BIOLOGICAL OVERVIEW

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose has also been identified in a genetic screen for modifiers of Hairless (H), a Notch pathway antagonist (Schreiber, 2002) and rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

Activation of Protein kinase A (PKA) at discrete intracellular sites facilitates oogenesis and development in Drosophila. Thus, PKA-anchor protein complexes may be involved in controlling these crucial biological processes. Evaluation of this proposition requires knowledge of PKA binding/targeting proteins in the fly. DAKAP550/Rugose is a large (>2300 amino acids) acidic protein that is maximally expressed in anterior tissues. Later in development it is found in neuroblasts and is enriched in neurons. It binds regulatory subunits (RII) of both mammalian and Drosophila PKAII isoforms. The anchor protein is expressed in many cells in nearly all tissues throughout the lifespan of the fly. However, DAKAP550 is highly enriched and asymmetrically positioned in subpopulations of neurons and in apical portions of cells in gut and trachea. The combination of RII (PKAII) binding activity with differential expression and polarized localization is consistent with a role for DAKAP550 in creating target loci for the reception of signals carried by cAMP (Han, 1997).

Genetic studies in Drosophila have shown that cAMP and PKA (A kinase or protein kinase A)-mediated signaling is required in a variety of processes, including oogenesis, establishment of tissue polarity in the embryo, imaginal disc morphogenesis, and synaptic function. In the developing eye, PKA plays an important role in the initiation of pattern formation and morphogenesis through its interactions with Hedgehog (Hh), DPP, and Wingless (Wg). Signaling by Sev, Egfr, N, and the cAMP-PKA pathways leads to the activation of a variety of downstream transcription factors. Because these signaling pathways regulate a multitude of cellular functions in different tissues and due to the number of transcription factors involved, it became apparent that a single signaling pathway alone could not specify particular cell fates. Recent studies have demonstrated that the determination of specific cell fates involves the integration of inputs from multiple pathways and a combinatorial code of activated transcription factors generates specific cell types. A kinase anchor proteins (AKAPs) are important components of the cAMP-PKA-mediated signal transduction pathway. AKAPs modulate the specificity of PKA function by targeting and compartmentalization of PKA to specific subcellular structures (Pawson, 1997; Scott, 2000). Studies on mammalian systems have led to the identification and molecular characterization of a large number of AKAPs, which mediate targeting of the PKA to various cellular organelles (Gray, 1998; Eide, 1998; Vo, 1998). Tissue and cell type-specific isoforms of AKAPs have recently been characterized (Dong, 1998). In Drosophila, biochemical characterization and molecular cloning led to the identification of two AKAPs, DAKAP 550 and DAKAP 200; however, no physiological roles have been reported for these proteins (Han, 1997; Li, 1999). Data from the genetic and phenotypic analyses of rugose mutants implicate DAKAP 550 in retinal pattern formation. Data on the genetic interactions of rugose suggest that rugose interacts with components of the signaling pathway mediated by the Egfr and N (Shamloula, 2002 and references therein).

The wild-type Drosophila eye is a hexagonal array of about 800 ommatidia. This arrangement of the ommatidia gives a smooth appearance to the external surface of the eye. Any perturbation of the reiterated pattern of the ommatidia results in a rough eye surface. In the wild-type eye each ommatidium contains eight photoreceptor cells (R cells), which occupy stereotypic positions. The plasma membrane of the R cells is multiply folded to form a tightly compacted array of microvilli known as the rhabdomere. The rhabdomeres of the cells R1–R6 are arranged in an asymmetric trapezoidal manner and extend the length of the retina. The rhabdomeres of the two central cells, R7 and R8, are smaller in profile and shorter in length than the R1–R6 rhabdomeres. They are positioned in the same axial plane as the distal R7 rhabdomere, on top of the more basal R8 rhabdomere. The lens-secreting cone cells are found on top of the apical surface of the R cells. The cone cells extend into thin bag-like structures between the R cells and terminate at the floor of the retina where their feet contribute to the formation of the fenestrated basement membrane. The pigment cells ensheathe the R cells and the cone cells. The anterior and posterior primary pigment cells form a cup-like structure around the cone cells. The secondary and tertiary pigment cells form a hexagonal lattice enclosing the ommatidia within. Each ommatidium shares six secondary pigment cells and three tertiary cells with its neighbors. The feet of the secondary and tertiary pigment cells flatten into the fenestrated membrane and form the floor of the retina. A scanning electron micrograph of the compound eye of a rg null allele (rg^gamma6) shows that the eyes of rg mutants are characterized by a rough external surface. The ommatidial facets of the rg mutants are irregular and not hexagonal as in the wild-type eyes. In addition, the mechano-sensory bristles are irregularly positioned and often multiple bristles are seen in place of a single bristle in each corner of the hexagonal facet seen in the wild type. Frequently in rg mutant eyes, the ommatidia exhibit abnormal numbers of R cells. The precise positions of the R cells are altered and the pigment cell lattice loses its hexagonal arrangement leading to the disorganization of the retina. The missing retinal photoreceptors can be seen in the region below the fenestrated basement membrane as seen in transverse sections of the compound eyes. This mispositioning of the R cells may be due to the defects in the fenestrated basement membrane caused by the abnormal number and organization of the cone cells and the disorganized pigment cell lattice (Shamloula, 2002).

To understand the cellular basis of the rough eye phenotype of the rg mutants the number and organization of the cone cells in wild type and rg mutants was examined. The number of cone cells can be determined by staining pupal stage eyes with cobalt sulfide. The cone cell number and organization in the various rugose mutant alleles has been determined. In the wild type, four cone cells are apical to the R cells and the individual cone cells can be identified by their specific position. On the basis of their positions, the four cone cells can be identified as an anterior cone cell, a posterior cone cell, an equatorial cone cell, and a polar cone cell. The eyes of rg mutants show abnormal numbers of cone cells when compared to the wild type. The number of cone cells varies in the different mutant alleles and is dependent on the level of the residual rg gene function. Partial loss-of-function alleles such as rg^gamma10 show two or three cone cells in the majority of the ommatidia, while strong alleles that behave as genetic nulls have either one or no cone cells in the majority of the ommatidia. Some of the rg mutant alleles also exhibit aberrant numbers of secondary/tertiary pigment cells (Shamloula, 2002).

Cut protein expression was used to identify the differentiating cone cells in the developing eye. Cut protein is a transcription factor and its expression in the developing eye is specific to cells that assume a cone cell fate. To localize cone cells in the developing eye, 60-hr pupal eyes were stained with the anti-Cut antibody. In the wild-type pupal eye, four cone cells are labeled with the anti-Cut antibody in each ommatidium whereas the hypomorphic rugose allele rg^gamma10 shows either one or two Cut positive cells. In the null allele, rg^gamma6, no Cut positive cells are seen showing the lack of differentiating cone cells (Shamloula, 2002).

To ascertain if the aberrant number of cone cells seen in rg mutants is due to failure of cone cell differentiation, developing pupal stage eyes were examined using transmission electron microscopy. In the developing eye, the R7 cell is the last photoreceptor to differentiate, resulting in a mature eight-cell cluster. The cone cells differentiate during the late third larval instar stages of development. Once the R7 cell nucleus completes its apical migration, two cone cell nuclei move up from the basal region and flank the R cell cluster. The cone cells spread over the apical tips of the photoreceptor cells and become the anterior and posterior cone cells. These cone cells contact each other centrally above the R cells. Subsequently, two more cells, the equatorial and polar cone cells, are added resulting in four cone cells. During the early pupal stages, in a wild-type eye a stereotypic arrangement of the R cells and the cone cells can be seen at the four-cone cell stage. In rugose mutants, cone cell specification is abnormal and consequently some of the cone cells fail to differentiate. In the hypomorphic allele, rg^gamma10, a single cone cell is seen, while in the null allele rg^gamma3 no cone cells are seen. In all of the mutant alleles tested, an undifferentiated precursor cell occupies the position of the cone cell and fails to differentiate (Shamloula, 2002).

To determine if mutations at the rugose locus affect R cell differentiation, the sequential specification of the R cells was followed in the third instar eye discs. Transmission electron microscopic analysis showed normal differentiation of R cell clusters in both wild-type and rugose mutant eye discs. In addition, immunochemical experiments involving a variety of R cell-specific probes also showed normal specification of the R cells in the developing third instar eye imaginal discs. This further supports the conclusion that rugose mutations do not affect R cell differentiation (Shamloula, 2002).

In addition to phenotypes in the eye, rugose mutants exhibit wing vein defects and embryonic lethality with partial penetrance. The penetrance is variable depending on the particular rugose allele. Loss of rugose function leads to an incomplete wing vein L5. rugose mutants also exhibit an embryonic semilethal phenotype. The extent of lethality varies with the strength of the rugose allele. In the null alleles rg^gamma6 and rg^gamma3, for example, 20%–40% of the fertilized eggs fail to hatch, suggesting defects in the embryonic stages. The cellular and molecular basis for the lethality is not clear at this point. The pleiotropic nature of rugose mutations is consistent with the multiple roles of cAMP-PKA-mediated signaling in Drosophila (Shamloula, 2002).

Thus rg is required for correct retinal pattern formation and mutations at the rugose locus result in rough eyes due to an aberrant ommatidial organization. This phenotype may be the consequence of a malformed retinal basement membrane and a defective pigment cell lattice. Analyses of the cellular basis of this phenotype revealed that, in rugose mutants, cone cell differentiation is abnormal, leading either to the lack of or to reduced numbers of cone cells. Cone cell number is sensitive to the rugose gene dosage, suggesting that rugose function is required for normal differentiation of the cone cells. Developmental studies employing both light and transmission electron microscopy have revealed that, in rugose mutants, cone cell development is defective even at the earliest stages and suggest that rugose is a positive effector of the cone cell determination. Cone cells belong to the R7 equivalence group and begin to differentiate immediately following R7 cell specification. In rugose mutants, the number of secondary/tertiary pigment cells is increased. This may be due to the alternative recruitment of the cone cell precursors to the pigment cell fate. Although the ommatidia are disorganized in rugose mutants, photoreceptor cell specification and differentiation are unaffected (Shamloula, 2002).

Direct evidence for the role of AKAPs in the regulation of signaling comes from studies in mammalian systems and has led to the characterization of a large family of AKAPs. Altered localization of PKA by ectopic expression in tissue culture cells results in the altered pattern of phosphorylation of PKA substrates such as ion channels and CREB (Feliciello, 1997; Gao, 1997; Gray, 1998). The data presented in this study suggest that rugose, a Drosophila AKAP, is important for correct retinal pattern formation in the developing eye, a function likely to be mediated through its effects on the subcellular localization of PKA. If true, mutations in rugose would be expected to result in the mislocalization of PKA, leading to altered signaling. Determining the cellular and subcellular distribution of PKA in rugose mutants will test this. At this point the precise role of the cAMP-PKA signaling pathway in cell fate specification in the Drosophila eye is unclear. The cAMP-PKA signal cascade may play a role in cell fate determination through its interactions with the Notch and Egfr pathways. Recent studies have shown that cell fate determination in the developing eye requires the integration of inputs from multiple signaling pathways and a combinatorial code involving the activation of specific transcription factors. The data from the genetic modifier screen show that rugose interacts with components of the Egfr and Notch signaling pathways in a dosage-dependent manner. Mutations that reduce either Egfr or N signaling behave as dominant enhancers of the rough eye phenotype. Conversely, gain-of-function mutations that increase Egfr or N signaling act as dominant suppressors of the rugose phenotype. The molecular basis of these observed interactions is not clear at the present time. It is proposed that these interactions between PKA signaling and the Egfr and Notch pathways are likely to be indirect (Shamloula, 2002).

In a separate study, Huang (2000) identified AKAP 200, the second Drosophila AKAP (Li, 1999), in a misexpression screen for genes that modulate RAS1 signaling. This suggests interactions between the RAS1 signaling and the cAMP-PKA pathways. Others have also reported interactions between the cAMP-PKA, RAS-MAPK, and the Notch pathways in a variety of systems. However, molecular details of interactions between the various pathways are not yet clear. It is possible that rugose interacts with the Egfr and N pathway components at the protein level. In this model, Rugose, as an AKAP, may be involved in anchoring components of other signaling pathways. Although initially it was thought that AKAPs were specific for PKA, it has been shown subsequently that AKAPs are multivalent and can anchor a wide variety of signaling components (Klauck, 1996; Pawson, 1997). Taken together the results suggest that rugose could exert its effects on cone cell specification and retinal pattern formation through its interactions with the Egfr and Notch pathways by facilitating the cross-talk between the different signaling pathways (Shamloula, 2002).

During the course of embryogenesis Drosophila expresses several RII-binding proteins with sizes ranging from 70,000 to >200,000 kDa. As the fly matures, the levels of the smaller binding proteins are diminished, thereby suggesting that these polypeptides play specialized roles in early development. In contrast, DAKAP550/Rugose is evident in many cells and nearly all tissues throughout development and becomes the principal anchor protein in adult flies. Although DAKAP550 accumulates in multiple cell types, the content of the anchor protein is regulated. DAKAP550 is significantly enriched in subpopulations of peripheral and central neurons (e.g., photoreceptor neurons) as well as in cells of the gut and trachea. Moreover, immunocytochemical staining revealed that DAKAP550 accumulates asymmetrically (e.g., apical localization) in certain neurons and portions of the gut and trachea. This pattern could potentially facilitate polarized cAMP signaling and is consistent with the previous published observations that targeted PKA activity is essential for anterior/posterior patterning, oocyte development, and differential segregation of mRNAs (Han, 1997).

Biochemical analysis has documented that the content of DAKAP550 is markedly elevated in anterior tissues of Drosophila. A similar pattern of enrichment was reported for Drosophila RII (PKAII) (Foster, 1984). Moreover, the anchor protein was recovered in the cytosolic fraction of tissue homogenates. This result does not preclude a tight association of DAKAP550 with cytoskeleton. Many components of highly-organized cytoskeletal structures appear in cytosol (e.g., microtubule-associated protein-2, actin-binding proteins, and paxillin) when tissues are homogenized in standard, low ionic strength buffers in the absence of specific stabilizing agents. Development of stabilizing conditions for Drosophila cytoskeleton will be essential to accurately monitor the distribution of the anchor protein with various subcellular fractions in future studies. Finally, it is also possible that DAKAP550 serves as a 'cytoplasmic anchor' or 'scaffold' to either (1) focus cAMP signaling to target proteins in cytoplasm, (2) inhibit signaling at certain membranes or cytoskeletal sites, or (3) co-assemble tethered PKAII with substrate/effector proteins directly bound to other domains of the anchor protein (Han, 1997).

GENE STRUCTURE

Han (1997) first reported the cloning and sequence of a 7.2-kb partial cDNA for DAKAP 550. The complete sequence of the DAKAP 550 has been reported in the annotation of the Drosophila genome by the Drosophila genome project (gene ID no. CG6775). The DAKAP 550 gene is composed of 26 exons in a 29-kb transcription unit and the complete cDNA is 11.2 kb in size. The DNA sequence data analysis shows that the 1.9-kb cDNA RgcD10 is derived from exons 4–10 of DAKAP 550 (Shamloula, 2002).

PROTEIN STRUCTURE

Amino Acids - 2359

Structural Domains

The predicted partial DAKAP550 (Rugose) polypeptide sequence includes>2,350 amino acids. Like mammalian anchor proteins, partial DAKAP550 is acidic (predicted pI = 4.96) and bears a substantial net negative charge (approximately 100) at neutral pH. DAKAP550 is not related to previously-characterized proteins. However, substantial portions of the fly anchor protein are homologous with segments of a predicted C. elegans protein designated F10F2.1 and a derived polypeptide encoded by a recently discovered human gene named 'beige-like'. Sequences homologous with portions of C. elegans F10F2.1 appear as discrete modules that are inserted at several points within the DAKAP550 polypeptide chain. For example, long (>175 amino acid residues), sharply demarcated N-terminal, central and near C-terminal segments of DAKAP550 are 40%-50% identical with portions of the C. elegans protein. Central and C-terminal regions of DAKAP550 also have counterparts in the human beige-like protein. A segment of DAKAP550 bounded by amino acids 1693 and 1929 is highly conserved in both the human (69% identity) and C. elegans (42% identity) proteins (Han, 1997).

EVOLUTIONARY HOMOLOGS

Neurobeachin is a neuron-specific multidomain protein of 327 kDa with a high-affinity binding site [K(d), 10 nm] for the type II regulatory subunit of protein kinase A (PKA RII). Neurobeachin is peripherally associated with pleomorphic tubulovesicular endomembranes near the trans sides of Golgi stacks and throughout the cell body and cell processes. It is also found in a subpopulation of synapses, where it is concentrated at the postsynaptic plasma membrane. In live cells, perinuclear neurobeachin is dispersed by brefeldin A (BFA) within 1 min, and in permeabilized cells a recruitment of neurobeachin from cytosol to Golgi-near membranes is stimulated by GTPgammaS and prevented by brefeldin A. Spots of neurobeachin recruitment are close to but distinct from recruitment sites of COP-I, AP-1, and AP-3 coat proteins involved in vesicle budding. These observations indicate that neurobeachin binding to membranes close to the trans-Golgi requires an ADP-ribosylation factor-like GTPase, possibly in association with a novel type of protein coat. A neurobeachin isoform that does not bind RII, beige-like protein BGL), is expressed in many tissues. Neurobeachin, BGL, and approximately 10 other mammalian gene products share a characteristic C-terminal BEACH-WD40 sequence module, which is also present in gene products of invertebrates, plants, protozoans, and yeasts, thus defining a new protein family. The prototype member of this family of BEACH domain proteins, lysosomal trafficking regulator (LYST), is deficient in genetic defects of protein sorting in lysosome biogenesis (the beige mouse and Chediak-Higashi syndrome). Neurobeachin's subcellular localization, its coat protein-like membrane recruitment, and its sequence similarity to LYST suggest an involvement in neuronal post-Golgi membrane traffic, one of its functions being to recruit protein kinase A to the membranes with which it associates (Wang, 2000).

Mutations in chs1/beige result in a deficiency in intracellular transport of vesicles that leads to a generalized immunodeficiency in mice and humans. The function of NK cells, CTL, and granulocytes is impaired by these mutations, indicating that polarized trafficking of vesicles is controlled by CHS1/beige proteins. However, a molecular explanation for this defect has not been identified. A novel gene with orthologs in mice, humans, and flies is described that contains key features of both chs1/beige and A kinase anchor genes. This novel gene has been designated lba for LPS-responsive, beige-like anchor gene. Expression of lba is induced after LPS stimulation of B cells and macrophages. In addition, lba is expressed in many other tissues in the body and has three distinct mRNA isoforms that are differentially expressed in various tissues. Strikingly, LBA-green-fluorescent protein (GFP) fusion proteins are localized to vesicles after LPS stimulation. Confocal microscopy indicates this protein is colocalized with the trans-Golgi complex and some lysosomes. Further analysis by immunoelectron microscopy demonstrates that LBA-GFP fusion protein can localize to endoplasmic reticulum, plasma membrane, and endocytosis vesicles in addition to the trans-Golgi complex and lysosomes. It is hypothesized that LBA/CHS1/BG proteins function in polarized vesicle trafficking by guiding intracellular vesicles to activated receptor complexes and thus facilitate polarized secretion and/or membrane deposition of immune effector molecules (Wang, 2001).

date revised: 3 August 2002

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