Gene name - corkscrew
Cytological map position - 2D3--2D3
Function - Protein tyrosine phosphatase, Receptor substrate assembly
Symbol - csw
Genetic map position - 1-[0.5]
Classification - Tyrosine specific protein phosphatase, Src homology 2 (SH2) domain
Cellular location - cytoplasmic
|Recent literature||Breitkopf, S. B., Yang, X., et al. (2016). A cross-species study of PI3K protein-protein interactions reveals the direct interaction of P85 and SHP2. Sci Rep 6: 20471. PubMed ID: 26839216
A fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome was conducted in a Drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. These data revealed an unexpected direct binding of Corkscrew, the Drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (human GAB2 but not Drosophila Dos). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.
Corkscrew, a component of the Ras signaling pathway, is a protein tyrosine phosphatase (PTP). PTPs, it should be kept in mind, remove (or lyse) phosphates, as the name phosphatase implies. csw was initially identified as a maternal effect mutation of the terminal system: CSW functions in the same signaling pathway as the receptor tyrosine kinase (RTK) Torso (Perkins, 1992).
In a signaling pathway, RTKs signal for action -- the binding of phosphates: RTKs are said to have positive or upward regulatory effects. PTPs are thought to function in a complementary, albeit antagonistic manner to RTKs, signaling for quiescence, or the removal of phosphates: they are said to have negative, or downward regulatory effects. Subsequent studies of CSW reveal a paradox: CSW appears to initiate positive activity (that is, it aids in sending a signal for phosphate binding) in the Ras pathway phosphorylation cascade. This apparent paradox offers insight into the functioning of the early stages of Ras pathway signaling.
The phosphatase CSW, unlike the RTK Torso, is required during zygotic development. Two factors contribute to the belief that CSW has zygotic roles. First, zygotically acting csw mutations, unlike tor, result in embryonic lethality. Secondly, unlike tor, the csw maternal effect phenotype is partially paternally rescuable. For example, the posterior midgut invagination, and hence the midgut, is entirely deleted in null embryos, whereas in paternally rescued embryos the midgut is malformed and reduced in size. CSW also functions in the development of ventral cell fate in the embryo. Significantly fewer cells stain with Fas III in csw mutant embryos, relative to wild type. This apparent loss of ventral cell fate is accompanied by a concomitant expansion of lateral cell fates. For example, the laterally positioned cells of the tracheal pits are closer to the midline in csw mutants. Likewise, the ventrolaterally positioned Keilin's organs appear closer together than normal in csw mutants (Perkins, 1996).
CSW also plays a role in embryonic CNS development. In null csw embryos, which display a more severe phenotype, the horizontal commissures are collapsed. This is in contrast to paternally rescued csw embryos that display the less severe phenotype: the commissures are separated along the ventral midline. In both paternally rescued and null mutant embryos the longitudinal axon tracts are rudimentary and discontinuous. Similar phenotypes have been reported in embryos mutant for EGF Receptor (Perkins, 1996).
To determine whether CSW is involved in signaling from the Breathless receptor tyrosine kinase during tracheal development, phenotypes of mutant csw embryos were examined using a tracheal-specific marker. In mutant embryos, tracheal cell precursors are produced normally, but their subsequet migration, which generates the tracheal tree, appears defective. An incomplete and disconnected system of tracheal branches is the final result. Thus it appears that CSW operates positively in BTL signaling for the formation of the mature larval tracheal network (Perkins, 1996).
CSW also plays a role in the development of adult structures. Mutant csw adults reveal consistent defects including absence of one or both of the distal-most antennal segments, the aristae; lack of one or more of the distal-most leg segments, the tarsal claws; incomplete formation of distal portions of wing vein L5 (and often loss of L4), and eyes with disorganized, reduced in numbers ommatidia and ommatidial bristles. The phenotypes of csw mutant adults are similar to those reported for viable EGF-R mutations, suggesting that csw functions positively during imaginal development in the EGF-R signaling pathway (Perkins, 1996).
CSW is also required during oogeneis for normal follicle cell development. Follicle cell development is known to be stimulated by an asymmetrically localized signal from Gurken, originating in the oocyte. Phenotypes of eggs derived from partially zygotically rescued csw females were examined to determine whether CSW plays any role in EGF-R signaling in dorsal follicle cells. Such females lay eggs with fused dorsal appendages that correspond to an expansion of ventral chorionic cell fates at the expense of dorsal chorionic cell fates. This effect is similar to the role of EGF-R during oogenesis and suggests that CSW operates downstram of EGF-R in the establishment of dorsal follicle cell fates (Perkins, 1996).
RTK signaling still operates in the absence of CSW activity, as evidenced by the incomplete phenotype of genetically null csw alleles compared to torso mutants. In addition, the role of CSW is positive, expediting the RTK signals in response to ligand binding. What then is the role of CSW in RTK signaling? It may help to think of CSW as a docking protein. The presence of two SH2 domains suggests that they could directly bind activated receptor tyrosine kinases. This model is supported by studies with the mammalian phosphatase SHP-2, which physically associates with PDGF and EGF receptors and the insulin receptor substrate IRS-1. Further, upon binding to the EGF or PDGF receptors, SHP-2 becomes tyrosine phosphorylated. One of the sites of tyrosine phosphorylation within SHP-2 provides a binding site for Grb2. These findings suggest a mechanism whereby upon PDGF receptor activation, SHP-2 is recruited to the receptor and then becomes tyrosine phosphorylated, which in turn recruits the GRb2/Sos complex to the membrane, thereby activating Ras. The binding of SHP-2 to PDGF and EGF receptors is analagous to the binding of CSW to EGF-R in Drosophila. In both cases the phosphatase helps recruit additional proteins involved in the signaling process. However, this model still does not account for the PTPase catalytic activity of CSW. It is likely that the phosphatase activity acts only after the assembly of the whole signaling complex, and then the phosphatase activity tones down the strength of the signal, the conventional role ascribed to phosphatases (Perkins, 1996 and references).
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).
Two transcripts, each one longer than the prevalent 4.7 kb form (6.0 and 7.2 kb), are first observed during late embryogenesis, but upon longer exposure are also observed throughout larval, pupal and adult stages (Perkins, 1992).
Bases in 5' UTR - 140
Exons - 2
Bases in 3' UTR - 1866
The csw gene encodes a putative nonreceptor protein tyrosine phosphatase covalently linked to two N-terminal SH2 domains, which is similar to the mammalian PTP1C protein (Perkins, 1992).
The CSW PTPase domain is similar to that of PTP1C, with which it shares 58% identity. The CSW PTPase domain is unusual among the known PTPase proteins, since it is interrupted by a hydrophilic serine- and cysteine-rich stretch of approximately 150 aa, the PTPase insert, which shares no homologies with other PTPase proteins. The N-terminal SH2 domains of the CSW protein share between 45% and 65% identity with the next most closely related SH2 domains, those from the mammalian phosphatase PTP1C (Perkins, 1992).
date revised: 17 Dec 96
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