SHP-2 acts in vivo as a positive transducer of vertebrate receptor tyrosine kinases. Also referred to as PTP1D, PTP2C, SHPTP-2, SHPTP-3, and/or Syp, this vertebrate PTPase is structurally related to Corkscrew and rescues csw mutant phenotypes in Drosophila. It has therefore been proposed that CSW/ SHP-2 is an integral part of the evolutionarily conserved cassette of signal transducers that operate downstream of all Drosophila RTKs (Perkins, 1996).

NOTE: SHP-2, Syp, PTP1D and other terms are used almost interchangeably in the literature when referring to the putative corkscrew vertebrate homolog. In the references cited below, author's usage has been retained.

SHP-2/Corkscrew homolog PTP-2 in C. elegans

Genetic analysis has shown that dos/soc-1/Gab1 functions positively in receptor tyrosine kinase (RTK) stimulated Ras/Map kinase signaling, through the recruitment of csw/ptp-2/Shp2. Using sensitised assays in C. elegans for let-23/Egfr and daf-2/InsR (Insulin receptor-like) signaling, it has been shown that soc-1/Gab1 inhibits phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3'-kinase (PI3K) mediated signaling. Furthermore, as well as stimulating Ras/Map kinase signaling, soc-1/Gab1 stimulates a poorly defined signaling pathway that represses class 2 daf-2 phenotypes. In addition, it is shown that SOC-1 binds the C-terminal SH3 domain of SEM-5. This binding is likely to be functional because the sem-5(n2195)G201R mutation, which disrupts SOC-1 binding, behaves in a qualitatively similar manner to a soc-1 null allele in all assays for let-23/Egfr and daf-2/InsR signaling examined. Further genetic analysis suggests that ptp-2/Shp2 mediates the negative function of soc-1/Gab1 in PI3K mediated signaling, as well as the positive function in Ras/Map kinase signaling. Other effectors of soc-1/Gab1 are likely to inhibit PLCgamma mediated signaling and stimulate the poorly defined signaling pathway that represses class 2 daf-2 phenotypes. Thus, the recruitment of soc-1/Gab1, and its effectors, into the RTK signaling complex modifies the cellular response by enhancing Ras/Map kinase signaling while inhibiting PI3K and PLCgamma mediated signaling (Hopper, 2006).

SHP-2 structure

The structure of the SHP-2 tyrosine phosphatase, determined at 2.0 angstroms resolution, shows how its catalytic activity is regulated by its two SH2 domains. In the absence of a tyrosine-phosphorylated binding partner, the N-terminal SH2 domain binds the phosphatase domain and directly blocks its active site. This interaction alters the structure of the N-SH2 domain, disrupting its phosphopeptide-binding cleft. Conversely, interaction of the N-SH2 domain with phosphopeptide disrupts its phosphatase recognition surface. Thus, the N-SH2 domain is a conformational switch; it either binds and inhibits the phosphatase, or it binds phosphoproteins and activates the enzyme. Recognition of bisphosphorylated ligands by the tandem SH2 domains is an integral element of this switch; the C-terminal SH2 domain contributes binding energy and specificity, but it does not have a direct role in activation (Hof, 1998).

SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase-linked receptors. Recent three-dimensional structures suggest mechanisms by which tandem SH2 domains might confer higher specificity than individual SH2 domains. To test this, binding studies were conducted with tandem domains from the five signaling enzymes: phosphatidylinositol 3-kinase p85, ZAP-70, Syk, SHP-2, and phospholipase C-gamma1. Bisphosphorylated TAMs (tyrosine-based activation motifs) were derived from biologically relevant sites in platelet-derived growth factor, T cell, B cell, and high affinity IgE receptors and the receptor substrates IRS-1 (insulin receptor substrate-1) and SHPS-1/SIRP. Each tandem SH2 domain binds a distinct TAM corresponding to its appropriate biological partner with highest affinity (0.5-3.0 nM). Alternative TAMs bind the tandem SH2 domains with 1,000- to >10,000-fold lower affinity than biologically relevant TAMs. This level of specificity is significantly greater than the approximately 20-50-fold typically seen for individual SH2 domains. It is concluded that high biological specificity is conferred by the simultaneous interaction of two SH2 domains in a signaling enzyme with bisphosphorylated TAMs in activated receptors and substrates (Ottinger, 1998).

SHP-2 and phosphorylation

Because the protein-tyrosine phosphatase (PTP) Syp, the vertebrate Corkscrew homolog, associates with the tyrosine-phosphorylated platelet-derived growth factor beta receptor (beta PDGFR), the beta PDGFR is a likely Syp substrate. The beta PDGFR has been phosphorylated at multiple tyrosine residues in an in vitro kinase assay and then incubated with increasing concentrations of either Syp or PTP1B. While the receptor is nearly completely dephosphorylated by high concentrations of PTP1B, receptor dephosphorylation by Syp plateaus at approximately 50%. Syp displays a clear preference for certain receptor phosphorylation sites; the most efficiently dephosphorylated sites are phosphotyrosines In contrast, PTP1B displays no selectivity for the various beta PDGFR tyrosine phosphorylation sites and dephosphorylates all of them with comparable efficiency. A Syp construct that lacks the SH2 domains is still able to discriminate between the various receptor phosphorylation sites, although less effectively than full-length Syp. A beta PDGFR mutant (F1009) that associates poorly with Syp, has a much slower in vivo rate of receptor dephosphorylation than the wild type receptor. In addition, the GTPase-activating protein of Ras (GAP) and phosphatidylinositol 3-kinase are less stably associated with the wild type beta PDGFR than with the F1009 receptor. Syp prefers to dephosphorylate sites on the beta PDGFR that are important for binding phosphatidylinositol 3-kinase. Syp is a substrate-selective PTP: both the catalytic domain and the SH2 domains contribute to Syp's ability to choose substrates. It appears that Syp plays a role in PDGF-dependent intracellular signal relay by selectively dephosphorylating the beta PDGFR and thereby regulating the binding of a distinct group of receptor-associated signal relay enzymes (Klinghoffer, 1995).

Regulation of cell proliferation, differentiation, and metabolic homeostasis is associated with the phosphorylation and dephosphorylation of specific tyrosine residues of key regulatory proteins. The phosphotyrosine phosphatase 1D (PTP 1D) contains two amino terminally located Src homology 2 (SH2) domains and is similar to the Drosophila Corkscrew protein, which positively regulates theTorso tyrosine kinase signal transduction pathway. PTP activity is found to be regulated by physical interaction with a protein tyrosine kinase. PTP 1D does not dephosphorylate receptor tyrosine kinases, despite the fact that it associates with the epidermal growth factor receptor and chimeric receptors containing the extracellular domain of the epidermal growth factor receptor and the cytoplasmic domain of either the HER2-neu, kit-SCF, or platelet-derived growth factor beta (beta PDGF) receptors. PTP 1D is phosphorylated on tyrosine in cells overexpressing the beta PDGF receptor kinase. This tyrosine phosphorylation correlates with an enhancement of its catalytic activity. Thus, protein tyrosine kinases and phosphatases do not simply oppose each other's action; rather, they may work in concert to maintain a fine balance of effector activation needed for the regulation of cell growth and differentiation (Vogel, 1993).

The specific activity of the Src tyrosine kinase is elevated in human colon carcinoma cells. To identify Src-binding proteins that might upregulate Src activity in these cells, a human colon carcinoma lambda gt11 expression library was screened with purified, 32P-labeled Src. The SH-PTP2 (Syp) tyrosine phosphatase was isolated and shown to associate with Src. In vitro studies demonstrate that: (1) transforming F527 Src phosphorylates Syp, and (2) Syp dephosphorylates Src at Tyr 527. Both events are known to upregulate enzyme activity. Overexpression of the receptor tyrosine phosphatase alpha in rat embryo fibroblasts results in Src activation by dephosphorylation of Tyr 527, cell transformation and tumorigenesis. Thus, transmembrane tyrosine phosphatases may be involved in cell transformation exerting at least some of their effects through activation of Src (Peng, 1995).

SHP-2 association with receptor tyrosine kinases

An important role forsrc homology 2 (SH2) domain-containing tyrosine phosphatase (SH-PTP2), the vertebrate homolog of Corkscrew, in signal transduction is suggested both by its intrinsic tyrosine phosphatase activity and by its association with the activated epidermal growth factor (EGF) and platelet-derived growth factor receptors, as well as the insulin receptor substrate 1 and growth-factor-receptor-bound protein 2. A positive role has been demonstrated for SH-PTP2 in growth-factor-mediated cell signaling. Paradoxically, SH-PTP2 also functions to negatively regulate EGF-mediated signal transduction in a human glioma cell line. Glioma cells lack the ability to proliferate in response to EGF but retain the ability to bind EGF and also activate the EGF receptor as well as allow for the association of SH-PTP2 with the phosphorylated receptor. Stable overexpression of an interfering SH-PTP2 mutant can restore the ability of these cells to proliferate in response to EGF (Reeves, 1995).

The interaction of PTP2C with the PDGF receptor was studied by examining the localization of both proteins after PDGF stimulation of cultured cells which stably express the human PDGF receptor. In resting cells, transiently expressed PTP2C is distributed throughout the cytoplasm. Upon stimulation with PDGF, PTP2C is translocated from the cytoplasm to membrane ruffles. Immunofluorescence examination reveals that PTP2C colocalized with actin, the PDGF receptors, and hyper-tyrosine- phosphorylated protein(s). Neither deletion of the SH2 domains nor point mutations at either the catalytic site or the major phosphorylation site affected membrane ruffling or the localization of PTP2C to the ruffles of PDGF-stimulated cells. However, the expression of a catalytically inactive mutant PTP2C substantially prolongs ruffling activity following PDGF stimulation. These results suggest that PTP2C is involved in the down-regulation of the membrane ruffling pathway, and in contrast to its positive function in the MAP kinase pathway, the phosphatase activity negatively regulates ruffling activity (Cossette, 1996).

The expression of a catalytically inactive mutant of SH-PTP2 (containing the mutation Cys-459-->Ser) in Chinese hamster ovary cells that overexpress human insulin receptors (CHO-IR cells) markedly attenuates insulin-stimulated Ras (See Drosophila Ras) activation. Expression of mutant SH-PTP2 also inhibits MAP kinase activation in response to insulin but not in response to phorbol ester. In contrast, the insulin-induced association of phosphoinositide 3-kinase activity with IRS-1 is not affected by the expression of inactive SH-PTP2. Furthermore, the expression of mutant SH-PTP2 had no effect on the binding of Grb2 to IRS-1, on the tyrosine phosphorylation of Shc, or on the formation of the complex between Shc and Grb2 in response to insulin. However, the amount of SH-PTP2 bound to IRS-1 in insulin-treated CHO-IR cells expressing mutant SH-PTP2 is greater than that observed in CHO-IR cells overexpressing wild-type SH-PTP2. Recombinant SH-PTP2 specifically dephosphorylates a synthetic phosphopeptide corresponding to the sequence surrounding Tyr-1172 of IRS-1, a putative binding site for SH-PTP2. Additionally, phenylarsine oxide, an inhibitor of protein-tyrosine phosphatases, inactivates SH-PTP2 in vitro and increases the insulin-induced association of SH-PTP2 with IRS-1. All this information suggests that SH-PTP2 may regulate an upstream element necessary for Ras activation in response to insulin and that this upstream element may be required for the Grb2- or Shc-dependent pathway. This is consistent with the notion that SH-PTP2 may bind to IRS-1 through its SH2 domains in response to insulin and dephosphorylate the phosphotyrosine residue to which it binds, thereby regulating its association with IRS-1 (Noguchi, 1994).

In response to appropriate growth factor stimulation, Syp becomes phosphorylated on tyrosine residues and associates with insulin receptor substrate 1 (IRS-1) and/or the corresponding growth factor receptor via its SH2 domains, leading to increased Syp activity. Insulin-, insulin-like growth factor-1-, and epidermal growth factor-stimulated DNA synthesis, are all dramatically decreased following microinjection of either an anti-Syp antibody (Ab) (65-85%) or a Syp GST-SH2 fusion protein. In addition, microinjection of an IRS-1-derived phosphonopeptide, which inhibits in vitro binding of Syp-SH2 to IRS-1, also decreases DNA synthesis by approximately 50-75%. Microinjection of the Syp Ab, Syp GST-SH2 fusion protein, or the phosphonopeptide have no effect on serum-stimulated DNA synthesis incorporation. Disruption of Syp function in living cells inhibits cell cycle progression in response to growth factor stimulation, indicating that Syp is a critical positive regulator of mitogenic signal transduction (Xiao, 1994).

Src homology 2 (SH2) domains are phosphotyrosine binding modules found within many cytoplasmic proteins. A major function of SH2 domains is to bring about the physical assembly of signaling complexes. Simultaneous occupancy of both SH2 domains of the phosphotyrosine phosphatase SH-PTP2 by a tethered peptide with two IRS-1-derived phosphorylation sites potently stimulates phosphatase activity. The concentration required for activation by the tethered peptide is 80-160-fold lower than either corresponding monophosphorylated peptide. Moreover, the diphosphorylated peptide stimulates catalytic activity 37-fold, compared with 9-16-fold for the monophosphorylated peptides. Mutational analyses of the SH2 domains of SH-PTP2 confirm that both SH2 domains participate in this effect. Binding studies with a tandem construct comprising the N- plus C-terminal SH2 domains show that the diphosphorylated peptide binds with 60-90-fold higher affinity than either monophosphorylated sequence. These results demonstrate that SH-PTP2 activity can be potently regulated by interacting (via both of its SH2 domains) with phosphoproteins having two cognate phosphorylation sites (Pluskey, 1995).

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The phosphorylation of proteins at tyrosine residues is critical in the growth signaling induced by Epo. This mechanism is regulated by the activities of both protein-tyrosine kinases and protein tyrosine phosphatases. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. Syp becomes phosphorylated on tyrosine after stimulation with Epo in M07ER cells engineered to express high levels of human EpoR. Syp is complexed with Grb2 in Epo-stimulated M07ER cells. Direct binding between Syp and Grb2 is also observed in vitro. Furthermore, Syp appears to bind directly to tyrosine-phosphorylated EpoR in M07ER cells. Both NH2-terminal and COOH-terminal SH2 domains of Syp are able to bind to the tyrosine-phosphorylated EpoR in vitro. These results suggest that Syp may be an important signaling component downstream of the EpoR and may regulate the proliferation and differentiation of hematopoietic cells (Tauchi, 1995).

Protein tyrosine phosphorylation and thus dephosphorylation are part of the interleukin (IL)-11 response in mouse 3T3-L1 cells. Addition of IL-11 to 3T3-L1 cells results in an increase in the tyrosine phosphorylation of Syp. The C-terminal SH2 domain of Syp was shown to precipitate several proteins of 70, 130, 150, and 200 kDa that are tyrosine phosphorylated in response to IL-11. Syp is inducibly associated with both gp130 and Janus kinase 2 (JAK2). A phosphopeptide containing the sequence for a potential Syp binding site (YXXV) was used to compete with the associations of Syp with gp130 and JAK2. The phosphopeptide reduces the Syp association with both gp130 and JAK2. To summarize, Syp has multiple interactions in IL-11 signal transduction. In addition to the IL-11-induced tyrosine phosphorylation of Syp, Syp coprecipitates with gp130, JAK2, and other tyrosine-phosphorylated proteins in response to IL-11 (Fuhrer, 1995).

The absence of CTLA-4 results in uncontrolled T cell proliferation. The T cell receptor-specific kinases FYN, LCK, and ZAP-70 as well as the RAS pathway have been found to be activated in T cells of Ctla-4-/- mutant mice. In addition, CTLA-4 specifically associates with the tyrosine phosphatase SYP, an interaction mediated by the SRC homology 2 (SH2) domains of SYP and the phosphotyrosine sequence Tyr-Val-Lys-Met within the CTLA-4 cytoplasmic tail. The CTLA-4-associated SYP had phosphatase activity toward the RAS regulator p52SHC. Thus, the RAS pathway and T cell activation through the T cell receptor are regulated by CTLA-4-associated SYP (Marengere, 1996).

TEK is a newly cloned receptor tyrosine kinase that is expressed predominantly in the endothelium of actively growing blood vessels. Disruption of TEK function in transgenic mice results in a profound defect in vascular development leading to embryonic lethality. TEK signaling is indispensable for the development of the embryonic vasculature and TEK signaling may also be required for the development of the tumor vasculature. Because the ligand for TEK has not been identified, it has been difficult to study signal transduction by this important endothelial receptor. To circumvent this problem, a soluble TEK kinase domain (GTEKH) was developed which could be easily purified, autophosphorylated, and radiolabeled. Using the autophosphorylated, radiolabeled GTEKH to probe a mouse embryo expression library only two candidate signaling molecules were isolated, SH-PTP2 and GRB2. Autophosphorylated GTEKH associates with GRB2 and SH-PTP2 from endothelial lysates and not with PI3 kinase or PLC gamma. The association of GRB2 and SH-PTP2 with TEK is highly dependent on specific tyrosine residues in the TEK c-tail. These studies identify GRB2 and SH-PTP2 as potentially important mediators of TEK signaling that may trigger crucial endothelial responses during embryonic vascular development and during pathologic vascular growth (Huang, 1995).

Since SH2 domains have been shown to target the association of signal-transducing molecules to activated tyrosine kinases, experiments were performed to determine whether Syp might form specific complexes with p210bcr-abl (See Drosophila Abl oncogene), a fusion protein believed to be involved in the pathogenesis of chronic myelogenous leukemia and, thus, possibly alter or mediate p210bcr-abl tyrosine kinase activity. Syp was highly and constitutively tyrosine phosphorylated in three different murine cell lines transfected with a p210bcr-abl expression vector. Furthermore, p210bcr-abl, Syp, and Grb2 formed stable complexes in BCR-ABL-expressing cells. Complex formation between p210bcr-abl and Syp is mediated in vitro by the NH2-terminal SH2 domain of Syp. P210bcr-abl tyrosine kinase is effectively dephosphorylated by Syp in vitro. These results suggest an interaction between Syp and BCR-ABL protein, which might play a role in cellular transformation of BCR-ABL (Tauchi, 1994).

Src homology 2-containing phosphotyrosine phosphatase (Shp2) functions as a positive effector in receptor tyrosine kinase (RTK) signaling immediately proximal to activated receptors. However, neither its physiological substrate(s) nor its mechanism of action in RTK signaling has been defined. In this study, Sprouty (Spry) is demonstrated to be a possible target of Shp2. Spry acts as a conserved inhibitor of RTK signaling, and tyrosine phosphorylation of Spry is indispensable for its inhibitory activity. Shp2 is able to dephosphorylate fibroblast growth factor receptor-induced phosphotyrosines on Spry both in vivo and in vitro. Shp2-mediated dephosphorylation of Spry results in dissociation of Spry from Grb2. Furthermore, Shp2 can reverse the inhibitory effect of Spry on FGF-induced neurite outgrowth and MAP kinase activation. These findings suggest that Shp2 acts as a positive regulator in RTK signaling by dephosphorylating and inactivating Spry (Hanafusa, 2004).

Shp2 and Ras activation

SHP2 down-regulates PI3K activation by dephosphorylating; however, the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, whether SHP2 dephosphorylates them to facilitate Ras activation was tested. A Gab1 construct preventing SHP2 recruitment promotes membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restores the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppresses RasGAP redistribution and restores the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 interacts with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr(317) are severely affected in RasGAP binding and are the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. Thus a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding has been identified and an important SHP2 function has been demonstrated to down-regulate this site's phosphorylation to disengage RasGAP and sustain Ras activation (Montigner, 2005).

Shp2 and the insulin signaling pathway

The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms. However, in an in vitro system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), only Ser-318, but not Ser-307 phosphorylation was detected, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. Since the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, the role of Shp2 in this context was examined. Phorbol ester-induced Ser-307 phosphorylation is reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2-/-) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depends on the presence of Shp2. Furthermore, Shp2-dependent phorbol ester effects on Ser-307 are blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. This suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1 (Mussig, 2005).

SHP-2 interaction with Gab1 and Gab2

Gab1 has structural similarities to Drosophila Dos (Daughter of sevenless); Dos is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and Dos have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. Gab1 is tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-alpha), and IFN-gamma. Upon the stimulation of IL-6 or IL-3, Gab1 is found to form a complex with phosphatidylinositol PI3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, reveals that neither tyrosine residues of gp130 nor its carboxy terminus is required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhances gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogates the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation is inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation (Takahashi-Tezuka, 1998).

A novel human adapter molecule is described containing a pleckstrin homology (PH) domain at the N terminus that is closely related to human Grb2-associated binder 1 (Gab1) and Drosophila Daughter of sevenless. This protein has been designated as Gab2. Northern blot analysis indicates that Gab2 is widely expressed and has an overlapping but distinctive expression pattern as compared with Gab1, with high levels of Gab2 mRNA detected in the heart, brain, placenta, spleen, ovary, peripheral blood leukocytes, and spinal cord. Upon tyrosine phosphorylation, Gab2 physically interacts with Shp2 tyrosine phosphatase and Grb2 adapter protein. Strikingly, Gab2 has an inhibitory effect on the activation of Elk-1-dependent transcription triggered by a dominant active Ras mutant (RasV12) or under growth factor stimulation, whereas Gab1 acts to potentiate slightly the Elk-1 activity in the same system. In contrast to the reciprocal effects of Gab1 and Gab2 in mediating Elk-1 induction, these two molecules have a similar function in extracellular signal-regulated kinase activation induced by either oncogenic Ras or growth factor stimulation. Taken together, these results argue that Gab1 and Gab2, two closely related PH-containing adapter proteins, might have distinct roles in coupling cytoplasmic-nuclear signal transduction. This is the first evidence that an intracellular molecule with a PH domain operates as a negative effector in signal relay to the regulation of gene expression (Zhao, 1999).

Although the physiological function of Gab1 has not been fully understood, it apparently acts to promote cell growth and transformation. Overexpression of Gab1 leads to enhanced cell division and anchorage-independent cell growth in soft agar. Tyrosine phosphorylation of Gab1 and its association with Grb2 might also play an important role in cellular transformation by Tpr-Met, an oncoprotein consisting of the catalytic domain of hepatocyte growth factor receptor tyrosine kinase fused with sequences encoded by the tpr gene. A mutant Tpr-Met protein (Tyr489 to Phe) that fails to bind to Grb2 shows significantly impaired transforming activity. Although it is unclear how Gab1 functions in cytoplasmic signaling, the molecular architecture of this protein strongly suggests that Gab1 is tyrosine phosphorylated at numerous sites and therefore is engaged in the formation of a complex that consists of many proteins, including phospholipase Cgamma, Shp2, and phosphatidylinositol 3-kinase. In this regard, Gab1 and Gab2 might work in cells in a similar manner as the family of insulin receptor substrates, IRS1-4, by coupling to multiple signaling pathways. However, the difference is the possession of a PTB domain following the PH domain in the IRS proteins: this is missing from Gab1 and Gab2. Without a PTB domain, Gab1/2 might interact with growth factor receptors indirectly via other SH2-containing molecules, such as Grb2 (Zhao, 1999).

Gab1 and Gab2 share structural similarity with the Drosophila Daughter of sevenless protein. Not only is Daughter of sevenless physically associated with Corkscrew, but Daughter of sevenless is the major phosphoprotein trapped by a catalytically inactive mutant of Corkscrew that is overexpressed in cells. Both Gab1 and Gab2 have been detected in association with Shp2, the mammalian homolog of Drosophila Corkscrew. Now the critical issue is to determine whether they are the physiological substrates of Shp2 and how this dephosphorylation event could contribute to the Ras signaling pathway. It is possible that these two molecules may serve as substrates of the ubiquitously expressed Shp2 phosphatase in different cell types. Further, these proteins may also be potential substrates of another Shp2-related phosphatase, Shp1, which is predominantly expressed in hematopoietic cells (Zhao, 1999).

The most striking finding in this report is the reciprocal effects of Gab1 and Gab2 in mediating Ras-responsive Elk-1 activation in cells. Gab2 does not seem to block the ERK kinase activation by either receptor tyrosine kinase or dominant active Ras, but rather it acts to uncouple signals from activated ERK to Elk-1, a member of the ternary complex factors family. Expression of murine p97/Gab2 in Ba/F3 cells also leads to a suppression of IL-3-induced Elk-1 activity, whereas ERK1 kinase activation is not affected. This inhibitory effect of mouse Gab2 on IL-3-stimulated Elk-1 activity is exacerbated by the introduction of a C-terminal truncation mutation that abolishes its binding to Shp2 (Zhao, 1999 and references).

A similar uncoupling effect on ERK kinase to Elk-1 stimulation was recently observed for the kinase suppressor of Ras. When overexpressed, the kinase suppressor of Ras blocks EGF and the Ras induction of Elk-1-dependent transcription. Therefore, the Gab2 and kinase suppressor of Ras might represent components of a novel and poorly understood pathway that modulates the signal relay from the Ras-ERK kinase to the activation of nuclear ternary complex factors. Although the mechanism for its suppression on Elk-1 activation has yet to be understood, the Gab2 protein without any catalytic activities acts apparently by complexing with other enzymes, such as kinases and phosphatases, that contain SH2 and SH3 domains. Further investigation of the unique function of Gab2 will provide fundamental insight into the molecular mechanism by which the Ras-signaling scheme is concerted (Zhao, 1999 and references).

Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. The protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1, compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. These results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner (Zhang, 2002).

Focal adhesion kinase is a substrate and downstream effector of SHP-2

Infection with cagA-positive Helicobacter pylori (H. pylori) is associated with atrophic gastritis, peptic ulcer, and gastric adenocarcinoma. The cagA gene product CagA is translocated from H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation by Src family kinases (SFKs). Tyrosine-phosphorylated CagA binds and activates SHP-2 phosphatase and the C-terminal Src kinase (Csk) while inducing an elongated cell shape termed the 'hummingbird phenotype.' CagA reduces the level of focal adhesion kinase (FAK) tyrosine phosphorylation in gastric epithelial cells. The decrease in phosphorylated FAK is due to SHP-2-mediated dephosphorylation of FAK at the activating phosphorylation sites, not due to Csk-dependent inhibition of SFKs, which phosphorylate FAK. Coexpression of constitutively active FAK with CagA inhibits induction of the hummingbird phenotype, whereas expression of dominant-negative FAK elicits an elongated cell shape characteristic of the hummingbird phenotype. These results indicate that inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. Impaired cell adhesion and increased motility by CagA may be involved in the development of gastric lesions associated with cagA-positive H. pylori infection (Tsutsumi, 2006).

Phosphatidylinositol-3 kinase is a substrate of SHP-2

Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) migration and the PI-3 kinase pathway plays an important role in mediating the IGF-I induced migratory response. Prior studies have shown that the tyrosine phosphatase SHP-2 is necessary to activate PI-3 kinase in response to growth factors and expression of a phosphatase inactive form of SHP-2 (SHP-2/C459S) impairs IGF-I stimulated cell migration. However, the mechanism by which SHP-2 phosphatase activity or the recruitment of SHP-2 to other signaling molecules contributes to IGF-I stimulated PI-3 kinase activation has not been determined. SMCs that have stable expression of SHP-2/C459S, have reduced cell migration and Akt activation in response to IGF-I compared with SMC expressing native SHP-2. Similarly in cells expressing native SHP-2, IGF-I induces SHP-2 binding to p85, whereas in cells expressing SHP-2/C459S there is no increase. Since the C459S substitution results in loss of the ability of SHP-2 to disassociate from its substrates, making it inaccessible not only to p85 but also the other proteins, a p85 mutant in which tyrosines 528 and 556 were changed to phenylalanines was prepared to determine if this would disrupt the p85/SHP-2 interaction and if the loss of this specific interaction would alter IGF-I stimulated the cell migration. Substitution for these tyrosines in p85 resulted in loss of SHP-2 recruitment and was associated with a reduction in association of the p85/p110 complex with IRS-1. Cells stably expressing this p85 mutant also showed a decrease in IGF-I stimulated PI-3 kinase activity and cell migration. Pre-incubation of cells with a cell permeable peptide that contains the tyrosine556 motif of p85 also disrupts SHP-2 binding to p85 and inhibits the IGF-I induced increase in cell migration. The findings indicate that tyrosines 528 and 556 in p85 are required for SHP-2 association. SHP-2 recruitment to p85 is required for IGF-I stimulated association of the p85/p110 complex with IRS-1 and for the subsequent activation of the PI-3 kinase pathway leading to increased cell migration (Kwon, 2005).

SHP2 and Leptin signaling

Although leptin is known to induce proliferative response in gastric cancer cells, the mechanism(s) underlying this action remains poorly understood. Evidence is provided that leptin-induced gastric cancer cell proliferation involves activation of STAT and ERK2 signaling pathways. Leptin-induced STAT3 phosphorylation is independent of ERK2 activation. Leptin increases SHP2 phosphorylation and enhances binding of Grb2 to SHP2. Inhibition of SHP2 expression with siRNA but not SHP2 phosphatase activity abolishes leptin-induced ERK2 activation. While JAK inhibition with AG490 significantly reduces leptin-induced ERK2, STAT3 phosphorylation, and cell proliferation, SHP2 inhibition only partially reduces cancer cell proliferation. Immunostained gastric cancer tissues display local overexpression of leptin and its receptor indicating that leptin might be produced and act locally in a paracrine or autocrine manner. These findings indicate that leptin promotes cancer growth by activating multiple signaling pathways; therefore, blocking leptin action at the receptor level could be a rational therapeutic strategy (Pai, 2005).

Mutation of SHP-2: LEOPARD and Noonan syndromes

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. The functional effects were compared for three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation (Fragale, 2004).

Noonan syndrome is a common human autosomal dominant birth defect, characterized by short stature, facial abnormalities, heart defects and possibly increased risk of leukemia. Mutations of Ptpn11 (also known as Shp2), which encodes the protein-tyrosine phosphatase Shp2, occur in approximately 50% of individuals with Noonan syndrome, but their molecular, cellular and developmental effects, and the relationship between Noonan syndrome and leukemia, are unclear. Mice were generated expressing the Noonan syndrome-associated mutant D61G. When homozygous, the D61G mutant is embryonic lethal, whereas heterozygotes have decreased viability. Surviving Ptpn11(D61G/+) embryos (approximately 50%) have short stature, craniofacial abnormalities similar to those in Noonan syndrome, and myeloproliferative disease. Severely affected Ptpn11(D61G/+) embryos ( approximately 50%) have multiple cardiac defects similar to those in mice lacking the Ras-GAP protein neurofibromin. Their endocardial cushions have increased Erk activation, but Erk hyperactivation is cell and pathway specific. These results clarify the relationship between Noonan syndrome and leukemia and show that a single Ptpn11 gain-of-function mutation evokes all major features of Noonan syndrome by acting on multiple developmental lineages in a gene dosage-dependent and pathway-selective manner (Araki, 2004).

LEOPARD (LS) and Noonan (NS) are overlapping syndromes associated with distinct mutations of SHP-2. Whereas NS mutations enhance SHP-2 catalytic activity, the activity of three representative LS mutants is undetectable when assayed using a standard protein tyrosine phosphatase (PTP) substrate. A different assay using a specific SHP-2 substrate confirms their decreased PTP activity, but also reveals a significant activity of the T468M mutant. In transfected cells stimulated with epidermal growth factor, the least active LS mutants promote Gab1/PI3K binding, validating the in vitro data. LS mutants thus display a reduced PTP activity both in vitro and in transfected cells (Hanna, 2006).

Brain-derived neurotrophic factor (BDNF) and other neurotrophins induce a unique prolonged activation of mitogen-activated protein kinase (MAPK) compared with growth factors. Characterization and kinetic and spatial modeling of the signaling pathways underlying this prolonged MAPK activation by BDNF will be important in understanding the physiological role of BDNF in many complex systems in the nervous system. In addition to Shc, fibroblast growth factor receptor substrate 2 (FRS2) is required for the BDNF-induced activation of MAPK. BDNF induces phosphorylation of FRS2. However, BDNF does not induce phosphorylation of FRS2 in cells expressing a deletion mutant of TrkB (TrkBDeltaPTB) missing the juxtamembrane NPXY motif. This motif is the binding site for SHC. NPXY is the consensus sequence for phosphotyrosine binding (PTB) domains, and notably, FRS2 and SHC contain PTB domains. This NPXY motif, which contains tyrosine 484 of TrkB, is therefore the binding site for both FRS2 and SHC. Moreover, the proline containing region (VIENP) of the NPXY motif is also required for FRS2 and SHC phosphorylation, which indicates this region is an important component of FRS2 and SHC recognition by TrkB. The phosphorylation of FRS2 induces association of FRS2 and growth factor receptor binding protein 2 (Grb2). Intriguing data indicates BDNF induces association of the SH2 domain containing protein tyrosine phosphatase, Shp2, with FRS2. Moreover, the PTB association motif of TrkB containing tyrosine 484 is required for the BDNF-induced association of Shp2 with FRS2 and the phosphorylation of Shp2. These results imply that FRS2 and Shp2 are in a BDNF signaling pathway. Shp2 is required for complete MAPK activation by BDNF, as expression of a dominant negative Shp2 in cells attenuates BDNF-induced activation of MAPK. Moreover, expression of a dominant negative Shp2 attenuates Ras activation showing that the protein tyrosine phosphatase is required for complete activation of MAPKs by BDNF. In conclusion, Shp2 regulates BDNF signaling through the MAPK pathway by regulating either Ras directly or alternatively, by signaling components upstream of Ras. Characterization of MAPK signaling controlled by BDNF is likely to be required to understand the complex physiological role of BDNF in neuronal systems ranging from the regulation of neuronal growth and survival to the regulation of synapses (Easton, 2006).

Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. A panel of LS mutants were examined, including the two most common alleles. Surprisingly, it was found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinase-mediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. These results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation (Kontaridis, 2006).

Within the developing mammalian CNS, growth factors direct multipotent precursors to generate neurons versus glia, a process that if perturbed might lead to neural dysfunction. In this regard, genetic mutations resulting in constitutive activation of the protein tyrosine phosphatase SHP-2 cause Noonan Syndrome (NS), which is associated with learning disabilities and mental retardation. Genetic knockdown of SHP-2 in cultured cortical precursors or in the embryonic cortex inhibits basal neurogenesis and causes enhanced and precocious astrocyte formation. Conversely, expression of an NS SHP-2 mutant promotes neurogenesis and inhibits astrogenesis. Neural cell-fate decisions are similarly perturbed in a mouse knockin model that phenocopies human NS. Thus, SHP-2 instructs precursors to make neurons and not astrocytes during the neurogenic period, and perturbations in the relative ratios of these two cell types upon constitutive SHP-2 activation may contribute to the cognitive impairments in NS patients (Gauthier, 2007).

Mutation of SHP-2 induces hematopoietic malignancy

Mutations in SHP-2 phosphatase that cause hyper-activation of its catalytic activity have been identified in human leukemias, particularly, juvenile myelomonocytic leukemia which is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce hematopoietic malignancies are not fully understood. SHP-2 plays an essential role in IL-3 signal transduction in both catalytic-dependent and -independent manners; the overexpression (5-to-6 fold) of wild type (WT) SHP-2 attenuates IL-3 mediated hematopoietic cell function through accelerated dephosphorylation of STAT5. These results raised the possibility that SHP-2-associated leukemias are not solely attributed to the increased catalytic activity of GOF mutant SHP-2. GOF mutant SHP-2 must have gained additional capacities. To test this possibility, effects of a GOF mutation of SHP-2 (SHP-2 E76K) on hematopoietic cell function and IL-3 signal transduction were investigated by comparing with those of overexpressed WT SHP-2. The results show that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreases hematopoietic potential of the transduced cells in recipient animals. The E76K mutation in the N-terminal Src homology 2 domain increases interactions of mutant SHP-2 with Grb2, Gab2, and p85, leading to hyperactivation of IL-3-induced Erk and PI3 kinase pathways. In addition, despite the substantial increase in the catalytic activity, dephosphorylation of STAT5 by SHP-2 E76K was dampened. Furthermore, catalytically inactive SHP-2 E76K with an additional C459S mutation retains the capability to increase the interaction with Gab2 and to enhance the activation of the PI3 kinase pathway. Taken together, these studies suggest that in addition to the elevated catalytic activity, fundamental changes in physical and functional interactions between GOF mutant SHP-2 and signaling partners also play an important role in SHP-2-related leukemigenesis (Yu, 2005).

Sprouty2-modulated Kras signaling rescues Shp2 deficiency during lens and lacrimal gland development

Shp2/Ptpn11 tyrosine phosphatase is a general regulator of the RTK pathways. By genetic ablation, it was demonstrated that Shp2 is required for lacrimal gland budding, lens cell proliferation, survival and differentiation. Shp2 deletion disrupts ERK signaling and cell cycle regulation, which could be partially compensated by activated Kras signaling, confirming that Ras signaling is the main downstream target of Shp2 in lens and lacrimal gland development. It was also shown that Sprouty2, a general suppressor of Ras signaling, is regulated by Shp2 positively at the transcriptional level and negatively at the post-translational level. Only in the absence of Sprouty2 can activated Kras signaling robustly rescue the lens proliferation and lacrimal-gland-budding defects in the Shp2 mutants. It is proposed that the dynamic regulation of Sprouty by Shp2 might be important not only for modulating Ras signaling in lens and lacrimal gland development, but also for RTK signaling in general (Pan, 2010).

SHP-2 is a mediator of activity-dependent neuronal excitotoxicity

Calcium influx can promote neuronal differentiation and survival, at least in part by activating Ras and its downstream targets, including the Erk pathway. However, excessive calcium influx can initiate molecular signals leading to neuronal death during excitotoxicity or in neurodegenerative diseases. A new signaling pathway associated with calcium influx is described that contributes to neuronal cell death in cerebellar neurons. Influx of calcium, mediated either by L-type voltage-sensitive calcium channels or glutamate receptors, is associated with the suppression of brain-derived neurotrophic factor (BDNF) activation of Ras and its effectors Erk and Akt. This is the result of enhanced association of the tyrosine phosphatase Shp-2 with TrkB receptors that inhibits BDNF-induced TrkB autophosphorylation and activation. Deletion of the Shp2 gene in neuronal cultures reverses inhibition of TrkB function and increases neuronal survival after extended depolarization or glutamate treatment. These findings implicate Shp-2 in a feedback system initiated by calcium that negatively regulates neurotrophin signaling and sensitizes neurons to excitotoxicity (Rusanescu, 2005).

SHP-2, cell biology and development

SH-PTP2, the vertebrate homolog of Drosophila Corkscrew, associates with several activated growth factor receptors, but its biological function is unknown. The effects of wild-type and mutant SH-PTP2 RNA were examined on Xenopus embryogenesis. An internal phosphatase domain deletion (delta P) acts as a dominant negative mutant, causing severe posterior truncations. This phenotype is rescued by SH-PTP2, but not by the closely related SH-PTP1. In ectodermal explants, delta P blocks fibroblast growth factor (FGF)- and activin-mediated induction of mesoderm and FGF-induced mitogen-activated protein (MAP) kinase activation. These results indicate that SH-PTP2 is required for early vertebrate development, acting as a positive component in FGF signaling downstream of the FGF receptor and upstream of MAP kinase (Tang, 1995).

SH-PTP2 is a protein-tyrosine phosphatase with Src homology-2 (SH2) domains, shown to be highly expressed in the rat brain. Specific immunoreactivity is widely distributed, most abundant in neurophil, weak in neuronal somata, and absent from white matter. Intense labeling is observed on synapses and concentrated in the pre- and post-synaptic plasma membranes. In subcellular fractionation analysis of brain, SH-PTP2 is mainly observed in the particulate fraction, especially in myelin and synaptosomes. SH-PTP2 is further recovered in the synaptic plasma membrane. These results suggest that SH-PTP2 associates with synaptic membranes and may play a role in the synaptic communications in the brain (Suzuki, 1995).

Syp, a mouse phosphotyrosine phosphatase containing two Src homology 2 (SH2) domains binds to autophosphorylated epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors through its SH2 domains and is rapidly phosphorylated on tyrosine in PDGF- and EGF-stimulated cells. Syp is constitutively phosphorylated on tyrosine in cells transformed by v-src. This mammalian phosphatase is most closely related, especially in its SH2 domains, to the corkscrew gene product of Drosophila, which is required for signal transduction downstream of the Torso receptor tyrosine kinase. The Syp gene is widely expressed throughout embryonic mouse development and in adult tissues. Thus, Syp may function in mammalian embryonic development and as a common target of both receptor and nonreceptor tyrosine kinases (Feng, 1993).

In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Using a dominant negative mutant approach, it has been shown that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, novel, activated mutants of SHP-2 have been generated. Expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. These results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. These activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems (O'Reilly, 2000).

Shp2, a Src homology 2-containing tyrosine phosphatase, has been implicated in a variety of growth factor or cytokine signaling pathways. However, it is conceivable that this enzyme acts predominantly in one pathway versus the others in a cell, depending on the cellular context. To determine the putative functions of Shp2 in the adult brain, Shp2 was selectively deleted in postmitotic forebrain neurons by crossing CaMKIIalpha-Cre transgenic mice with a conditional Shp2 mutant [Shp2(flox)] strain. Surprisingly, a prominent phenotype of the mutant [CaMKIIalpha-Cre:Shp2(flox/flox)] or CaSKO) mice was the development of early-onset obesity, with increased serum levels of leptin, insulin, glucose, and triglycerides. The mutant mice were not hyperphagic but developed enlarged and steatotic liver. Consistent with in vitro data, it was found that Shp2 down-regulates Jak2/Stat3 activation by leptin in the hypothalamus. However, Jak2/Stat3 down-regulation is offset by a dominant Shp2 promotion of the leptin-stimulated Erk pathway, leading to induction rather than suppression of leptin resistance upon Shp2 deletion in the brain. Collectively, these results suggest that a primary function of Shp2 in postmitotic forebrain neurons is to control energy balance and metabolism, and that this phosphatase is a critical signaling component of leptin receptor ObRb in the hypothalamus. Shp2 shows potential as a neuronal target for pharmaceutical sensitization of obese patients to leptin action (Zhang, 2004).

Early development of the lens and retina depends upon reciprocal inductive interactions between the embryonic surface ectoderm and the underlying neuroepithelium of the optic vesicle. FGF signaling has been implicated in this signal exchange. The docking protein FRS2alpha is a major mediator of FGF signaling by providing a link between FGF receptors (FGFRs) and a variety of intracellular signaling pathways. After FGF stimulation, tyrosine-phosphorylated FRS2alpha recruits four molecules of the adaptor protein Grb2 and two molecules of the protein tyrosine phosphatase Shp2, resulting in activation of the Ras/extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase/Akt signaling pathways. This report explores the role of signaling pathways downstream of FRS2alpha in eye development by analyzing the phenotypes of mice that carry point mutations in either the Grb2-[Frs2alpha(4F)] or the Shp2-binding sites [Frs2alpha(2F)] of FRS2alpha. Although Frs2alpha(4F/4F) mice exhibited normal early eye development, all Frs2alpha(2F/2F) embryos were defective in eye development and showed anophthalmia or microphthalmia. Consistent with the critical role of FRS2alpha in FGF signaling, the level of activated extracellular signal-regulated kinase in Frs2alpha(2F/2F) embryos was significantly lower than that observed in wild-type embryos. Furthermore, expression of Pax6 and Six3, molecular markers for lens induction, were decreased in the Frs2alpha(2F/2F) presumptive lens ectoderm. Similarly, the expression of Chx10 and Bmp4, genes required for retinal precursor proliferation and for lens development, respectively, was also decreased in the optic vesicles of Frs2alpha(2F/2F) mice. These experiments demonstrate that intracellular signals that depend on specific tyrosine residues in FRS2alpha lie upstream of gene products critical for induction of lens and retina (Gotoh, 2004).

SHP-2, a tyrosine phosphatase implicated in diverse signaling pathways induced by growth factors and cytokines, is also involved in DNA damage-triggered signaling and cellular responses. SHP-2 plays an important role in DNA damage-induced apoptosis and G(2)/M cell cycle checkpoint. Evidence is provided that SHP-2 functions in DNA damage apoptosis and G(2)/M arrest in catalytically dependent and independent manners, respectively. Mutant embryonic fibroblasts with the Exon 3 deletion mutation in SHP-2 show decreased apoptosis and diminished G(2)/M arrest in response to cisplatin treatment. Wild type (WT), but not catalytically inactive mutant SHP-2 (SHP-2 C459S), rescues the apoptotic response of the mutant cells. Interestingly, both WT and SHP-2 C459S efficiently restore the G(2)/M arrest response. Furthermore, inhibition of the catalytic activity of endogenous SHP-2 in WT cells by overexpression of SHP-2 C459S greatly decreases cell death but not G(2)/M arrest induced by cisplatin. Biochemical analyses revealed that activation of c-Abl kinase is decreased in SHP-2 C459S-overexpressing cells. However, DNA damage-induced translocation of Cdc25C from the nucleus to the cytoplasm is fully restored in both WT and SHP-2 C459S 'rescued' cells. Additionally, it is demonstrated that the role of SHP-2 in DNA damage-induced cellular responses is independent of the tumor suppressor p53. Embryonic stem cells with the SHP-2 deletion mutation show markedly decreased sensitivity to cisplatin-induced apoptosis, attributable to impaired induction of p73 but not p53. In agreement with these results, DNA damage-induced apoptosis and G(2)/M arrest are also decreased in SHP-2/p53 double mutant embryonic fibroblasts. Collectively, these studies have further defined the mechanisms by which SHP-2 phosphatase regulates DNA damage responses (Yuan, 2005).

Mammalian corticogenesis occurs through a complex process that includes neurogenesis, in which neural progenitor cells proliferate, differentiate, and migrate. Neurogenesis occurs in the subventricular zone (SVZ), a region that has been thought to be the primary site of gliogenesis. In the SVZ, intermediate progenitor cells, derived from radial glial cells that are multipotent neural stem cells, produce only neurons. However, the molecular mechanisms underlying the regulation of neural stem cells and intermediate progenitor cells as well as their contribution to overall corticogenesis remain unknown. The docking protein FRS2alpha is a major mediator of signaling by means of FGFs and neurotrophins. FRS2alpha mediates many of its pleiotropic cellular responses by recruiting the adaptor protein Grb2 and the protein tyrosine phosphatase Shp2 upon ligand stimulation. Targeted disruption of Shp2-binding sites in FRS2alpha leads to severe impairment in cerebral cortex development in mutant mice. The defect in corticogenesis appears to be due at least in part to abnormalities in intermediate progenitor cells. Genetic evidence is provided that FRS2alpha plays critical roles in the maintenance of intermediate progenitor cells and in neurogenesis in the cerebral cortex. Moreover, FGF2-responsive neurospheres, which are cell aggregates derived from neural stem/progenitor cells (NSPCs), from FRS2alpha mutant mice are smaller than those of WT mice. However, mutant NSPCs are able to self-renew, demonstrating that Shp2-binding sites on FRS2alpha play an important role in NSPC proliferation but are dispensable for NSPC self-renewing capacity after FGF2 stimulation (Yamamoto, 2005).

Shp-2 is a member of a small family of cytoplasmic Src homology 2 (SH2) domain-containing protein tyrosine phosphatases. Although Shp-2 has been shown to be necessary for hematopoiesis using a mouse model expressing a mutant residual protein (Shp-2Delta/Delta), siRNA was used to reduce Shp-2 expression, and the consequences on embryonic stem (ES) cell-derived hemangioblast, primitive, and definitive hematopoietic development were examined. Shp-2 siRNA at a concentration of 50 nM effectively diminishes Shp-2 expression in differentiating embryoid bodies (EBs). Hemangioblast, primitive, and definitive hematopoietic progenitor formation is decreased significantly following transfection with Shp-2 siRNA, but not with scrambled siRNA. Since Shp-2 is involved in signals emanating from the bFGF receptor, it was asked whether Shp-2 functions in bFGF-mediated hemangioblast development. Reduction of Shp-2 expression using siRNA, but not scrambled siRNA, blocks the bFGF-induced increase in hemangioblast development. Using siRNA as an independent method of reducing Shp-2 function, in contrast to the mutant mouse model (Shp-2Delta/Delta) previously utilized, it has been demonstrated that Shp-2 is required in hemangioblast, primitive, and definitive progenitor hematopoietic development and that Shp-2 is integrally necessary for bFGF-mediated hemangioblast production (Zou, 2005).

Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must be tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. An important function has been established for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. Shp2 is specifically expressed in embryonic lung epithelial buds, and loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, direct evidence is provided that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung is associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation is not affected. These results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis (Tefft, 2005).

The 'signal regulatory protein' SIRPalpha is an Ig superfamily, transmembrane glycoprotein with a pair of cytoplasmic domains that can bind the phosphatase SHP-2 when phosphorylated on tyrosine. SIRPalpha is prominent in growth cones of rat cortical neurons and located, together with the tetraspanin CD81, in the growth cone periphery. SIRPalpha is dynamically associated with Triton-X-100-sensitive, but Brij-98-resistant, lipid microdomains, which also contain CD81. Challenge of growth cones with the integrin-binding extracellular-matrix (ECM) protein, laminin, or with the growth factors, IGF-1 or BDNF, increases SIRPalpha phosphorylation and SHP-2 binding rapidly and transiently, via Src family kinase activation; phosphorylated SIRPalpha dissociates from the lipid microdomains. A cytoplasmic tail fragment of SIRPalpha (cSIRPalpha), when expressed in primary cortical neurons, also is phosphorylated and binds SHP-2. Expression of wild-type cSIRPalpha, but not of a phosphorylation-deficient mutant, substantially decreases IGF-1-stimulated axonal growth on laminin. On poly-D-lysine and in control conditions, axonal growth is slower than on laminin, but there is no further reduction in growth rate induced by the expression of cSIRPalpha. Thus, the effect of cSIRPalpha on axon growth is dependent upon integrin activation by laminin. These results suggest that SIRPalpha functions in the modulation of axonal growth by ECM molecules, such as laminin (Wang, 2006).

Little is known about how growth factors control tissue stem cell survival and proliferation. Mice with a null mutation of Shp2 (Ptpn11), a key component of receptor tyrosine kinase signaling, were analyzed. Null embryos die peri-implantation, much earlier than mice that express an Shp2 truncation. Shp2 null blastocysts initially develop normally, but they subsequently exhibit inner cell mass death, diminished numbers of trophoblast giant cells, and failure to yield trophoblast stem (TS) cell lines. Molecular markers reveal that the trophoblast lineage, which requires fibroblast growth factor-4 (FGF4), is specified but fails to expand normally. Moreover, deletion of Shp2 in TS cells causes rapid apoptosis. Shp2 is required for FGF4-evoked activation of the Src/Ras/Erk pathway that culminates in phosphorylation and destabilization of the proapoptotic protein Bim. Bim depletion substantially blocks apoptosis and significantly restores Shp2 null TS cell proliferation, thereby establishing a key mechanism by which FGF4 controls stem cell survival (Yang, 2006).

The isolation and culturing of cardiac progenitor cells has demonstrated that growth factor signaling is required to maintain cardiac cell survival and proliferation. This study demonstrates in Xenopus that SHP-2 activity is required for the maintenance of cardiac precursors in vivo. In the absence of SHP-2 signaling, cardiac progenitor cells downregulate genes associated with early heart development and fail to initiate cardiac differentiation. This requirement for SHP-2 is restricted to cardiac precursor cells undergoing active proliferation. By demonstrating that SHP-2 is phosphorylated on Y542/Y580 and that it binds to FRS-2, SHP-2 is placed in the FGF pathway during early embryonic heart development. Furthermore, inhibition of FGF signaling mimics the cellular and biochemical effects of SHP-2 inhibition, and these effects can be rescued by constitutively active/Noonan-syndrome-associated forms of SHP-2. Collectively, these results show that SHP-2 functions within the FGF/MAPK pathway to maintain survival of proliferating populations of cardiac progenitor cells (Langdon, 2007).

corkscrew continued: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.