corkscrew

DEVELOPMENTAL BIOLOGY

Embryonic, Larval and Adult

Maternal CSW functions in terminal development. Zygotically expressed csw is implicated in the establishment of ventral cell fates, plays a role in commissure formation in the central nervous system, is required for tracheal development, and is involved in the formation of adult structures, including eyes, aristae (the terminal antennal segment), wing veins, tarsal claws, specific macrochaetae, male sex combs and female genital disc derivatives. In addition, CSW is required during oogenesis in follicle cell development (Perkins, 1996 ). This is also discussed in the Biological Overviewsection of corkscrew.

Effects of Mutation or Deletion

corkscrew is maternally required for normal determination of cell fates at the termini of the embryo. Determination of terminal cell fates is mediated by a signal transduction pathway that involves Torso (a receptor tyrosine kinase), components of the Ras pathway, and the transcription factors Tailless and Huckebein. Double mutant and cellular analyses among csw, torso, D-raf and tailless indicate that csw acts downstream of torso and in concert with D-raf to positively transduce the Torso signal via tailless, to downstream terminal genes (Perkins, 1992).

csw is required maternally, since embryos derived from females lacking csw activity during oogeneisis die during embryogenesis. Externally these embryos, referred to as csw mutant embryos, though twisted or U-shaped, appear like wild type. However, mutant embryos show abdnormal development of their internal terminals structures that include disruption anterior to the cephalopharyngeal skeleton and dorsal bridge and posterior to the posterior midgut and Malpighian tubules (Perkins, 1992).

The role in patterning of quantitative variations of MAPK activity in signaling from the Drosophila Torso (Tor) receptor tyrosine kinase (RTK) has been examined. Activation of Tor at the embryonic termini leads to differential expression of the genes tailless and huckebein. Using a series of mutations in the signal transducers Corkscrew/SHP-2 and D-Raf, it has been demonstrated that quantitative variations in the magnitude of MAPK activity trigger both qualitatively and quantitatively distinct transcriptional responses. When terminal activity is progressively removed, there is a corresponding progressive malformation and eventual loss of terminal cuticular structures. The first terminal cuticular elements that are malformed or lost require the highest terminal activation (e.g., the anal tuft and posterior spiracles visualized by the presence of Filzkorper material). The next elements that are malformed or lost require intermediate levels of terminal signal (e.g., the abdominal 8 (A8) denticle belt and the posterior spiracles). Finally, the last elements that are malformed or lost require the lowest levels of terminal activity (e.g., posterior A7). While in the absence of D-raf activity, no activated MAPK (dp-ERK) is observed at the posterior pole. In csw null mutant embryos, where the tll and Hb expression domains are present though mispositioned, reduced levels of dp-ERK reactivity are observed. Collectively, these results reveal that a precise transcriptional response translates into a specific cell identity (Ghiglione, 1999).

Two chimeric receptors, Torextracellular-Egfrcytoplasmic and Torextracellular-Sevcytoplasmic, cannot fully functionally replace the wild-type Tor receptor, revealing that the precise activation of MAPK involves not only the number of activated RTK molecules but also the magnitude of the signal generated by the RTK cytoplasmic domain. For example, analysis of Torextracellular-Egfrcytoplasmic reveals that the posterior domain of Hunchback does not retract from the posterior pole, but rather remains as a terminal cap. Further, the anterior border of this posterior Hb domain is shifted posteriorly. Altogether, these results illustrate how a gradient of MAPK activity controls differential gene expression and thus, the establishment of various cell fates. The roles of quantitative mechanisms in defining RTK specificity are discussed. It is possible that in some instances, the generation of differing magnitudes of activity from the cytoplasmic domains of specific RTKs might be dependent on the specific affinities of the downstream signal transducers to the receptor. Csw binds through one of its SH2 domains to only one phosphotyrosine on Tor. Perhaps a higher or lower affinity of Csw to this site, or addition of another site that would also engage the second SH2 domain of Csw, would increase or decrease signal output. Presumably, in each individual cell there exists a mechanism built into the enhancer elements of the promoters of both tll and hkb that acts to read directly the magnitude of Tor signaling. In the tll promoter, a Tor-response element that mediates the repression of tll has been identified, indicating that the Tor signal activates tll by a mechanism of derepression. A putative candidate for this repressor activity is encoded by the transcription factor Grainyhead. Grainyhead binds to the Tor-response element and can be directly phosphorylated by MAPK in vitro: a decrease in Gh activity has been shown to cause tll expansion in early embryos. Further, the transcriptional corepressor Groucho is required for terminal patterning. Further characterization of how Gh and/or Gro activities are regulated by activated MAPK should clairify how differing levels of phosphorylation translate into derepression of terminal target genes (Ghiglione, 1999).

An Enhancer of sevenless mutation acts as a dominantly inhibiting allele of csw. csw function is essential for Sevenless signaling. Expression of a membrane-targeted form of CSW can drive R7 photoreceptor development in the absence of sevenless function. The dominantly inhibiting CSW shows a substitution of glutamate for glycine at codon 547. In mutant eye discs, prepared by transducing the dominantly inhibiting CSW, elav expression appeares to be normal at the stage when R8, R2 and R5 express elav. However, subsequent staining for Elav is abnormal. Staining of cells occupying the normal position of R3 and R4 was only rarely observed. Transduction of normal CSW suppresses the dominant negative mutant. The dominantly inhibiting csw allele was used to examine the role of CSW during signaling by activated forms of Ras1 and Raf. csw function is still required during signaling by activated Ras1 and Raf proteins. These results define a function for CSW that is either downstream of Ras1 activation or in a parallel pathway that acts with activated Ras1/Raf to specify R7 photoreceptor development (Allard, 1996).

Vertebrate Src can be activated by specific mutations to become oncogenic. Analogous mutations in Drosophila Src oncogene 1 induce abnormal differentiation of photoreceptor cells when expressed ectopically in the developing Drosophila adult eye. The roles played in this process by the adapter protein Downstream of receptor kinases (Drk), and the SH2 domain-containing tyrosine phosphatase Corkscrew (Csw), have been examined. Dominant-negative mutations in either the drk or csw genes ameliorate the developmental abnormalities induced by activated Src64. This suggests that Drk and Csw are required downstream of, or parallel to, Src64. Csw does not act solely as an upstream activator of Scr64. The results are discussed in relation to potential roles for the vertebrate homologs of Drk and Csw (Grb2 and SHP2, respectively) in the transformation of fibroblasts by vertebrate Src (Cooper, 1996).

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, Csw^src90 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-csw^src90 in several tissues phenocopies gain-of-function mutations and constructs in positive signaling genes in the Egfr pathway. Moreover, tissue-specific expression of csw^src90 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 csw^src90 construct to study Csw function in RTK pathways. csw^src90, 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-csw^src90 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-ras1^ACT) in the embryonic mesoderm, but not by expression of UAS-ras1^ACT alone. This result, seen with two copies of UAS-csw^src90, might reveal a nonphysiological ability for Csw^src90 to bypass the need for Wg signaling at the transcriptional level during myogenesis (Hamlet, 2001).

While the possibility that Csw^src90 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-csw^src90 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-csw^src90 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-csw^WT) was expressed using twi-Gal4 in various genetic backgrounds. While this could be due to the extent to which UAS-csw^WT 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 Csw^src90 protein is already in an active state, which results in hyperactivation of the RTK pathway. Csw^src90 is hence insensitive to the normal downregulation of the RTK signal that occurs. The mechanism of action of Csw^src90 is unknown, but it is possible that membrane localization either provides constitutive access to substrates or changes the conformation of Csw^src90 such that the N-terminal SH2 domain is unable to bind to the catalytic domain to block its function. Nevertheless, the phenotypes produced by csw^src90 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 Csw^src90 rescues VA2 precursor cell formation in spi, rho, and twi-Gal4/+; UAS-Egfr^DNDER mutant embryos. This latter finding places the interaction of Csw^src90 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 csw^src90 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 csw^VA199 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 Csw^src90 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 csw^src90 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 Csw^src90 is able to function downstream of D-Raf, it is possible that Csw^src90 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 Csw^src90. 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, Csw^src90 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).

Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS), an autosomal dominant disorder with pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker than leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, transgenic fruitflies were generated with GAL4-inducible expression of wild type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles was lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous expression of the weaker N308D allele causes ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses have established that the csw^N308D ectopic wing vein phenotype requires intact EGF ligand and receptor, and that this transgene interacts genetically with Notch, DPP and JAK/STAT signaling. Expression of the mutant csw transgenes increases RAS-MAP kinase activation, which is necessary but not sufficient for transducing their phenotypes. The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers (Oishi, 2006).

CSW is the Drosophila orthologue of SHP-2 and works as a positive regulator of multiple RTK pathways. The amino acid sequence of SHP-2's PTP domain is 63% identical to CSW excluding an insertion of unknown consequence in the latter and is 76% similar in the SH2 domains. Since the amino acids altered by the mutations in PTPN11 are conserved in the fly, it was hypothesized that transgenic Drosophila expressing mutant CSW would model the GOF effects on signal transduction observed in cell culture and frog animal cap systems. This study demonstrated that the mutant CSW proteins were biologically active in the eye rescue experiment and that they altered development. One NS transgene, N308D, had a GOF effect on wing vein formation, while two stronger GOF transgenes caused lethality when expressed ubiquitously (Oishi, 2006).

Among patients with NS who harbor PTPN11 mutations, no significant correlation between genotype and phenotype has been established, although the statistical power of those studies has been limited. Biochemical and cell physiologic studies, however, have documented that the GOF effects of NS-associated SHP-2 mutants vary, with the N308D allele being weakest. More strikingly, there are clinical, genetic and biochemical data showing that mutant SHP-2's causing hematopoietic proliferative disorders tend to have greater GOF effects than those associated with isolated NS. Juvenile myelomonocytic leukemia (JMML) occurring in the context of NS can be a milder disease than when it occurs in an otherwise normal child. NS with JMML is caused by specific germline PTPN11 mutations (i.e., most NS-associated PTPN11 mutations including the commonest N308D allele have never been associated with JMML). In contrast, somatic PTPN11 mutations result in JMML, ALL and AML. The molecular lesions also differ from those in NS with generally less conservative amino acid substitutions that are almost entirely restricted to the N-SH2 domain. In vitro biochemical analyses revealed that the phosphatase activities of the leukemia-associated SHP-2 proteins are generally greater than the NS-associated ones. Finally, the leukemia-associated PTPN11 mutations appear to result in embryonic lethality in humans when transmitted through the germline. Thus, it has been hypothesized that the GOF effects of PTPN11 mutation on signal transduction would be graded with leukemia mutants --> NS/JMML mutants --> NS mutants. The results of the present work with the transgenic csw GOF fly models are consistent with this hypothesis (Oishi, 2006).

The leukemic allele, E76K, caused the earliest lethality when expressed ubiquitously. The A72S allele, which has been shown to be a relatively strong NS allele biochemically, was also lethal. The weakest NS allele, N308D, was not lethal until the dose was increased through transgene homozygosity. Similarly, exposing developing embryos to lower ambient temperatures, which decreases transgene expression, shifted the lethality of the A72S allele to later stages and permitted a few to survive to adulthood; of interest, surviving A72S adult flies had ectopic wing veins. The molecular basis of the GOF strength differences among SHP-2 mutants has been attributed to relative effects on the molecular switching mechanism, an idea that still awaits experimental confirmation. The data presented here provide the first proof that the disease-associated PTPN11 mutations have graded effects on development (Oishi, 2006).

Ubiquitous expression of the csw^N308D transgene results in ectopic wing vein formation. This closely resembles the phenotype resulting from an Egfr GOF allele. Consistent with the well-defined role of EGFR signaling and its activation by ligand stimulation in wing vein development, LOF alleles of nearly all extra- and intra-cellular members of the EGFR-RAS-MAP kinase signaling cascade that promote signaling suppress the csw^N308D-associated phenotype, while loss of negative regulators enhance it. Despite the fact that most disease-associated SHP-2 mutant proteins including N308D have increased basal activity, the data from the fly model reveals that the N308D-induced perturbation of wing development requires an intact ligand and RTK. This is consistent with findings from in vitro studies showing Egf-dependent and Gab1-mediated ERK2 prolonged kinase activity, as well as from frog animal cap studies in which expression of a dominant-negative Fgfr quenched the induction of dorsolateral mesoderm from mutant SHP-2 expression. While the precise basis for this reliance on ligand-induced RTK stimulation has not been elucidated, it is speculated that there are two critical factors. (1) Signaling through RTKs such as EGFR requires ligand-induced phosphorylation of the receptor that then results in recruitment of numerous signaling and docking proteins including SHP-2 and CSW to the plasma membrane. In this context, basally activated SHP-2 or CSW is not physically associated with its requisite signaling partners unless and until the relevant RTK engages ligand. (2) SHP-2 is a positive regulator of signal transduction for many RTK-RAS-MAP kinase pathways, which primarily rely on phosphorylation to propagate their signals. This is true despite the fact that SHP-2 and CSW dephosphorylate RTKs such as PDGFR and Torso. While the elucidation of the full range of bona fide SHP-2 substrates continues to be pursued, two defined SHP-2 substrates, PAG/cbp and Paxillin, negatively regulate RTK-RAS signaling. SHP-2 dephosphorylates these substrates, preventing them from recruiting , which, in turn, is a negative regulator of Src family kinases. While loss of SHP-2 activity reduces RAS signaling, SHP-2 GOF should not be sufficient to initiate it. Taken together, these two factors provide a rationale for the finding that reduction of EGFR or its ligand, Vein, suppresses the formation of ectopic wing veins from N308D (Oishi, 2006).

Another striking finding observed with the csw GOF alleles was the pleomorphism of effects among RTK pathways in which CSW participates. Development of the trachea, dorsal tube and photoreceptor R7, structures dependant upon Breathless, Heartless and Sevenless signaling, respectively, were not altered. The domains of tailless expression in the anterior and posterior poles, which have been used as a read out of Torso signaling and diminish with loss of maternal CSW, showed no augmentation from CSW GOF. The pleomorphism was also present among structures whose development is EGFR-signaling dependent: N308D expression altered wing vein formation but did not affect photoreceptor development. Thus, it is concluded that the CSW GOF mutants alter signal transduction in a selective and specific manner. This is consistent with the findings in NS, in which the GOF SHP-2 protein is ubiquitously expressed but the phenotype reflects developmental abnormalities occurring in a tissue- and stage-specific manner. In addition to that specificity, increased MAP kinase activation in the Ptpn11D61G knock-in model mice was noted only in specific tissues (Oishi, 2006).

What is the basis for the specificity in developmental abnormalities arising from SHP-2/CSW GOF? (1) GOF SHP-2 might dephosphorylate substrates promiscuously, thereby recruiting non-canonical signaling affecting specific developmental processes. In support of this, MAP kinase activation proved necessary but not sufficient for generating the CSW GOF phenotypes. Moreover, N308D expression recruited JAK/STAT signaling in the context of wing vein development, a non-canonical interaction. Of note, a similar phenomenon was observed for Torso signaling in which the effects of a torso GOF allele required STAT activation even though the JAK/STAT pathway plays no role in Torso signaling ordinarily. (2) There may be a critical developmental window that is dependent upon the dosage of SHP-2/CSW and/or MAP kinase activity. The tolerance to variability in these activities may differ among signaling pathways and between tissues for a given pathway. A comparison of the phenotypes observed in the N308D transgenic and the GOF Egfr^E1 flies provides an example of the latter. Although both alleles increase MAP kinase activation in the context of EGFR signaling, the former resulted in ectopic wing veins while the latter exhibited ectopic veins and a rough eye phenotype. The rough eye phenotype in Egfr^E1 flies is a LOF EGFR phenotype as a result of negative feedback caused by high expression of a negative regulator, Argos, which is induced by GOF EGFR activity. Argos inhibits EGFR signaling by binding to an activating ligand, Spitz. In contrast, the NS GOF CSW appears to cause high, nonconstitutive EGFR signal activation, which apparently does not increase the argos expression level enough to induce the EGFR LOF phenotype during photoreceptor development. It is likely that there is a distinct difference (although it may be subtle) in the levels of EGFR signaling activity between these two models and it determines the phenotypic pattern. In particular, specificity of the developmental abnormality seems to be established by the level of increased signaling activity caused by the NS alleles in a given pathway and by a specific requirement of a fine-tuned signaling activity for normal development in a given tissue and/or cell type. This idea is supported by work showing that there are multiple distinct thresholds of required EGFR signaling activity in different cell types during retinal development. In contrast, EGFR signaling in wing vein specification predominantly uses the ligand, Vein, a neuregulinlike molecule. Vein is a relatively weak ligand, which is proposed to be required for the tight basal control of the level of EGFR/RAS/MAP kinase pathway signaling during wing vein specification. Thus, the more modest increased EGFR signaling induced by CSW GOF alleles is still sufficient to induce ectopic veins (Oishi, 2006).

The N308D allele interacts genetically with several genes from the EGFR/RAS/MAP kinase pathway as well as some from the Notch, DPP (BMP), and JAK/STAT pathways. The degree of the suppression or enhancement of the interacting genes varies. While some of this variability could be attributed to allelism (e.g., null vs hypomorphic alleles for a single gene), it is apparent that there are differences in the intensity of the epistatic effects between interacting genes. A fuller elucidation of these epistatic genes might provide clues for understanding the clinical variability in NS. The phenotype of NS is variable among patients sharing the same PTPN11 mutation, even within families. Phenotypic variability was also observed in Ptpn11D61G knock-in mice, in which the cardiac phenotype was dependent upon the genetic background. While stochastic effects may explain some of the variability in NS patients, the results of epistatic studies underscore the fact that the PTPN11 mutant alleles are interacting with a variety of signal pathways, some finely balanced, such that relatively minor differences in gene expression or protein activities might significantly alter the phenotype (Oishi, 2006).

Congenital heart disease is a prominent feature of NS. Pulmonary valve stenosis is the most common cardiac defect and is particularly prevalent among NS patients with PTPN11 mutations. To date, the specific molecular mechanism underlying pulmonary stenosis in NS is unknown. Recent cardiac embryologic studies have shown that a delicate regulation of a variety of signaling pathways including VEGF, Notch, Wnt/ß-catenin, TGFß, BMP, and EGFR are important for valvulogenesis. Altered signaling leads to aberrant endothelial-mesenchymal transformation during cardiac valve cushion formation or remodeling of those cushions. Interestingly, the critical pathways for valvulogenesis are conserved in Drosophila and play essential roles for wing vein development (Oishi, 2006).

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date revised: 20 November 2006



 

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