (Lorenzen, 2001). The suppression of integrin-related phenotypes in fly wings by moleskin mutations led to an examination a potential connection between integrins and the regulation of ERK import in a Drosophila cell culture system, and the results suggest that DIM-7 may represent a novel nexus of integrin and RTK signaling (James, 2007).

This study shows that a vertebrate homologue of DIM-7 can rescue the ERK localization phenotype of DIM-7 dsRNA treated cells. Thus, ERK nuclear translocation is a property of members of the Importin-7 family of proteins generally. This function cannot necessarily be extended to other MAP kinases; for example, no change is seen in nuclear localization of the p38 MAP kinase in S2 cells grown in DIM-7 dsRNA, although c-Jun NH2-terminal kinase transport does seem to involve DIM-7 (James, 2007).

The ability of growth factors to activate ERK signaling is often linked to integrins; however, specific integrin functions typically have not been examined. The reduced levels of nuclear dpERK in cells expressing βPS-G1 (which contains a frameshift mutation in the cytoplasmic domain that eliminates the two NPXY motifs that are critical for interaction with a number of cytoplasmic proteins, including talin) or βPS-G4 (which has a mutation in the second serine of the MIDAS domain (DXSXS), which would be expected to inhibit extracellular ligand binding) show that ERK signaling seen in Drosophila S2 cells is dependent on functional integrins and that it is not due solely to changes in cell adhesion or shape. Specifically, both the extracellular and cytoplasmic integrin domains must be able to interact properly with ECM or intracellular components for the integrins to support high levels of nuclear dpERK (James, 2007).

A simple model in which soluble dpERK (activated by integrins or growth factors) finds DIM-7 ('activated' by integrins) for import cannot explain all of the data. The experiments that examine total ERK distribution in the integrin mutants suggest that most of the activated ERK cannot enter the nucleus even after translocation into the DIM-7-rich perinuclear region. One would expect that the importins here are actively working with various other cargos and that a significant fraction of the DIM-7 is generally capable of import. This result suggests a model in which ERK phosphorylation must be spatially coupled to DIM-7 activation to make a complex that can be imported efficiently (see Model for the role of integrins in ERK nuclear import in S2 cells). Interestingly, a specific membrane localization of DIM-7 has been suggested as a regulatory mechanism in developing Drosophila eyes (Vrailas, 2006); however, in this case the targeting to apical epithelia has been seen as an inhibitory mechanism (James, 2007).

moleskin (DIM-7) function is required for normal cell proliferation in animals, where patches of mutant cells disappear in developing epithelia (Baker, 2002; Vrailas, 2006; Pepple, 2007). Examples in which a 50% reduction in DIM-7 function has produced phenotypes in developing flies have involved circumstances in which a signaling pathway has been stimulated to high levels by gene overexpression (Baker, 2002; Pepple, 2007). In the current study DIM-7 levels are reduced significantly, but they are not eliminated, and the cells show no obvious phenotype during normal growth. However, clear effects are seen after acute stimulation of the ERK pathway. The integrin-mediated activation of DIM-7 may be especially important as a regulatory component in such cases of acute, high-level signaling (James, 2007).

Barberis (2000) found that mouse embryo fibroblasts expressing a β1 mutant similar to βPS-G1 transiently display elevated phospho-ERK after stimulation with growth factor, but subsequently the same groups (Hirsch, 2002) reported that ERK does not necessarily enter the nucleus after plating on fibronectin. Interestingly, the import defect seen in the integrin mutants can be rescued by adding constitutively active Rac. Although multiple pathways may couple integrins to ERK activation and transport in different cell types, Importin-7 family members are likely to be a common feature of dpERK nuclear translocation, and it will be interesting to see whether various pathways leading from integrins to import converge at this protein (James, 2007).

Perhaps most intriguing with respect to the current studies is the work on a natural human β1 variant. β1C is an alternatively spliced form that, like βPS-G1, replaces the cytoplasmic NPXY motifs with other sequence. Cells that express β1C show reduced proliferation and reduced activation of the Ras-ERK pathway, relative to cells expressing the more common β1A. β1A-containing integrins seem to form a complex that includes insulin-like growth factor-I receptor and the insulin receptor substrate-1 (IRS-1), and the addition of insulin leads to cell proliferation and inhibition of adhesion to laminin. In contrast, β1C expression leads to decreased proliferation and increased adhesion, and these effects seem to be mediated by a complex that includes Gab1 and SHP-2, but not IRS-1. There is no β1C variant naturally in Drosophila, but the βPS-G1 mutant does show some dominant-negative effects in flies, and the work reported in this study suggests that this might result at least in part from disruptive effects on intracellular signaling (James, 2007 and references therein).

Significant amounts of cortical DIM-7 are found where integrins are located at the spreading edges of cells. Consistent with a role of integrins in peripheral DIM-7 localization, cells that are protease treated, heat shocked to induce integrin expression, and spread in serum-free media (where spreading is integrin independent), DIM-7 is not found at the periphery in fully spread cells at early times, but it appears when integrin expression is detected after a few hours. One striking feature of the peripheral DIM-7 is that it does not colocalize with integrins in more organized cell-substratum adhesion sites. Thus, peripheral integrin-DIM-7 associations seem to depend on the functional state of the integrins (James, 2007).

Vertebrate SHP-2 is necessary in many contexts for growth factor activation of ERK, and SHP-2 has also been shown to be involved in integrin-dependent signaling. However, the data from different cell types fail to paint a simple, cohesive picture of SHP-2 molecular function, especially with respect to signaling downstream of integrins. Previous biochemical data indicate that DIM-7 binds Drosophila Corkscrew (SHP-2) (Lorenzen, 2001). Corkscrew is generally an essential component in signaling via receptor tyrosine kinases; however, Corkscrew lacking tyrosine phosphatase activity can rescue some phenotypes when reintroduced into corkscrew mutants, suggesting that in some contexts Corkscrew functions primarily as a scaffolding protein. The role of Corkscrew in DIM-7 activation may be largely as a scaffold, because DIM-7 disappears from the periphery when Corkscrew is depleted. This does not seem to be an indirect result of a defect in DIM-7 activation, because cortical DIM-7 remains in other situations that affect its ability to import dpERK, for example, in both of the integrin mutants tested (James, 2007 and references therein).

Interestingly, the screen that identified moleskin (DIM-7) as a suppressor of Blistermaker assayed only ~40% of the Drosophila genome (the third chromosome) (Baker, 2002). Further elucidation of the molecular mechanisms underlying the DIM-7/integrin connection is likely to be facilitated by the identification of additional Blistermaker suppressors on other chromosomes. Screens for such loci are in progress (James, 2007).

Moleskin is essential for the formation of the myotendinous junction in Drosophila

It is the precise connectivity between skeletal muscles and their corresponding tendon cells to form a functional myotendinous junction (MTJ) that allows for the force generation required for muscle contraction and organismal movement. The Drosophila MTJ is composed of secreted extracellular matrix (ECM) proteins deposited between integrin-mediated hemi-adherens junctions on the surface of muscle and tendon cells. This paper identifies a novel, cytoplasmic role for the canonical nuclear import protein Moleskin (Msk) in Drosophila embryonic somatic muscle attachment. Msk protein is enriched at muscle attachment sites in late embryogenesis and msk mutant embryos exhibit a failure in muscle-tendon cell attachment. Although the muscle-tendon attachment sites are reduced in size, components of the integrin complexes and ECM proteins are properly localized in msk mutant embryos. However, msk mutants fail to localize phosphorylated focal adhesion kinase (pFAK) to the sites of muscle-tendon cell junctions. In addition, the tendon cell specific proteins Stripe (Sr) and activated mitogen-activated protein kinase (MAPK) are reduced in msk mutant embryos. Rescue experiments demonstrate that Msk is required in the muscle cell, but not in the tendon cells. Moreover, muscle attachment defects due to loss of Msk are rescued by an activated form of MAPK or the secreted epidermal growth factor receptor (Egfr) ligand Vein. Taken together, these findings provide strong evidence that Msk signals non-autonomously through the Vein-Egfr signaling pathway for late tendon cell late differentiation and/or maintenance (Liu, 2011).

In Drosophila, the formation of a stable myotendinous junction is essential to withstand the force of muscle contraction required for larval hatching. Proper formation of the MTJ requires proper integrin heterodimer formation at the junctions between both muscle cells and tendon cells for a permanent linkage to the ECM proteins deposited between these two cell types. The precise mechanism by which the muscle cells signal to the tendon cells to form and maintain the semi-adherens junctions that comprise the stable MTJ is still being elucidated. This study shows that the canonical nuclear import protein Msk is essential for Drosophila somatic muscle attachment. Moreover, a model is provided explaining how Msk may function in non-cell autonomously from the muscle to the tendon cell for proper MTJ maintenance. Though vein mRNA is produced in the myotubes, Vein protein is secreted and is restricted to the junctions at muscle-tendon attachment sites. Msk signals through the secreted Egfr ligand Vein to mediate cross-talk between the muscle and tendon cells. The binding of Vein to the tendon-expressed Egfr activates a signaling cascade through activated MAPK. Activated MAPK translocates to the nucleus and with SrA, activates downstream genes to induce terminal differentiation in the tendon cells. An inability of the tendon cells to maintain activated MAPK and Sr activity would affect the amounts of target proteins required to maintain stable muscle-tendon adhesion. For example, a decrease in Tsp deposition into the ECM would result in smaller attachment sites and an inability to maintain a tight integrin-ECM association (Liu, 2011).

Msk plays a general role in myogenesis as defects in msk mutant embryos were observed in all hemisegments and affected all muscle groups. The variable penetrance, which was classified as either major (Class I) or moderate (Class II) muscle detachment phenotypes, present in msk mutant embryos is likely due to the presence of maternal msk transcript. Attempts to further knockdown Msk levels by removal of maternal load resulted in non-viable egg chambers, consistent with a requirement for Msk function in cell viability. It is possible that a further decrease, but not complete loss in Msk function, could result in earlier defects in myogenesis, since muscle patterning defects, predominantly characterized by missing muscles, were observed in a subset of msk mutant embryos (Liu, 2011).

After cell fate determination is established in somatic muscle development, myoblast fusion and myotube migration begin to proceed simultaneously in stage 13 embryos until the final muscle pattern is completed. As the migrating myotubes approach their target tendon cells, the αPS2ΒPS integrin heterodimer begins to accumulate at the leading edge of the muscle. This integrin complex is required for at least two separate events: (1) to serve as a transmembrane link between the internal actin cytoskeleton and the ECM components Tsp and Tig; and (2) for the proper localization and/or accumulation of Vein. The accumulation of Vein at the sites of muscle-tendon interactions is necessary for activation of the Egfr pathway and subsequent late tendon cell differentiation. In these mature, muscle-linked tendon cells, SrB expression is positively regulated. SrB also turns on the downstream transcriptional target Tsp, resulting in more Tsp secretion and subsequent strengthening of the MTJ through integrin binding (Liu, 2011).

The results suggest that Msk affects the later stages of tendon cell maturation and MTJ formation and/or maintenance. (1) No obvious defects were observed in myoblast fusion or the guidance of muscles to their correct target tendon cell. (2) Msk protein expression, visualized by both antibody immunolocalization and a fluorescently-tagged Msk fusion protein, demonstrates that enrichment of Msk protein at the future muscle-tendon attachment sites occurs after stage 15. The appearance of Msk protein localization corresponds to the timing of MTJ junction formation, but it does not rule out the possibility that Msk has a role in earlier myogenic events. (3) The muscle detachment phenotypes observed in msk mutant embryos are consistent with the myospheroid phenotype observed for other genes, including myospheroid (βPS int), inflated (αPS2 int), and rhea (Talin), which are well-characterized for their role in embryonic muscle attachment. Finally, in all msk mutants examined, regardless of the severity of the muscle detachment phenotypes, MTJ formation occurred in the correct location. Although the muscle attachment sites were smaller in msk mutants than in WT embryos, they were initially formed correctly, but not capable of reaching their mature size. These data taken together indicate that the muscle-specific αPS2βPS integrin complex initially forms an attachment to the ECM proteins Tig and Tsp. However, as mature tendon cell induction is compromised in msk mutant embryos, whereby the tendon cells are not able to produce and secrete proper levels of Tsp protein. Thus, the size of the mature MTJ is reduced and results in a decreased affinity at the muscle-tendon junctions (Liu, 2011).

Sr is a key factor in tendon cell differentiation in the embryo and fly thorax. In the embryo, SrB is essential for early tendon cell induction and SrA is activated for later tendon cell maturation. Furthermore, ectopic expression of Sr can act as a guidance cue for migrating myotubes as ectopic expression of Sr in epidermal cells, the salivary glands, or the CNS results in muscle patterning defects where muscles take the incorrect route and/or become attached to ectopic cells expressing Sr. Even though the data shows that Msk is required for nuclear Sr in the tendon cells, ectopic expression of Msk is not sufficient for tendon cell induction based upon three lines of experimentation. First, ectopic Msk expression in either the muscle or epidermis resulted in aberrant muscle attachment, but not misguided myotubes. All muscles were found to be in the correct position, regardless of Msk expression in domains outside of the normal hemisegments. Second, ectopic Msk expression was not sufficient to induce either early or elevated Sr levels. Third, expression of Msk in the salivary glands or CNS did not result in misguided muscles toward these locations (Liu, 2011).

The tissue-specific rescue experiments show that Msk is required in the muscle cell for proper muscle-tendon attachment to occur. Thus, two mechanisms that are not mutually exclusive are proposed by which Msk may be functioning in the muscle cells to exert its effect on tendon cell maturation. First, Msk may be required directly and/or indirectly for the localization and/or accumulation of secreted Vein. As antibodies against Vein were not available for testing this possibility, it was shown that reintroducing Vein in msk mutants could rescue muscle attachment defects. Second, Msk may act via an integrin-dependent mechanism to modulate adhesion, which is explained in detail below (Liu, 2011).

From these studies, Msk localizes to the ends of muscles at the sites of muscle attachment. It is proposed that this localization of Msk recruits other proteins to the sites of muscle attachment to sequester proteins near the cell periphery and/or to modulate integrin affinity at the muscle attachment site. First, the absence of pFAK localization in msk mutants strongly suggests that Msk is essential for pFAK localization to the muscle-tendon attachment site. Surprisingly, mutations in FAK do not result in embryonic muscle attachment defects. However, pFAK localization is also lost in integrin mutants, suggesting that pFAK is involved in undefined events in myogenesis. If Msk serves as a scaffold protein to localize pFAK and/or other molecules to the sites of muscle-tendon cell attachment, these proteins may play an accessory role in integrin-mediated adhesion. One idea is that a Msk-pFAK complex may serve to limit the signaling function of integrins so its adhesive role predominates in MTJ formation. It is well-established that integrins play both adhesive and signaling roles in cell migration and development. Clustering of the cytoplasmic tails of βPS-integrins initiates a downstream signaling pathway that regulates gene expression in the Drosophila midgut, but is not sufficient to induce tendon cell differentiation in formation of the MTJ. This suggests that integrin-mediated adhesion is required to assemble ECM components and influence the ability of Vein to activate the Egfr pathway. While loss of FAK activity does not result in somatic muscle defects, overexpression of FAK does. Muscles that have detached from the epidermis as a result of FAK overexpression still retain βPS2 integrins at the muscle ends. As observed in mammalian systems, this raises the possibility that pFAK may play a role in integrin complex disassembly. Excess pFAK may either displace proteins that bind to the cytoplasmic domain of integrins or excessively phosphorylate proteins resulting in integrin complex turnover and a decrease in stable adhesion. Alternatively, pFAK may exhibit redundancy with another protein at the attachment sites. There is precedence for this in the fly as pFAK functions redundantly with the tyrosine kinase Src downstream of integrins in the larval neuromuscular junctions (NMJs) to restrict NMJ growth. Furthermore, in FAK mutants, phosphotyrosine signal is still observed at the sites of muscle attachment, supporting the idea that another tyrosine kinase is functional. A viable candidate may be Src42A, as it is also expressed at the sites of muscle attachment in the embryo (Liu, 2011).

The canonical role for Msk is to import proteins, such as activated MAPK into the nucleus. However, not activated MAPK or nuclear Msk were detected in the nuclei of muscle cells. As this study has shown, strong Msk immunolocalization is detected at the MTJ in stage 16 embryos, but not in the nuclei of developing muscles in stages 13-15. It cannot be ruled out that Msk is present at low levels in the muscle, and was not detectable. It is true that the YFP-Msk fusion protein was detected in the nucleus, but this may be due to the over-expression of the protein. While it is possible that nuclear Msk and MAPK in muscles are required for some aspects of myogenesis, this requires further analysis (Liu, 2011).

Future experiments will determine the precise mechanism by which Msk influences Vein secretion, localization, and/or accumulation at muscle-tendon cell attachment sites. Is it mediated through integrins? Is phosphorylation of Msk essential for activity? Msk is tyrosine phosphorylated in response to insulin and PS integrins, although the kinase remains unknown. FAK may be an example of a kinase that can phosphorylate Msk at the muscle attachment sites (Liu, 2011).

In mammalian studies, the canonical role for Importin-7 is in nuclear import. Other roles for cytoplasmic Importin-7 have not been examined. Thus, it will be interesting to uncover new roles for Importin-7, specifically in vertebrate muscle development (Liu, 2011).

Drosophila Importin-7 functions upstream of the Elmo signaling module to mediate the formation and stability of muscle attachments

Establishment and maintenance of stable muscle attachments is essential for coordinated body movement. Studies in Drosophila have pioneered a molecular understanding of the morphological events in the conserved process of muscle attachment formation, including myofiber migration, muscle-tendon signaling, and stable junctional adhesion between muscle cells and their corresponding target insertion sites. In both Drosophila and vertebrate models, integrin complexes play a key role in the biogenesis and stability of muscle attachments through the interactions of integrins with extracellular matrix (ECM) ligands. This study shows that Drosophila Importin7 (Dim7) is an upstream regulator of the conserved Elmo-Mbc-->Rac signaling pathway in the formation of embryonic muscle attachment sites (MASs). Dim7 is encoded by the moleskin (msk) locus and was identified as an Elmo-interacting protein. Both Dim7 and Elmo localize to the ends of myofibers coincident with the timing of muscle-tendon attachment in late myogenesis. Phenotypic analysis of elmo mutants reveal muscle attachment defects similar to that previously described for integrin mutants. Furthermore, Elmo and Dim7 interact both biochemically and genetically in the developing musculature. The muscle detachment phenotype resulting from mutations in the msk locus can be rescued by components in the Elmo-signaling pathway, including the Elmo-Mbc complex, an activated Elmo variant, or a constitutively active form of Rac. In larval muscles, the localization of Dim7 and activated Elmo to the sites of muscle attachment is attenuated upon RNAi knockdown of integrin heterodimer complex components. These results show that integrins function as upstream signals to mediate Dim7-Elmo enrichment to the MASs (Liu, 2013).

Previous studies have shown that Dim7 localizes to developing muscle-tendon insertion sites and removal of Dim7 has severe consequences in muscle attachment maintenance (Liu, 2011). The current studies extend these observations to elucidate the functional contribution of Dim7-Elmo in regulating Drosophila muscle attachment. The results show that Dim7 is an upstream adaptor protein that recruits Elmo in response to integrin adhesion and/or signaling. Thus, it is proposed that the spatial and temporal regulation Elmo-Mbc activity results in regulation of the Rac-mediated actin cytoskeleton changes at the MASs (Liu, 2013).

The 'myospheroid' phenotype in elmo or msk mutants resemble attachment defects first characterized in mutated genes that encode for integrins, ILK and Talin, and is not due to earlier developmental defects in myogenesis. A similar number of cells expressing the muscle differentiation factor DMef2 was present in elmo or msk mutants, indicating that muscle specification was not affected (Geisbrecht, 2008; Liu, 2011). Genes essential for muscle migration and targeting also lead to detached muscles. For example, in kon/perd or grip mutants, the early arrest of migrating myotubes resulting from defective migration eventually leads to a linkage failure between the muscle and tendon cells. In mutant embryos with reduced levels of Elmo or Dim7, the muscle detachment phenotype did not appear to result from muscle migration defects. First, the spatial-temporal accumulation of Elmo and Dim7 is developmentally regulated. Both proteins are not detected at the leading edges of migrating muscles, but begin to accumulate at MASs after stage 15. Second, failure of muscle ends contacting their corresponding attachment sites was not observed in elmo or msk mutants at late stage 15, when muscle migration was almost complete (Liu and Geisbrecht, 2011) (Liu, 2013).

Both membrane localization and Rac-dependent cell spreading of the uninhibited, active version of Elmo is enhanced compared to native WT Elmo in cultured mammalian cells (Patel, 2010). These in vitro results are in agreement with the current in vivo analysis, where ElmoEDE (a mutation that prevents the autoinhibitory interaction of Elmo) is enriched at larval muscle ends compared to the poor accumulation of full-length Elmo-YFP. This may reflect a potential regulatory mechanism controlling the subcellular localization of Elmo from the cytoplasm to the muscle ends upon the release of Elmo autoinhibition. Within different cells or tissues, various proteins may regulate Elmo localization to the cell periphery, or other sites where active Elmo is needed. In cultured mammalian epithelial cells, membrane recruitment of the Elmo-Dock180 complex is dependent on active RhoG for cell spreading. Consistent with a functional role for membrane-targeted Elmo, active Elmo promotes cell elongation in Hela cells, when co-expressed with RhoG (Patel, 2010; Liu, 2013 and references therein).

The data argues that adaptor proteins may be required in muscle cells for activated Elmo membrane recruitment. Decreased levels of ElmoEDE are observed at the polarized ends of muscle insertion sites when Dim7 levels are decreased. It is still not clear if Dim7 binding is required for the conformational change that results in Elmo activation or if an activated Dim7-Elmo complex already exists within the cell and gets recruited as a complex upon integrin activation. Furthermore, complete loss of Elmo-EDE protein levels is not observed, suggesting that either Dim7 protein levels are not depleted enough or other proteins in addition to Dim7 play a role in Elmo membrane recruitment. Alternatively, post-translational modification(s), such as phosphorylation, could be an additional mechanism for the relief of Elmo autoinhibition. Thus, it is concluded that in muscle, Dim 7 is an essential adaptor protein for the polarized membrane localization of active Elmo or the active Elmo-Mbc complex downstream of integrin signaling pathway (Liu, 2013).

What is the relationship between the integrin adhesome and the Dim7-Elmo complex? Two explanations are proposed that are not mutually exclusive. One possibility is that the Dim7-Elmo-Mbc complex assembles at MASs via integrin-mediated 'outside-in' signaling. Upon ligand binding to ECM molecules, integrin activation results in Dim7-Elmo-Mbc complex localization for the spatial-temporal regulation of Rac activity to maintain dynamic actin filament adhesion at the MASs. It is predicted that localization of activated Elmo to the MASs is a prerequisite regulatory mechanism for actin cytoskeleton remodeling via Rac to maintain stable attachments. This hypothesis is supported by three lines of evidence: (1) muscle attachment defects upon loss of Dim7 or Elmo are only observed after the establishment of the integrin adhesion complex and onset of muscle contraction; (2) muscle detachment in msk mutants can be rescued by expressing low levels of activated Rac; and (3) the enrichment of Dim7 and Elmo-EDE proteins at the ends of muscle fibers is greatly reduced in integrin-deficient larvae (Liu, 2013).

It is also possible that accumulation of the Dim7-Elmo complex to the ends of muscles regulates 'inside-out' signaling to dynamically regulate integrin affinity for strong ligand binding and stable muscle attachments. Previously, it was reported that Dim7 acts upstream of the Vein-Egfr signaling pathway in muscle to tendon cell signaling (Liu, 2011). Combined with previous results that muscle-specific Vein secretion is dependent on the adhesive role of βPS integrin, the Dim7-Elmo complex may be internally required for integrins to regulate Vein secretion. A decrease in Vein-Egfr signaling and loss of tendon cell terminal fate results in a reduction in ECM secretion and weakened integrin-ECM attachment. This is consistent with the observation that msk or elmo mutants phenocopy embryos with reduced or excessive amounts of the αPSβPS integrin complex, where pointed muscle ends result in smaller muscle attachments. Future studies analyzing Dim7-Elmo-Mbc complex localization and function in the background of integrin deletion constructs which separate the 'inside-out' and 'outside-in' signaling pathways will be essential to uncover more detailed molecular mechanisms (Liu, 2013).

What is the relationship between the Dim7-Elmo-Mbc-->Rac signaling pathway and the integrin mediated adhesome complex assembly (including the Talin, IPP [integrin linked kinase (ILK)-PINCH-Parvin-α) complex]? It is proposed that actin filaments within the muscle cell are anchored to the muscle cell membrane via the IPP complex, while regulation of MAS-actin remodeling iscontrolled by the Dim7-Elmo-Mbc-->Rac pathway. The data suggests that these two complexes assemble independently at the muscle ends. In msk mutant embryos, both ILK and Talin properly accumulate at the MASs, suggesting that Dim7 is not responsible for their localization (Liu, 2011). Similarly, both MAS-enriched Dim7 and active Elmo can be detected at two ends of the muscles in ILK-deficient larva, even in fully detached muscles. In a vertebrate cell culture model, Elmo2 was found to physically interact with ILK for the establishment for cell polarity (Ho and Dagnino, 2012; Ho, 2009). Thus, it is possible that the current approaches have not fully knocked down Ilk levels or that the Dim7-Elmo recruitment by Ilk is redundant with another attachment site protein. Alternatively, an upstream scaffold protein may function to recruit both the IPP and Dim7-Elmo complex to the MASs. It is likely that these two complexes are temporally regulated in embryogenesis, where the actin remodeling complex is not needed until initial muscle-tendon initiation has been established (Liu, 2013).

A genetic screen in Drosophila for genes interacting with senseless during neuronal development identifies the importin moleskin: Alleles of msk are suppressors of lz

Senseless (Sens) is a conserved transcription factor required for normal development of the Drosophila peripheral nervous system. In the Drosophila retina, sens is necessary and sufficient for differentiation of R8 photoreceptors and interommatidial bristles (IOBs). When Sens is expressed in undifferentiated cells posterior to the morphogenetic furrow, ectopic IOBs are formed. This phenotype was used to identify new members of the sens pathway in a dominant modifier screen. Seven suppressor and three enhancer complementation groups were isolated. Three groups from the screen are the known genes Delta, lilliputian, and moleskin/DIM-7 (msk), while the remaining seven groups represent novel genes with previously undefined functions in neural development. The nuclear import gene msk was identified as a potent suppressor of the ectopic interommatidial bristle phenotype. In addition, msk mutant adult eyes are extremely disrupted with defects in multiple cell types. Reminiscent of the sens mutant phenotype, msk eyes demonstrate reductions in the number of R8 photoreceptors due to an R8 to R2,5 fate switch, providing genetic evidence that Msk is a component of the sens pathway. Interestingly, in msk tissue, the loss of R8 fate occurs earlier than with sens and suggests a previously unidentified stage of R8 development between atonal and sens (Pepple, 2007).

Sens, along with its homologs Gfi-1 and Pag-3, comprises a conserved family of proteins required for normal neural development. In Drosophila, sens is both necessary and sufficient for development of the PNS. In mice, loss of Gfi-1 leads to neurodegeneration of cerebellar Purkinje cells and sensoneural deafness due to loss of inner ear hair cells. Despite the obvious importance of the GPS proteins in normal neural development and their place near the top of the neuronal development cascade, few targets of these proteins in the process of neurogenesis are known. To identify members of this pathway required in neurogenesis, an F1 dominant modifier screen was performed using an ectopic Sens phenotype in Drosophila. Advantage was taken of a dominant, modifiable phenotype generated by ectopic expression of Sens in undifferentiated cells posterior to the morphogenetic furrow. This ectopic Sens led to the recruitment of undifferentiated cells to the bristle fate (Pepple, 2007).

Both known and novel genes have been identified as potential members of the sens pathway by their ability to modify an ectopic Sens phenotype. The Notch signaling pathway is known to regulate Sens function during the resolution of the proneural cluster. This interaction was identified in the screen by the ability of heterozygous loss of Dl to enhance the ectopic Sens phenotype. The nuclear import gene moleskin (msk) was able to strongly suppress the effect of ectopic Sens. msk plays a role in normal eye development and R8 photoreceptor differentiation. Identification of the genes that are represented in the remaining complementation groups will lead to a better understanding of the GPS pathway and normal neural development. It is likely that the remaining complementation groups represent components of the Sens pathway due to their specific effect on lz and not the secondary screens as well as their requirement for normal bristle development in adult thoracic clones. Further characterization of these genes will offer new insight into the highly conserved Sens pathway (Pepple, 2007).

Alleles of msk were found to be suppressors of lz (the expression of UAS-sens in undifferentiated cells by the lozenge-GAL4 driver) with the highest frequency of any complementation group in the EMS screen. Usually such high representation of alleles indicates that the gene has an important role in the phenotype being tested and/or is readily mutagenized. The results presented here suggest a model in which Msk plays a role in the sens pathway. Initial observations of the effect of Msk on the lz phenotype suggested that Msk was needed to maintain high levels of Sens expression. It is possible that in this ectopic situation, Msk contributes to Sens import, but more likely Msk contributes to Sens expression indirectly by importing another component of the pathway that regulates Sens expression. Characterization of the ey-GAL4, UAS-flp (EGUF); msk phenotype strongly suggests that Msk is not the only import factor involved in the Sens pathway during normal development. Clearly, there is functional redundancy with another importin since complete loss of Msk function during early eye development does not remove Sens expression in all R8 cells. In third instar discs, Msk appears to play a role in the maintenance of the R8 cell fate very early in development. Little is known about the early stages of R8 differentiation after specification by Atonal. Previous work on R8 specification and development outlined a hierarchy of events in which Atonal is expressed first and appears to simultaneously activate expression of the downstream targets sens and sca-lacZ. Work on the sens phenotype determined that sca-lacZ expression is still present in sens clones, thereby establishing an epistatic relationship between sca-lacZ expression and sens. The data indicate that there is yet another step in the relationship between Atonal and these two downstream factors. The data suggest that in the msk eye, after specification of the R8 by Atonal but before the onset of sca-lacZ expression, R8 development is disrupted in some clusters, leading to an R2,5 fate switch. This is the first genetic evidence for factors positioned between ato and sca-LacZ/sens (Pepple, 2007).

Nuclear transport is required for the viability of all cells. Interestingly, the loss or decrease in function of some importins can cause specific defects during development. For example, the nuclear exportin Dcas is required for the export of Importin α3 in Drosophila. While null mutants in dcas are not viable, hypomorphs lead to specific cell fate changes in mechanosensory bristles. This phenotype is likely due to extreme sensitivity of Notch signaling to disruption of nuclear transport of one of its pathway members by Importin α3. It is possible that the Msk/Sens interaction was detectable for a similar reason. In the Sens gain-of-function situation, the high level of Sens required to generate ectopic bristles is very sensitive to decreased Msk levels, while during wild-type SOP differentiation, Sens is far less sensitive to Msk levels and exhibits only sporadic effects (Pepple, 2007).

One question still remains: How does the EGUF; msk eye survive at all given the important cargo that Msk is known to transport? The functional redundancy in the Importin family likely provides the cell with enough transport for survival and development in the absence of Msk. However, this idea raises a new question: Why was only Msk identified in the screen and no other importins? A model is proposed in which Msk is the key importin utilized by the cell for high levels of signaling. The lz phenotype requires high levels of signaling to generate ectopic bristles, and this model would explain why an effect with Msk and no other importin was detected. The model does not preclude the ability of other importins to provide transport redundancy for Msk cargos, and in fact evidence is seen for this redundancy in the ability of the EGUF; msk eye to survive and produce some normal ommatidia. Another importin must have the ability to import some level of Sens, pMAPK, and other unidentified factors into the nucleus. Data existst that indirectly support such a model for the role of Msk. In the Atonal intermediate groups within the morphogenetic furrow, Msk must be sequestered away from the nucleus to prevent the very high levels of cytoplasmic pMAPK from entering the nucleus. Although whether other nuclear importins are also sequestered to block pMAPK nuclear entry was not tested, overexpression of Msk in the intermediate groups allows pMAPK to enter the nucleus and affect nuclear signaling. The fact that the cell needs to sequester Msk to prevent high levels of EGFR pathway signaling supports a model in which Msk is important for high levels of signaling (Pepple, 2007).

It has been suggested in other developmental systems that importins are part of a mechanism that regulates the nuclear protein composition of transcription factors and chromatin remodeling factors. In Drosophila, Msk has been shown to import two other developmentally significant cargos, pMAPK and Caudal. In addition to these previously defined roles, the additional data that Msk and nucleocytoplasmic transport play an important role in Sens expression and R8 development. Perhaps more importantly, the fact that abnormalities seen in msk mutant eye discs arise between Atonal and Senseless expression suggests roles for as-yet undiscovered factors and new modes of regulation in this critical pathway (Pepple, 2007).

Msk is required for nuclear import of TGF-β/BMP-activated Smads

Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor β (TGF-β) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, a whole-genome RNAi screening was carried out in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-β. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. Thus, evolutionarily conserved proteins have been identified that are important in the signal transduction of TGF-ß and BMP into the nucleus (Xu, 2007).

Genome-wide RNAi screening in this study offers a genetic approach to uncover new elements in TGF-ß signal transduction. Msk and its mammalian orthologues Imp7 and 8 are critical components in transporting TGF-ß-activated Smads into the nucleus. Biochemical evidence further suggests that Msk/Imp7/8 directly import phospho-Smads as cargoes (Xu, 2007).

Although there appears to be some discrepancy between these new findings and previous reports that importins are dispensable for the nuclear import of Smads, these observations can be reconciled (Xu, 2002, Xu, 2003). The present and previous studies, based on different approaches, may have revealed different nuclear import mechanisms used by basal and activated Smads to enter the nucleus. There are important differences comparing Smads import with or without TGF-ß stimulation. Unphosphorylated Smads are monomers, but phosphorylated Smads are assembled into complexes with Smad4 and are thus much larger in size. Moreover, as phospho-Smads accumulate in the nucleus they have to move across the nuclear pore against an ascending concentration gradient of Smads already in the nucleus, whereas unphosphorylated Smads never reach a higher concentration in the nucleus than in the cytoplasm. Thus, importing phospho-Smad complexes and unphosphorylated Smad monomers may entail different mechanisms, with or without the participation of importins. Indeed, RNAi data in both Drosophila and mammalian cells suggest that nuclear import of the two forms of Smads is very different regarding the requirement of Msk/Imp7/8. This type of differential requirement for import factors is not unique to Smads. In fact, STATs (signal transducers and activators of transcription) in the interferon pathway are another example in which the latent STATs are imported by an importin-independent mechanism, whereas the phosphorylated STATs depend on importins to accumulate in the nucleus. It is also interesting to note that phospho-Smads were still detected in the nucleus upon RNAi-mediated knockdown of Msk/Imp7/8. Although the trivial explanation that this may be due to incomplete depletion of the targeted proteins cannot be ruled out, this observation may also suggest additional import mechanisms for activated Smads. It is recognized that the previous finding of importin-independent nuclear import of Smads was largely based on an in vitro reconstituted nuclear import assay. Although this in vitro system is widely accepted, it may not fully recapitulate nuclear import of activated Smads in cells. Based on RNAi data, regarding the requirement of importins, the conclusion drawn from the in vitro import assay may not apply to phospho-Smads in intact cells. However, the current study does not necessarily contradict the previous suggestions that direct Smad-nucleoporin interaction is critical for nuclear import of Smads (Xu, 2007).

The data showed that Msk/Imp7/8 interacted with Smads regardless of their phosphorylation status; thus, additional factors must be involved to explain why only TGF-ß/BMP-activated Smads can accumulate in the nucleus. Because basal-state Smads are actively exported out of the nucleus, it is possible that retaining only phospho-Smads in the nucleus requires blocking Smads nuclear export, a scenario that has been demonstrated for Smad4. This hypothesis would be consistent with findings in live cells, in which TGF-ß signaling led to reduced mobility of Smad2 in the nucleus (Xu, 2007).

Because Msk, Imp7, and Imp8 are shown to be critical for targeting phospho-Smads into the nucleus, it is conceivable that regulatory inputs to this nuclear import factor would impact TGF-ß signaling. Although no changes were observed in subcellular localization of Msk or Imp7/8 in response to TGF-ß in cultured cells, during Drosophila embryonic development, Msk distribution changed between cytoplasm and nucleus in a dynamic fashion (Lorenzen, 2001). Moreover, Msk is phosphorylated on tyrosine residues with yet-unknown functional consequences (Lorenzen, 2001). If and how Msk localization is regulated and by what signals are completely open questions at present (Xu, 2007).

A number of mitogen-induced phosphorylation events in the linker region of Smad have been suggested to inhibit TGF-ß-induced nuclear translocation of Smads in Xenopus and mammalian cells. Because part of the Imp7/8 binding was mapped to the linker region of Smad3, it will be interesting to determine if linker phosphorylation would affect the interaction between Smads and Imp7/8 and hence the rate of nuclear import. It is also worth noting that Msk has been genetically implicated in the nuclear import of activated ERK in Drosophila. Such convergence on the same molecule for nuclear import raises the possibility of cross-talk between MAP kinase and TGF-ß pathways at the level of nuclear translocation of key signal transducers (Xu, 2007).

Specific nucleoporin requirement for Smad nuclear translocation

Cytoplasm-to-nucleus translocation of Smad is a fundamental step in transforming growth factor beta (TGF-beta) signal transduction. This study identified a subset of nucleoporins that, in conjunction with Moleskin (Msk, Drosophila Imp7/8), specifically mediate activation-induced nuclear translocation of MAD (Drosophila Smad1) but not the constitutive import of proteins harboring a classic nuclear localization signal (cNLS) or the spontaneous nuclear import of Medea (Drosophila Smad4). Surprisingly, many of these nucleoporins, including Sec13, Nup75, Nup93, and Nup205, are scaffold nucleoporins considered important for the overall integrity of the nuclear pore complex (NPC) but not known to have cargo-specific functions. The roles of these nucleoporins in supporting Smad nuclear import are separate from their previously assigned functions in NPC assembly. Furthermore, novel pathway-specific functions of Sec13 and Nup93 were uncovered; both Sec13 and Nup93 are able to preferentially interact with the phosphorylated/activated form of MAD, and Nup93 acts to recruit the importin Msk to the nuclear periphery. These findings, together with the observation that Sec13 and Nup93 could interact directly with Msk, suggest their direct involvement in the nuclear import of MAD. Thus, this study has delineated the nucleoporin requirement of MAD nuclear import, reflecting a unique trans-NPC mechanism (Chen, 2010).

Delta and Egfr expression are regulated by Importin-7/Moleskin in Drosophila wing development

Drosophila DIM-7 (encoded by the moleskin gene, msk) is the orthologue of vertebrate Importin-7. Both Importin-7 and Msk/DIM-7 function as nuclear import cofactors, and have been implicated in the control of multiple signal transduction pathways, including the direct nuclear import of the activated (phosphorylated) form of MAP kinase. Two genetic deficiency screens were performed to identify deficiencies that similarly modified Msk overexpression phenotypes in both eyes and wings. Eleven total deficiencies were identifed, one of which removes the Delta locus. This report shows that Delta loss-of-function alleles dominantly suppress Msk gain-of-function phenotypes in the developing wing. Msk overexpression increases both Delta protein expression and Delta transcription, though Msk expression alone is not sufficient to activate Delta protein function. It was also found that Msk overexpression increases Egfr protein levels, and that msk gene function is required for proper Egfr expression in both developing wings and eyes. These results indicate a novel function for Msk in Egfr expression. The implications of these data are discussed with respect to the integration of Egfr and Delta/Notch signaling, specifically through the control of MAP kinase subcellular localization (Vrailas-Mortimer, 2007).

This study has focused on the effects of mutation in Delta, which was identified in this screen. Loss-of-function mutations in Delta dominantly suppress Msk over-expression phenotypes in developing wings. Further, Delta transcription and Delta protein expression is increased in areas over-expressing Msk protein in developing wing discs. Interestingly, the increased Delta protein induced by Msk over-expression is not competent to activate Notch signaling in adjacent cells. Thus, some mechanism must either be inhibiting this induced Dl protein from signaling to adjacent cells, or the induced Dl protein itself is non-functional for signaling (Vrailas-Mortimer, 2007).

Delta must be endocytosed in signal-sending cells in order to activate Notch in signal-receiving cells. Clones of cells that express Dl but are also deficient for Epsin, an adapter protein required for Clathrin-mediated endocytosis, similarly can not promote Notch signaling in adjacent cells. It has been proposed that the Delta protein must normally be endocytosed and mono-ubiquitinated in the signal-sending cells (Delta expressing cells), where it is then targeted to a special endocytic pathway where it acquires competency to activate Notch in signal receiving cells. Thus, over-expression of Msk may have some effect on the internalization and/or post-translational modification of Delta (mono-ubiquitination) to render it unable to signal to adjacent cells. Indeed, Msk protein expression in en::msk wing discs is in a pattern that is co-incident with disrupted Delta protein near the apical tips of cells in the wing disc. Msk expression has been observed in the apical tips of cells within the morphogenetic furrow in the developing eye disc (Vrailas, 2006), where this apical localization is proposed to functionally inactivate Msk nuclear translocation function. Thus, in en::msk wing discs, apical localization of Msk protein may disrupt important cellular functions at this localization in the cell, such as Dl internalization and/or compartmentalization (Vrailas-Mortimer, 2007).

It has been shown that levels of over-expressed exogenous Delta in clones of cells is several fold higher than normal peak levels of endogenous Dl protein expression, and this over-expression autonomously inhibits Notch activation within these clones. This study also observed autonomous inhibition of Notch activation in posterior compartment cells that over-express Dl (UAS:Dl) with en:GAL4 (en::Dl), as measured by decreased Ct protein expression. Thus, the increased levels of Dl protein observed in en::msk wing discs may also explain the decrease in Ct protein expression in these wing pouches. However, increased Ct protein expression was also observed in posterior/dorsal cells when both exogenous Msk and exogenous Dl are over-expressed simultaneously. What can explain these apparently paradoxical results (Vrailas-Mortimer, 2007)?

It is known that the ectopic Dl protein induced by Msk over-expression in wings is unable to signal to adjacent cells. However, if this ectopic Dl expression is sufficient to autonomously inhibit Notch signaling in these cells (as observed by a decrease in Ct protein expression), it may function in a dominant-negative fashion in some cells but not in others. Thus, when both Msk and Dl are over-expressed, two things happen: (1) functional Dl protein is expressed that is competent to signal to adjacent cells (UAS:Dl); (2) non-functional Dl protein is expressed that is not competent to signal to adjacent cells, but is capable of autonomously inhibiting competent Dl protein (UAS:msk). There would then exist a situation within these cells where these two forms of Dl could compete for function. In those cells where competent Dl (UAS:Dl) wins, Ct expression is inhibited. In those cells where non-competent Dl (UAS:msk) wins, Ct expression can then be induced by competent Dl (UAS:Dl) expression in adjacent cells. This could account for the spotty appearance of Ct protein expression observed in these discs (Vrailas-Mortimer, 2007).

This study has shown that Egfr levels are decreased in msk clones in both larval wings and eyes, while Egfr levels are increased when Msk is over-express in larval tissues. These data suggest the possibility of a regulatory feedback mechanism on Egfr protein expression in this tissue. Thus, in cells where MAPK can move into the nucleus, the initial activation of the Egfr/Ras/MAPK pathway leads to the nuclear translocation of MAPK in these cells, which subsequently results in further upregulation of Egfr levels in those cells. This increased Egfr expression then further promotes even greater MAPK nuclear translocation in those cells. In cells where pMAPK is held in the cytoplasm, Egfr levels are decreased, and this may act as a feedback signal for continued hold of pMAPK within the cytoplasm of these cells. Indeed, Egfr mRNA expression is reduced in developing pupal wings after hyper-activation of Egfr signaling by rhomboid (rho) overexpression (rho encodes a protease required to activate the positive ligand spitz). The pMAPK induced by rho overexpression in developing pupal wings is also predominantly cytoplasmic, and leads to extra wing vein formation. Thus, the regulation of Egf receptor levels may be a mechanism by which subsequent MAPK subcellular localization is controlled (Vrailas-Mortimer, 2007).

How could the subcellular localization of MAPK relate to Dl expression and function in developing Drosophila tissues? In clones of spitz (which encodes for an activating ligand for the Egfr pathway) Dl expression is lost in the developing eye. Similarly, clones of cells mutant for the Egfr receptor itself show a loss of Dl expression in the developing pupal eye, although these clones show normal Cut protein expression. In the developing larval and pupal wing discs, Dl mRNA expression is absent in wing tissue double mutant for both rhomboid and vein (which effectively eliminates both the Egfr activating ligands spitz and vein in this tissue). Thus, Egfr activation and signaling are clearly required for Dl expression in these developing Drosophila tissues (Vrailas-Mortimer, 2007).

Dl expression is not lost in msk clones, suggesting: (1) the nuclear translocation of pMAPK is not required for Dl expression, (2) there is a redundant pMAPK nuclear transporter capable of importing pMAPK in these cells, (3) there is sufficient pMAPK nuclear translocation even in the absence of Msk protein to allow Dl expression to occur. Indeed, msk null clones posterior to the morphogenetic furrow in the developing eye retain many important Egfr/Ras pathway functions (Vrailas, 2006). Yet, over-expression of Msk increases both Dl protein expression and Dl transcription, suggesting that the nuclear translocation of pMAPK is at least sufficient to increase Dl protein levels. However, the Dl induced by Msk over-expression is not competent to activate Notch signaling in adjacent cells, suggesting that the nuclear translocation of pMAPK alone is not sufficient to induce Notch signaling in adjacent cells. In wild type wing cells, where high levels of competent, active Dl protein expression occur, high levels of phosporylated, cytoplasmic MAPK, and low levels of Egfr protein expression are also observed. Similarly, where high levels of Notch expression are observed, observe high levels of Egfr protein expression are also observed. Gain-of-function mutations in Notch, or hyper-activation of the downstream Notch protein Enhancer of split (E(spl)) decrease rho expression, while loss-of-function mutations in Notch, or expression of a dominant-negative form of Notch increases rho expression and induces extra vein formation. pMAPK expression is also lost upon loss of rho expression. Thus, Notch signaling represses pMAPK expression. As the pMAPK expression induced by rho signaling is predominantly cytoplasmic, it is suggested that it may be the cytoplasmic hold of pMAPK that is normally required for Dl protein signaling competence to activate Notch in adjacent cells. When competent Dl protein was overexpressed in the posterior compartment of developing wings (en:Gal4, UAS:Dl), Notch activation was induce in adjacent anterior/dorsal cells, and also increased expression of pMAPK was induced in the posterior compartment. It has previously been shown that pMAPK expression is lost in the posterior compartment of en::msk developing wing discs, since this pMAPK is ectopically translocated to the nucleus (Marenda, 2006). If pMAPK expression is required to induce Dl signaling competency, the difference in pMAPK expression observed between these two genotypes (en::msk and en::Dl) may explain the differences in Ct expression observed within these different genotypes as well (Vrailas-Mortimer, 2007).

Understanding how diverse signaling pathways integrate to regulate important biological processes is central to an understanding of the mechanisms of development. Understand these basic mechanisms of regulation and how they function to coordinately control different cellular processes are beginning to be understood. This report suggests that the subcellular localization of one pathway component (MAPK) as mediated by the nuclear import cofactor Msk, is an important factor in Egfr signal regulation through the control of the expression of the Egfr protein itself. It is further suggested that MAPK subcellular localization also plays an important role in the cross-talk between Egfr and Notch signaling pathways (Vrailas-Mortimer, 2007).

MAP kinase subcellular localization controls both pattern and proliferation in the developing Drosophila wing

Mitogen-activated protein kinases (MAPKs) phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK phosphorylation was always followed by rapid nuclear translocation. However, MAPK phosphorylation is not always sufficient for nuclear translocation in vivo. In the developing Drosophila wing, MAPK-mediated signaling is required both for patterning and for cell proliferation, although the mechanism of this differential control is not fully understood. This study shows that phosphorylated MAPK (pMAPK) is held in the cytoplasm in differentiating larval and pupal wing vein cells, and this cytoplasmic hold is required for vein cell fate. At the same time, MAPK does move into the nucleus of other wing cells where it promotes cell proliferation. A novel Ras pathway bifurcation is proposed in Drosophila and the results suggest a mechanism by which MAPK phosphorylation can signal two different cellular outcomes (differentiation versus proliferation) based on the subcellular localization of MAPK (Marenda, 2006).

Msk is a Drosophila homolog of importin 7 (encoded by the moleskin gene, msk), which is a MAPK nuclear import co-factor. Msk expression can facilitate the nuclear translocation of pMAPK in vivo. To observe the phenotypic consequences of continuous and long-term reduction of MAPK cytoplasmic hold, MSK was expressed in the posterior compartment of wing discs (en:GAL4; UAS:msk or en::msk). Since the GAL4/UAS system was used to overexpress Msk in these discs, MAPK-GAL4 (MG) cannot also be used to detect MAPK nuclear translocation. Therefore, to visualize MAPK, HSV epitope-tagged MAPK (hs:M) was used and stained for the epitope tag. In control wings, the tagged MAPK is expressed at low levels, and is not specifically concentrated in any one compartment. However, when Msk is overexpressed in the posterior compartment, tagged MAPK expression is visibly elevated. A closer analysis of this epitope shows that it is in many cell nuclei both where hold normally occurs (e.g., the wing margin), and also in areas where hold does not occur, confirming that Msk overexpression increases the rate of MAPK nuclear translocation in the wing (Marenda, 2006).

This posterior ectopic expression of Msk eliminates pMAPK antigen within pro-vein and margin cells in the posterior domain of the wing pouch. Surprisingly, posterior Msk expression disrupts the anteroposterior compartment boundary, as determined by GFP marking. High levels of cell death can disrupt development, and cause cells to cease to respect compartment boundaries. Since high levels of cell death are seen in Msk overexpression wings, it is suggested that this may explain the disruption. However, even when cell death is blocked with p35, loss of pMAPK is still observed in the posterior wing pouch, suggesting that cell death alone is not the cause of lost pMAPK in this genotype (Marenda, 2006).

Taken together, these experiments suggest that ectopic Msk can increase MAPK nuclear translocation and overcome cytoplasmic hold, and that some nuclear enzyme, most likely a phosphatase, then rapidly eliminates the pMAPK antigen (Marenda, 2006).

In cultured CCL39 cells, MAPK cytoplasmic tethering inhibits the ability of cells to enter S phase, suggesting that MAPK nuclear translocation is important for cell cycle entry. In the developing Drosophila larval wing, elevated Ras signaling similarly promotes G1/S progression, and MAPK loss-of-function mutations suppress this progression. Taken together, these data suggest that the G1/S transition in the developing larval wing may require MAPK nuclear translocation. Since cell proliferation in the wing is better understood in larval rather than pupal stages, the analyses were focused at this stage (Marenda, 2006).

In larval wing discs, margin cells are non-proliferative [the zone of non-proliferating cells (ZNC)], and markers of S-phase (BrdU) and M-phase (phospho-histone H3 antigen, pH3) are reduced in this territory. Similarly, MG-driven GFP is also reduced in margin territories, indicating that it too may be a marker for proliferation. However, MG-driven GFP is not in the same cells as either BrdU or pH3 (phospho-histone H3 antigen, a marker of M-phase). In the developing eye, MG-driven GFP follows the transcription of MG with a delay of 4-6 hours. Thus, the observed non-coincidence of GFP with either BrdU or pH3 in the developing wing may simply be due to this time lag (Marenda, 2006).

To analyze the cell cycle more precisely, FACS was used to determine the cell-cycle phase of those cells expressing MG-driven GFP, following a 1-hour induction and 6 hour recovery time. Sorting was performed for GFP and then the DNA content profiles of the two cell populations (GFP control cells with little or no MAPK nuclear translocation versus GFP+ cells where MAPK nuclear translocation has occurred) was compared. The GFP+ cell population has a slightly elevated fraction in G2 and M phase, mostly at the expense of the pool in G1. Although these results are consistent with a function of MAPK nuclear translocation in triggering proliferation, it remains possible that MG-driven GFP is a consequence, not a cause of proliferation. To test this, MAPK nuclear translocation was increased using NMG (MG fused with a SV40 nuclear localization sequence), while simultaneously driving GFP reporter expression (hs:NMG, UAS:GFP). NMG was induced for 1 hour, followed by 6 hours recovery, and a dramatic reduction was seen in the fraction of GFP+ cells in G1, while greatly raising the fraction in S and G2/M, suggesting that nuclear translocation of MAPK is sufficient to induce proliferation. These larvae were then allowed to recover for 24 hours, the fraction of GFP+ cells in G2/M rose, at the expense of the pool in G1 and S. This suggests that MAPK nuclear translocation is sufficient to induce S-phase transition in wing cells, and after the initial nuclear MAPK-induced transition to S-phase, cells then progress normally through the division cycle (at least as far as G2) (Marenda, 2006).

However, it could be that upon induction of NMG, a block in G2/M occurs, and this allows cells to build up in S phase. To rule this out, hs:MG and hs:NMG were expressed, and pH3 staining was analyzed. But no difference in pH3-positive nuclei was observed in hs:NMG discs versus hs:MG controls. Indeed, more pH3-positive nuclei are seen in hs:NMG wing pouches when compared with hs:MG controls, along with increased pH3 staining in the ZNC. These data are consistent with ectopic nuclear MAPK inducing cell proliferation, even in populations of cells that are normally non-proliferative (Marenda, 2006).

Continuous posterior-compartment driven Msk expression (en::msk) was used as a second test to determine the role of MAPK nuclear translocation in wing cell proliferation. Again, the fraction of GFP+, S-phase cells is increased (27% versus 16% for the control, anterior compartment GFP- cells), as is the fraction in G2/M (37% versus 32%), at the expense of cells in G1. Since Msk is continuously available in this experiment, this is interpreted as a summation of the transient 6 and 24 hour effects seen with NMG. Consistent with this, in en::msk discs, elevated posterior compartment expression of the S-phase limiting factor Cyclin E, the M-phase limiting factor String (stg:lacZ) and the S-phase marker BrdU, are seen. Taken together, these data suggest that MAPK nuclear translocation does indeed normally promote S-phase transition in developing wing cells (Marenda, 2006).

Elevated proliferation in the posterior compartment might be expected to produce adult wings with enlarged posteriors (the 'J.Lo wing'). However, prolonged and elevated expression of Msk induces caspase-dependent cell death and the resulting adult wings are severely disrupted, with nearly normal anterior compartments and severely reduced posteriors (the 'Twiggy wing'). These wings display loss of posterior tissue, including distal regions of veins L4 and L5, and fused posterior and anterior crossveins (Marenda, 2006).

Reduction of EGFR pathway function via loss of one copy of the gene encoding MAPK (rl10A) strongly suppresses the Msk-induced Twiggy wing, consistent with the Msk overexpression phenotype being dependant on MAPK. If Msk is limiting in the wing, then msk gene dose should affect vein formation. msk gain-of-function should suppress vein formation, while msk loss-of-function should enhance vein formation (Marenda, 2006).

To examine Msk gain-of-function, overexpression of the negative ligand Argos in the posterior compartment of the wing, which leads to vein loss 100% in vein L4, and 90% in vein L5, was examined. When Msk and Argos are co-expressed, Msk enhances the vein loss phenotype of Argos to 100% in L4 and 100% in L5. Similarly, overexpression of the nuclear ETS domain transcription factor Pointed P2 (PntP2, a positive MAPK effector) induces vein loss in 0% in L4 and 89% in L5, consistent with the suggestion that MAPK nuclear function antagonizes vein fate. Co-expression of Msk and PntP2 further enhances this vein loss to 97% in L4 and 100% in L5 (Marenda, 2006).

To examine msk loss of function, interactions were examined of a msk null allele (msk5) with a rho gain-of-function allele (hs-rho30a, and a rolled gain-of-function allele (rlSem), both of which dominantly cause extra vein formation. Trans-heterozygous hs-rho30a/msk5 wings show a strong enhancement of the rho extra-vein phenotype. Similarly, trans-heterozygous rlSem/msk5 wings also show enhancement of the rolled extra vein phenotype. Furthermore, Msk gain of function suppresses the extra veins caused by both hs-rho30a and UAS:rlSem expression (Marenda, 2006).

Though these effects may reflect additive genetic phenotypes as opposed to true genetic interactions, when taken together, gain-of-function and loss-of-function data suggest that Msk normally functions to restrict vein formation. It is suggested that this is because gain of msk function leads to increased nuclear MAPK (vein loss), while loss of msk leads to increased cytoplasmic MAPK (extra veins). These data are consistent with the suggestion that vein formation through MAPK occurs through a cytoplasmic, rather than a nuclear target (Marenda, 2006).

In summary this study has report the existence and contribution of MAPK cytoplasmic hold in the developing Drosophila wing. A difference was observed in cytoplasmic versus nuclear function of MAPK, and it is suggested that in the developing wing, MAPK subcellular localization controls the difference between vein specification (cytoplasmic MAPK) and proliferation (nuclear MAPK).

Perhaps vein differentiation is simply an indirect effect of repressing cell proliferation by inhibiting MAPK nuclear translocation. To address this, vein formation was analyzed in adult wings overexpressing both positive and negative cell cycle regulators. If vein formation is lost by inducing cell proliferation with effects other than forced nuclear MAPK, this would argue that the observed vein loss in en::msk is most probably due to an indirect effect of disrupting cell proliferation, as opposed to disrupting cytoplasmic pMAPK (Marenda, 2006).

Overexpression of either CycE or Stg leads to increased proliferation in Drosophila wings; however, there is little to no effect on vein formation, with no vein loss in either case. Similarly, inhibiting cell proliferation by over-expressing either the cyclin-dependant kinase inhibitor dacapo, or the S-phase inhibitor p21 had no significant effect on vein formation. This is consistent with a direct effect for MAPK cytoplasmic hold on vein differentiation (Marenda, 2006).

There are a number of known cytoplasmic targets of MAPK, including p90RSK, cPLA2 and Myosin light chain kinase (Ebisuya, 2005). However, it is important to consider that some cytoplasmic target proteins for MAPK may first be phosphorylated in the cytoplasm and then translocate to the nucleus, or be inhibited from doing so, such as SV40 T-antigen and Xenopus nucleoplasmin. In fact, it has recently been reported that the co-repressor Groucho is directly phosphorylated by MAPK, and this phosphorylation weakens its repressor activity, leading to extra veins (Hasson, 2005). Groucho, though it functions as a nuclear transcription factor, may be phosphorylated in the cytoplasm in pro-vein cells, where it can then translocate to the nucleus to affect changes in Notch transcription, leading to vein formation (Marenda, 2006).

Recent reports suggest that MAPK cytoplasmic hold may perform similar functions in mammals (Ebisuya, 2005). In vertebrate cells, expression of the death effector PEA-15 can sequester pMAPK in the cytoplasm. After treatment with Retinoic acid, embryonic stem and carcinoma cells stop proliferating, restrict the nuclear entry of pMAPK and differentiate into primitive endoderm (Smith, 2004). In the mouse embryo, pMAPK is detected in the cytoplasm rather than the nuclei of cells receiving FGF signals (Corson, 2003). A family of proteins called SEFs antagonize MAPK signaling (Fürthauer, 2002). More recently, SEF has been found to act directly to hold pMAPK in the cytoplasm, suggesting a mechanism for FGF pathway attenuation through MAPK cytoplasmic hold. No homolog of PEA-15 or SEF has been identified outside the chordates by conventional bioinformatic techniques. However, a fly protein with a function that is very similar to SEF would fit the MAPK cytoplasmic hold phenomena observed in the eye and wing (Marenda, 2006).

While anchoring of pMAPK has been shown to restrict MAPK nuclear entry in cell culture, it remains possible that pMAPK nuclear import could be prevented by removing a required nuclear import co-factor. Thus, by cytoplasmic sequestration of Msk (for example), pMAPK would be unable to translocate into the nucleus, and pMAPK cytoplasmic hold would be achieved (Marenda, 2006).

Regardless of the mechanism, MAPK cytoplasmic hold may be a conserved mechanism necessary for the differentiation of certain developing tissues in many taxa, and proper control of MAPK subcellular localization may act as a developmental signal to determine the proliferative state of a cell (Marenda, 2006).

Mammalian importin 7 is reported to import several proteins into the nucleus, including histone H1, core histones, HIV-1 reverse transcription complexes and the glucocorticoid receptor. However, the current data suggest that MAPK is a crucial target for the phenotypes observed in wings overexpressing Msk: (1) a null mutation in Drosophila MAPK strongly suppresses the en::msk adult wing phenotype; (2) increased nuclear MAPK is observed after overexpression of Msk in larval wings; (3) loss-of-function mutations in Drosophila Histone H1 [Su(var)205] have no effect on the en::msk phenotype; (4) loss-of-function mutations in members of other vein promoting pathways (thick veins, tkv8) have no effect on the en::msk adult wing (Marenda, 2006).

In the developing compound eye, breaking MAPK cytoplasmic hold in cells within the morphogenetic furrow results in reduced expression of Atonal, which is required for the initiation of differentiation in the developing eye. Taken together with new data from the developing wing, it is suggested that MAPK cytoplasmic hold may be generally required for the cell cycle arrest necessary for the initiation of differentiation, thus defining a novel bifurcation in the Ras pathway to control different cellular outcomes. Finally, the regulation of MAPK cytoplasmic hold may help to distinguish the MAPK signals for cell fate from those for cell proliferation (Marenda, 2006).

smoothened and thickveins regulate Moleskin/Importin 7-mediated MAP kinase signaling in the developing Drosophila eye

The Drosophila Mitogen Activated Protein Kinase (MAPK) Rolled is a key regulator of developmental signaling, relaying information from the cytoplasm into the nucleus. Cytoplasmic MEK phosphorylates MAPK (pMAPK), which then dimerizes and translocates to the nucleus where it regulates transcription factors. In cell culture, MAPK nuclear translocation directly follows phosphorylation, but in developing tissues pMAPK can be held in the cytoplasm for extended periods (hours). This study shows that Moleskin antigen (Drosophila Importin 7/Msk), a MAPK transport factor, is sequestered apically at a time when lateral inhibition is required for patterning in the developing eye. It is suggested that this apical restriction of Msk limits MAPK nuclear translocation and blocks Ras pathway nuclear signaling. Ectopic expression of Msk overcomes this block and disrupts patterning. Additionally, the MAPK cytoplasmic hold is genetically dependent on the presence of Decapentaplegic (Dpp) and Hedgehog receptors (Vrailas, 2006).

Early in eye development, all cells anterior to the furrow (phase 0) are primed for Ras-induced neural differentiation; ectopic activation of the pathway causes all cells to differentiate as photoreceptors, even without atonal. Normally these cells are thought to receive only low levels of Egfr-mediated Ras signaling, supporting proliferation but not differentiation. Later, in the furrow (phase 1), Delta-induced, Notch-mediated lateral inhibition progressively restricts Atonal expression to single founder cells. Suspension of Ras signaling is required for this inhibition in order to avoid premature neuronal differentiation, and it has been proposed that this inhibition is mediated by MAPK cytoplasmic hold. However, this block to the Ras pathway must be released in phase 2 (posterior to the furrow) to allow for developmental induction by the R8 cell. To better understand how MAPK cytoplasmic hold is maintained in phase 1, the role was examined of the pMAPK nuclear transport factor Drosophila Importin 7/Msk, in eye development (Vrailas, 2006).

It is suggested that in wild-type eye discs, the level of pMAPK antigen is a very misleading reporter of Egfr/Ras pathway activity, because cytoplasmic hold in phase 1 allows even a relatively low level of pathway activity to build up high levels of pMAPK antigen. A system has been developed to reveal MAPK nuclear translocation without the use of an antibody (MG-driven reporter gene expression that reveals MAPK nuclear translocation). [Note: MG (Mapk-Gal4vp16) contains the entire sequence of Rolled, followed by the yeast GAL4 DNA binding domain (which is not known to contain a nuclear localization signal) with an acidic activation domain from herpes simplex virus protein 16]. However, it has been since found that under all conditions tested, MG-driven reporter expression does not reveal nuclear MAPK in phase 0, where Ras pathway activation is required. MG-driven reporter expression is reliably see in phase 2, where there is thought to be high (or sustained) levels of Ras pathway activity. In phase 1, the level of pathway signaling may be insufficient for expression, and thus MG-driven reporter expression may reveal only high (or sustained) levels of nuclear MAPK. Alternatively, this could be caused by a technical limitation: the hsp70 promoter drives the expression of only low levels of MG protein. Therefore, two less direct assays were used, that together, are interpreted as revealing the loss of MAPK cytoplasmic hold in the furrow: (1) loss of Atonal expression (as previously demonstrated by fusing an SV40 NLS to MAPK and by the ectopic expression of Rasv12); and (2) loss of pMAPK antigen, which may be due to exposure to a nuclear phosphatase/protease (Vrailas, 2006).

The MAPK nuclear transport factor Drosophila Importin 7/Msk is apically sequestered in phase 1, the time when pMAPK nuclear access is blocked. Furthermore, ectopic Msk is sufficient to break the cytoplasmic hold in the furrow, as seen by loss of pMAPK antigen and suppression of the early stages of Atonal expression. However, this transient expression of Msk is unable to promote the precocious neural differentiation or the increase in rough expression, as has been seen with hs:rasv12 or nuclear-directed MAPK. Because ectopic rasv12 produces an increase in pMAPK, and the phosphorylation state of nuclear-directed MAPK is not required for nuclear translocation, it may be that the available pool of pMAPK that can be imported into the nucleus by Msk is enough to affect Atonal expression, but not to affect Elav or Rough expression. Genetic evidence shows that the MAPK cytoplasmic hold depends on the Hedgehog receptor Smo and is enhanced by the loss of the Dpp receptor Tkv. smo loss-of-function clones reduce Atonal and pMAPK expression, whereas tkv clones have much weaker effects. However, the loss of smo and tkv together completely abolishes both pMAPK and Atonal expression in the furrow. This is consistent with a previous report of the loss of Atonal expression in smo tkv clones. Additionally, MAPK cytoplasmic hold in smo tkv clones is rescued by the additional loss of msk. Thus, msk genetically antagonizes pMAPK levels in the morphogenetic furrow: msk gain-of-function reduces pMAPK and msk loss-of-function (in smo tkv clones) increases it (Vrailas, 2006).

Hedgehog signaling has also been reported as a positive regulator of Atonal on the anterior side of the furrow and as a negative regulator (perhaps through Rough or Bar) on the posterior side. However, the inductive effect of Hedgehog on Atonal appears to be independent of the Hedgehog pathway transcription factor Ci, which is consistent with an indirect effect through the MAPK cytoplasmic hold. smo tkv msk triple mutant clones were used to show that msk is genetically epistatic to smo and tkv in the furrow, and suggest that Msk sequestration in the furrow is required for MAPK cytoplasmic hold, and that smo and tkv are genetically upstream of this sequestration of Msk. Indeed, loss of smo and tkv results in a disruption of the actin cytoskeleton in the furrow, as well as of expression of Egfr and other signaling molecules. The loss of apical constriction may therefore disrupt Msk apical sequestration in such a way as to allow precocious Msk-mediated pMAPK nuclear import (Vrailas, 2006).

What is more surprising is that differentiation and ommatidial assembly, which are known to require Ras signaling and MAPK nuclear translocation, occur normally in the absence of Msk in phase 2. It may be that cytoplasmic MAPK targets are important for ommatidial assembly or that pMAPK can translocate into the nucleus by some Ran-independent mechanism. However, the possibility is favored that, in phase 2, other (possibly redundant) transport factors are expressed (Vrailas, 2006).

Like the Ras pathway, msk plays a role in ommatidial rotation but not chirality. It may be that in the absence of Msk, enough pMAPK can translocate into the nucleus for ommatidial assembly, but not enough for proper rotation. Additionally, in phase 0, Msk is found to be required for proliferation, which also requires Ras signaling. Therefore, Msk is required for some pMAPK nuclear translocation in phase 0 and phase 2, but is not necessary in phase 1, in order to allow for the initial specification of the Atonal-positive R8 (Vrailas, 2006).

To conclude, the apical sequestration of Drosophila Importin 7/Msk in the morphogenetic furrow has been identified and it is suggested that this may be required for the MAPK cytoplasmic hold in the developing eye. Cytoplasmic hold is required to allow initial patterning through lateral inhibition and the focusing of the proneural factor Atonal. It is further suggested that this is mediated by the combined action of Hedgehog and Dpp (Vrailas, 2006).

Genetic interaction between integrins and moleskin, a gene encoding a Drosophila homolog of Importin-7

The Drosophila PS1 and PS2 integrins are required to maintain the connection between the dorsal and ventral wing epithelia. αPS subunits are inappropriately expressed during early pupariation via the Blistermaker chromosome (containing a PS2 gene driven by the wing pouch enhancer trap, 684). Inappropriate expression of αPS2 results in the separation of epithelia, causing a wing blister. Two lines of evidence indicate that this apparent loss-of-function phenotype is not a dominant negative effect, but is due to inappropriate expression of functional integrins: (1) wing blisters are not generated efficiently by misexpression of loss-of-function αPS2 subunits with mutations that inhibit ligand binding, and (2) gain-of-function, hyperactivated mutant αPS2 proteins cause blistering at expression levels well below those required by wild-type proteins. A genetic screen was carried out for dominant suppressors of Blistermaker induced wing blisters. Suppression was induced by null alleles of a gene named moleskin, which encodes the protein DIM-7. DIM-7, a Drosophila homolog of vertebrate importin-7, has been shown to bind the SHP-2 tyrosine phosphatase homolog Corkscrew and to be important in the nuclear translocation of activated D-ERK (Rolled). Consistent with this latter finding, homozygous mutant clones of moleskin fail to grow in the wing. Genetic tests suggest that the moleskin suppression of wing blisters is not directly related to inhibition of D-ERK nuclear import (Baker, 2002).

The ß-importin family of proteins is principally linked with nuclear import of protein cargos. However, recently other functions have been associated with members of the importin superfamily. For example, importin-ß, in some cases with importin-α, functions in vertebrates to sequester microtubule polymerization factors early in mitosis. Mitotic microtubule formation can be triggered by the release of the polymerizaion regulators by RanGTP, just as RanGTP binding to importin-ß leads to release of cargos inside the nucleus. DIM-7 protein can be detected immunologically at the cell cortex, both in early Drosophila embryos and in S2 cells in culture. It thus seems reasonable to consider a more direct connection between the peripheral DIM-7 and integrin regulation. Additionally, it appears that a mutation in corkscrew, the Drosophila SHP-2 homolog, can also suppress Blistermaker and that Corkscrew protein binds directly to DIM-7. Although Corkscrew has been implicated primarily in signaling events downstream of receptor tyrosine kinases, vertebrate SHP-2 has been implicated in signaling via a host of growth factor receptors, cytokines, hormones, and antigens. Most relevant to this study, SHP-2, often in association with the membrane glycoproteins PECAM-1 or SHPS-1, has been shown to be involved in many integrin-dependent signaling events and also to be important in regulating integrin-mediated cell adhesion, spreading, or migration. While SHP-2 is a cytoplasmic tyrosine phosphatase, some experiments suggest that it can serve as a scaffolding protein at or near the plasma membrane. For example, a Corkscrew protein mutated in the phosphatase domain retains significant wild-type activity in situ, and this activity is increased if the protein is targeted to the plasma membrane (Baker, 2002).

It is likely therefore that cell surface receptors mediate a localized Corkscrew/SHP-2 activation of cortical DIM-7. This active DIM-7, in combination with associated factors such as D-ERK, could then function more directly in integrin regulation. A more direct connection between DIM-7 and integrin function is also consistent with the fact that moleskin mutations were especially common among the suppressors isolated in the screen. A key question for future work, therefore, will be defining the subcellular location at which DIM-7 functions with respect to integrin-related phenotypes (Baker, 2002).

Recently, evidence has begun to appear that integrin engagement with the ECM can regulate nuclear import of regulatory molecules. For example, there is an association between αLß2 and the c-Jun coactivator JAB1; this connection is suggested to regulate the nuclear localization of JAB1. More directly relevant to these results, ERK nuclear translocation in fibroblasts is dependent on an integrin-mediated event, also involving the actin cytoskeleton. Also, primary mouse embryo fibroblasts with a ß1 integrin cytoplasmic mutant show reduced nuclear translocation of phosphorylated ERK. Regardless of the importance of nuclear transport in Blistermaker suppression, the genetic data indicate a functional connection between integrins and a specific importin-ß that can transport activated ERK and suggest another potential molecular mechanism whereby integrin and growth factor signals can be integrated by the cell (Baker, 2002).

The moleskin gene product is essential for Caudal-mediated constitutive antifungal Drosomycin gene expression in Drosophila epithelia

The homeobox gene, Caudal, encodes the DNA-binding nuclear transcription factor that plays a crucial role during development and innate immune response. The Drosophila homologue of importin-7 (DIM-7), encoded by moleskin, was identified as a Caudal-interacting molecule during yeast two-hybrid screening. Both mutation of the minimal region of Caudal responsible for Moleskin binding and RNA interference (RNAi) of moleskin dramatically inhibited the Caudal nuclear localization. Furthermore, Caudal-mediated constitutive expression of antifungal Drosomycin gene was severely affected in the moleskin-RNAi flies, showing a local Drosomycin expression pattern indistinguishable from that of the Caudal-RNAi flies. These in vivo data suggest that DIM-7 mediates Caudal nuclear localization, which is important for the proper Caudal function necessary for regulating innate immune genes in Drosophila (Han, 2004).

Analysis of Corkscrew signaling in the Drosophila Epidermal growth factor receptor pathway during myogenesis

The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the Epidermal growth factor receptor (Egfr). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require Egfr function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the Egfr, spitz (spi), rhomboid (rho), daughter of sevenless (Dos), kinase-suppressor of ras (ksr), ras1, D-raf, pointed (pnt), and moleskin. The Egfr-dependent formation of VA2 muscle precursor cells was followed as a sensitive assay for these genetic interaction studies. Csw is shown to have a positive function during mesoderm development. Tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled (rl)/MAPK in the EGFR pathway. Levels of Egfr signaling in various mutant backgrounds during myogenesis could be inferred. This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the EGFR signaling pathway (Hamlet, 2001).

A variety of genetic interaction experiments between gain- and loss-of-function mutations and/or constructs in genes involved in Egfr signaling has resulted in three principal findings. (1) Consistent with findings in the developing retina, Cswsrc90 functions like a bona fide gain-of-function protein in several Egfr-initiated developmental processes during oogenesis, embryogenesis, and metamorphosis. (2) Csw plays a positive role in Egfr signaling during myogenesis. (3) Tracking the formation of VA2 precursor cells serves as a sensitive assay to infer levels of Egfr signaling in various mutant genetic backgrounds (Hamlet, 2001).

Expression of UAS-cswsrc90 in several tissues phenocopies gain-of-function mutations and constructs in positive signaling genes in the Egfr pathway. Moreover, tissue-specific expression of cswsrc90 is able to rescue VA2 precursor cell formation in loss-of-function csw mutant embryos. However, there are important considerations to be made regarding use of the cswsrc90 construct to study Csw function in RTK pathways. cswsrc90, being a synthetic mutation, may have neomorphic activity, the result of which is an artificial, nonspecific phenotype not correlating with wild-type Csw function. For instance, in embryos expressing two copies of UAS-cswsrc90 in the mesoderm, Eve-positive cells form outside of the normal boundaries previously prepatterned by Wg signaling. This phenotype resembles the effect seen by simultaneous overexpression of UAS-wingless, UAS-twist (Twist is a downstream target of Wg signaling), and activated ras1 (UAS-ras1ACT) in the embryonic mesoderm, but not by expression of UAS-ras1ACT alone. This result, seen with two copies of UAS-cswsrc90, might reveal a nonphysiological ability for Cswsrc90 to bypass the need for Wg signaling at the transcriptional level during myogenesis (Hamlet, 2001).

While the possibility that Cswsrc90 exhibits some neomorphic properties cannot be ruled out, it is notable that, in all developmental contexts examined, the phenotypes resulting from expression of one copy of UAS-cswsrc90 never differed from what was expected for a gain-of-function csw mutation. Therefore, phenotypes were examined only in embryos in which one copy of UAS-cswsrc90 was expressed (Hamlet, 2001).

Furthermore, the phenotypes do not reflect promiscuous phosphatase activity because membrane-targeted expression solely of the Csw phosphatase domain is embryonic lethal and results in cuticle phenotypes not reflecting a predicted gain-of-function csw mutation (Hamlet, 2001).

Interestingly, no phenotypes were observed when wild-type csw (UAS-cswWT) was expressed using twi-Gal4 in various genetic backgrounds. While this could be due to the extent to which UAS-cswWT was expressed, on the basis of what is known about the regulation of its vertebrate functional homolog SHP-2, an alternative explanation is that simply adding more wild-type Csw in an otherwise wild-type background is not sufficient to increase its activity (Hamlet, 2001).

The crystal structure SHP-2 has revealed that the N-terminal SH2 domain binds to the catalytic domain, which keeps SHP-2 inactive. Engagement of the N-terminal SH2 domain with a tyrosine-phosphorylated protein releases the block of the catalytic domain, resulting in SHP-2 activation (Hof, 1998). Thus, if the molecules that engage the SH2 domain of Csw are limiting in amount, exogenously expressed wild-type Csw protein would not be able to release the N-terminal SH2 domain from the catalytic domain, thereby keeping the exogenous wild-type Csw protein in an inactive state. However, the myristylated and thereby membrane-targeted Cswsrc90 protein is already in an active state, which results in hyperactivation of the RTK pathway. Cswsrc90 is hence insensitive to the normal downregulation of the RTK signal that occurs. The mechanism of action of Cswsrc90 is unknown, but it is possible that membrane localization either provides constitutive access to substrates or changes the conformation of Cswsrc90 such that the N-terminal SH2 domain is unable to bind to the catalytic domain to block its function. Nevertheless, the phenotypes produced by cswsrc90 are consistent with those expected for a gain-of-function csw mutation (Hamlet, 2001).

Within the context of VA2 precursor cell formation, this study enables the inference of the relative contribution of gene function to the Egfr signal. For example, complete loss-of-function mutations in spi, rho, and D-raf essentially eliminate VA2 precursor cells, supporting the idea that these proteins are absolutely essential for the propagation of the Egfr signal (Hamlet, 2001).

The phenotype of csw loss-of-function mutant embryos is not as severe as the phenotypes of loss-of-function mutations in other positive RTK transducers, suggesting that Csw, unlike spi, rho, and D-raf, is not needed to transduce the entire RTK signal. Further support for this finding comes from the similar levels, although <100%, to which Cswsrc90 rescues VA2 precursor cell formation in spi, rho, and twi-Gal4/+; UAS-EgfrDNDER mutant embryos. This latter finding places the interaction of Cswsrc90 with these upstream signaling components in a separate category from that of the other genes analyzed (Hamlet, 2001).

Genetic interaction data between csw and Dos are consistent with a model whereby a direct interaction between Csw and Dos is essential for Drosophila Egfr signaling. A Dos protein containing only the pTyr sites that bind to the Csw SH2 domains is sufficient to provide wild-type Dos function. A vertebrate Dos homolog, Gab1, and SHP-2 associate upon activation of the vertebrate Egfr, results in an increase in MAPK signaling (Hamlet, 2001 and references therein).

The readout from the putative Dos dominant-negative mutant embryos is in the same range as that of dominant-negative csw mutant embryos. The identical genetic interaction of csw and Dos with cswsrc90 places their function in a category separate from that of the other signaling genes analyzed and suggests that they both function at the same level in the Egfr pathway (Hamlet, 2001).

Interestingly, Dos mutant embryos phenocopy the putative dominant-negative csw mutant embryos but not the protein null csw mutant embryos. These results suggest that the dominant-negative csw mutant phenotype reflects loss of Dos function. Since the cswVA199 mutation generates a truncated Csw protein where only the SH2 domains are expressed, perhaps the SH2 domains still bind to and sequester Dos function away from the signaling pathway (Hamlet, 2001).

Loss-of-function mutations derived from females bearing germline clones in ras1, ksr, and D-raf result in 9%, 4.5%, and 1.2%, respectively, of hemisegments in which VA2 precursor cells form. The D-raf and spi mutant phenotypes are nearly the same, suggesting that Spi and D-raf are absolutely essential for Egfr signal propagation. However, the ras1 protein null phenotype is not as strong as the D-raf protein null phenotype, suggesting that Ras1 transduces <100% of the Egfr signal. These results correlate well with phenotypic analyses of ras1 and D-raf in the Torso pathway where loss-of-function ras1 mutant embryos maintain a low level of Torso signaling, whereas loss-of-function mutations in D-raf abolish Torso signaling. Hence, it can be inferred from these studies that in the Egfr pathway, as perhaps in the Torso pathway, there is also a Ras1-independent mechanism to activate D-Raf (Hamlet, 2001).

The loss-of-function ksr mutant phenotype suggests that Ksr contributes more function to the Egfr pathway than Ras1 but less than D-Raf. Similarly, in the Torso pathway, the ksr loss-of-function mutant phenotype is more severe than the ras1 loss-of-function mutant phenotype. These data suggest that loss of Ksr function is more detrimental to transducing an RTK signal than is loss of Ras1 function. Ksr is thought to function as a scaffolding protein that binds Raf1, MEK, Rl/MAPK, and other signaling molecules to regulate a given RTK pathway. Therefore, the phenotype of embryos lacking Ksr function is more severe than that from loss of Ras1 because Ksr directly regulates not only Raf1, but also other crucial downstream molecules such as Rl/MAPK. It has been proposed that the scaffold function of Ksr may be analogous to the budding yeast scaffolding protein Ste5, which binds the Raf, MEK, and MAPK yeast homologs to facilitate MAPK-induced signaling in the mating response pathway (Hamlet, 2001 and references therein).

In the Egfr pathway Csw functions downstream of or parallel to Ras1, Ksr, and D-Raf. Introduction of Cswsrc90 into ras1, ksr, and D-raf loss-of-function mutant embryos derived from females bearing germline clones rescues each mutation to the same extent above basal levels. These levels of rescue are much lower than those for spi, rho, and Egfr mutant embryos. One reason for these lower levels of rescue might be that since D-Raf is the major feed-in molecule at this level of the signaling pathway, its absence or the absence of one or more of its activators will severely block any downstream signaling. Nevertheless, these results suggest that a portion of the Egfr signal requires Csw downstream of, or parallel to, Ras1, Ksr, and D-Raf (Hamlet, 2001).

The similar genetic interactions of ras1, ksr, and D-raf with cswsrc90 place their functions in a category separate from that of the other signaling genes analyzed and suggest roles for Csw both upstream and downstream of these intermediate signaling components (Hamlet, 2001).

Since Cswsrc90 is able to function downstream of D-Raf, it is possible that Cswsrc90 is able to facilitate Ras1-dependent, D-Raf-independent signaling, as is proposed to happen during RTK-dependent border cell migration. Alternatively, a portion of the Csw signal may contribute to a pathway functioning parallel to the D-Raf/MEK/MAPK pathway, perhaps by facilitating activation of other MAPK homologs, such as p38/MAPK. Mutations in licorne, a p38/MAPKK homolog, can phenocopy loss-of-function Egfr mutations and might affect Grk activity during oogenesis, implicating a role for p38/MAPK signaling in the Egfr pathway (Hamlet, 2001 and references therein).

It is possible that Csw can function downstream of D-Raf at the level of Rl/MAPK. Csw physically interacts with the nuclear import protein DIM-7, a member of the importin family of nuclear import proteins, which is thought to transport Rl/MAPK to the nucleus. A genetic interaction between csw and moleskin, the gene encoding DIM-7, has been demonstrated, since loss of DIM-7 suppresses the phenotype associated with Cswsrc90. This result is consistent with DIM-7 functioning downstream of Csw, as well as with DIM-7-dependent transport of Rl/MAPK into the nucleus (Hamlet, 2001 and references therein).

Pnt is a downstream target of Rl/MAPK signaling and functions as a transcriptional activator in many RTK pathways, including the Drosophila Egfr pathway. Deletion of both pnt transcripts (P1 and P2) results in 82% of hemisegments in which VA2 precursor cells form. This result suggests that Pnt contributes a small amount to the Egfr signal in this developmental context and that there are other Rl/MAPK target transcription factors whose activities are also required for proper VA2 precursor cell formation. The same partial pnt mutant phenotype is also seen in the context of Eve muscle progenitor specification. Moreover, pnt mutant embryos primarily lack the lateral longitudinal muscle 1 and several dorsal oblique muscles (DO3, DO4, and DO5), suggesting that certain muscle precursor cells are more sensitive to loss of Pnt function. Nevertheless, Cswsrc90 is unable to rescue loss of VA2 precursor cell formation in pnt mutant embryos, suggesting that all Csw function is upstream of Pnt and thereby placing Pnt function in a category separate from that of the other signaling genes analyzed. It should be noted that these data do not allow the placement of Csw function relative to the unidentified, positive transcription factors in this pathway (Hamlet, 2001 and references therein).

On the basis of this work, a model is proposed for Csw function in the Egfr pathway during myogenesis. Activation of the Egfr pathway by Spi binding to the receptor results in an association between Csw and Dos. The Csw/Dos complex might interact with the receptor either via Dos, since it has been demonstrated that the Dos homolog Gab1 binds to the vertebrate Egfr, or via Csw, since there is a binding site on the Drosophila Egfr in consensus to bind the N-terminal SH2 domain of Csw. Also contributing to the positive signal is the adapter protein Shc. Subsequently, the majority of Ras1 function leads to activation of D-Raf. However, on the basis of the ras1 null mutant phenotype, other molecules are capable of contributing to D-Raf activation. One of these molecules is likely Ksr, which binds to and regulates the Raf, MEK, and Rl/MAPK signaling cassette (Hamlet, 2001 and references therein).

Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin

The initiation of gene expression in response to Drosophila receptor tyrosine kinase signaling requires the nuclear import of the MAP kinase, Rolled. However, the molecular details of Rolled translocation are largely unknown. In this regard, D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin have been identified. DIM-7 exhibits a dynamic nuclear localization pattern that overlaps the spatial and temporal profile of nuclear, activated Rolled. Co-immunoprecipitation experiments show that DIM-7 associates with phosphorylated Rolled in Drosophila S2 cells. Furthermore, moleskin mutations enhance hypomorphic and suppress hypermorphic rolled mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated Rolled. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin beta homolog Ketel also reduce the nuclear localization of activated Rolled. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated Rolled (Lorenzen, 2001).

The activation of ERK represents the focal point of a conserved signaling module used by a diverse array of extracellular stimuli. The potency and duration of ERK activation and its accompanying translocation to the nucleus can profoundly affect the fate of a cell. This is apparent in PC12 cells where the decision to proliferate or differentiate depends upon the number and duration of receptors stimulated. Throughout development, cells respond to spatial and temporal signals and must interpret gradients to produce qualitative differences in gene expression. In Drosophila, the terminal system or Torso RTK signaling pathway illustrates one example whereby quantitative differences in D-ERK activity generate distinct cell fates. Distinct quantitative levels of D-ERK activity inside a cell may be achieved within the RTK pathway by modulating D-ERK phosphorylation. Moreover, it is apparent that mechanisms exist to control the localization of ERK activity by either regulating its retention in the cytoplasm and/or its nucleocytoplasmic shuttling. In this regard, nuclear translocation of dpERK is not always a compulsory consequence of RTK signaling. In Drosophila as photoreceptors are recruited into the developing retina, dpERK is held in the cytoplasm for up to several hours following EGFR and Sevenless RTK signaling. This raises the possibility that import is differentially controlled relative to D-ERK phosphorylation and/or dimerization. Interest in the active transport of dpERK also partly stems from the observation that dimer formation is a common property of mammalian MAP kinase family members (Lorenzen, 2001).

Presumably, D-ERK shares this property, since the residues involved in dimer formation have been conserved. Although monomeric dpERK can enter the nucleus passively, it has been shown that import of dimeric ERK is an active process. This study has clarified mechanistic issues in the nuclear relocalization of dpERK through the identification of DIM-7, the Drosophila homolog of importin 7 and member of the importin superfamily of nuclear transport proteins. DIM-7 exhibits several properties that establish its participation in RTK signaling. These include a physical interaction with CSW and dpERK, and the finding that DIM-7 is tyrosine phosphorylated in stimulated cells. Furthermore, alleles of msk, the gene that encodes DIM-7, dominantly interact with hypomorphic and hypermorphic alleles of D-ERK (Lorenzen, 2001).

The primary structure of DIM-7 originally suggested that it might play a role in nuclear transport. In addition to exhibiting significant sequence identity with its Xenopus and human homologs, DIM-7 also possesses a conserved Ran-binding domain. This latter property is a hallmark of nuclear transport proteins that display a RanGDP versus RanGTP regulated interaction with their cargo proteins. In addition to physically binding to phosphorylated D-ERK, DIM-7 and dpERK have overlapping nuclear localization patterns in developing tissues subject to RTK regulation. This made dpERK an attractive candidate cargo for DIM-7. To address this possibility during embryogenesis and in the absence of functional DIM-7, tracheal placodes and tracheal pits were assayed for defects in the accumulation of dpERK. Significantly, embryos in which the genomic interval encoding the msk gene is deleted or mutated exhibit a fivefold reduction in the number of dpERK-positive nuclei. Importantly and demonstrating that DIM-7 is essential for nuclear uptake of dpERK, expression of wild-type DIM-7 in a msk mutant background restores dpERK nuclear accumulation. These findings reinforce the idea that not only is dpERK actively transported through the nuclear pore complex but also that DIM-7 functions as either the transport receptor and/or adapter for dpERK (Lorenzen, 2001).

Finally, this study has implicated KET in the nuclear import of dpERK. As vertebrate importin 7 and importin b form an abundant heterodimeric complex, it was asked whether the Drosophila homolog of importin b, KET participates in the dpERK transport mechanism. Supporting a model whereby DIM-7 and KET function together in the nuclear transport of dpERK, it was found that nuclear localization of dpERK is impaired in homozygous ket mutant embryos. Although there are other possibilities, two models are envisioned by which DIM-7 could function in the nuclear import cycle of dpERK. In one, DIM-7 and KET could function together in the same import cycle, where DIM-7 and KET serve as the import adapter and import receptor, respectively. Alternatively, DIM-7 and KET could function independently of each other in separate import cycles that could be, at least partially, redundant. However, it is unlikely that DIM-7 and KET serve totally redundant functions for two reasons. First, each individual locus when deleted (msk) or mutated (msk and ket) reduces substantially the number of dpERK-positive nuclei, and, second, both msk and ket mutations, alone, are lethal (Lorenzen, 2001).

In the literature there appears to be an increasing number of transport receptors with complex cargo specificities. If importin 7 is the functional vertebrate homolog of DIM-7, then this transport factor can import at least three different proteins, dpERK, histone H1 and rpL23a. An additional point concerning specificity of DIM-7 regards its nearest homolog, Nmd5p, in Saccharomyces cerevisiae. Nmd5p is essential for the nuclear import of HOG1, a p38 MAP kinase family member that is activated in response to osmotic stress. Interestingly, the movement of HOG1 into the nucleus does not require the importin b homolog, RSL1. This work raises the possibility that DIM-7 might also mediate the nuclear transport of one or more other Drosophila MAPkinase family members, D-p38a (Mpk2), D-p38b (Mpk34C) and D-JNK (JUN kinase). The combinatorial use of different transport factors may provide a means for specific recognition of the various MAP kinase family members. For example, DIM-7 alone may bind and import D-p38; however, recognition of dpERK may require the simultaneous pairing of DIM-7 and KET. Determining the mechanism(s) employed to establish recognition between a cargo and its transport receptor will require a precise molecular dissection of the interactions between several transport receptor/cargo pairs (Lorenzen, 2001).

CSW was first demonstrated to have a positive function during embryogenesis in the Torso RTK pathway. In this pathway CSW serves two functions. First, the adapter protein DRK does not bind Torso; instead, CSW functions as an adapter linking Torso to RAS. Second, CSW is able to dephosphorylate the Torso autophosphorylation site that binds RasGAP. The work presented in this paper suggests a third function for CSW as an adapter to facilitate the physical interaction of DIM-7 with its import cargo dpERK. When two, or three, of these CSW functions are used within one signaling module, interpreting the epistatic relationships of CSW with various signaling components could become problematic. For example, previous epistasis experiments have suggested that CSW carries out its function either upstream or downstream of RAS1 and/or D-RAF. Now it appears these differences could simply reflect the differential use of the various signaling capabilities of CSW within a given RTK pathway (Lorenzen, 2001).

Whether or not the association of DIM-7 with CSW constitutes part of a regulatory process at the level of the receptor that governs D-ERK redistribution has yet to be determined. However, it appears that nuclear import is a fundamental mechanism used by cells to modulate incoming signals throughout development. It is expected, then, that the development of reagents to modulate the nuclear entry of specific molecules may have profound effects for controling both disease and oncogenic states (Lorenzen, 2001).


REFERENCES

Search PubMed for articles about Drosophila Moleskin

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date revised: 5 December 2013

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