Gen-Sheng Feng

Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2

Shp‐1, Shp‐2 and corkscrew comprise a small family of cytoplasmic tyrosine phosphatases that possess two tandem SH2 domains. To investigate the biological functions of Shp‐2, a targeted mutation has been introduced into the murine Shp‐2 gene, which results in an internal deletion of residues 46–110 in the N‐terminal SH2 domain. Shp‐2 is required for embryonic development, as mice homozygous for the mutant allele die in utero at mid‐gestation. The Shp‐2 mutant embryos fail to gastrulate properly as evidenced by defects in the node, notochord and posterior elongation. Biochemical analysis of mutant cells indicates that Shp‐2 can function as either a positive or negative regulator of MAP kinase activation, depending on the specific receptor pathway stimulated. In particular, Shp‐2 is required for full and sustained activation of the MAP kinase pathway following stimulation with fibroblast growth factor (FGF), raising the possibility that the phenotype of Shp‐2 mutant embryos results from a defect in FGF‐receptor signalling. Thus, Shp‐2 modulates tyrosine kinase signalling in vivo and is crucial for gastrulation during mammalian development.

Protein-tyrosine Phosphatase Shp-2 Regulates Cell Spreading, Migration, and Focal Adhesion

Shp-2, a widely expressed cytoplasmic tyrosine phosphatase with two SH2 domains, is believed to participate in signal relay downstream of growth factor receptors. We show here that this phosphatase also plays an important role in the control of cell spreading, migration, and cytoskeletal architecture. Fibroblast cells lacking a functional Shp-2 were impaired in their ability to spread and migrate on fibronectin compared with wild-type cells. Furthermore, Shp-2 mutant cells displayed an increased number of focal adhesions and condensed F-actin aggregation at the cell periphery, properties reminiscent of focal adhesion kinase (FAK)-deficient cells. This is consistent with our previous observations in vivo that mice homozygous for the Shp-2 mutation died at midgestation with similar phenotype to FAK and fibronectin-deficient embryos, having severe defects in mesodermal patterning, particularly the truncation of posterior structures. Biochemical analysis demonstrated that FAK dephosphorylation was significantly reduced in Shp-2 mutant cells in suspension. Furthermore, regulated association of Src SH2 domain with FAK and paxillin during cell attachment and detachment on fibronectin was disrupted in Shp-2 mutant cells. This report defines a unique role of the Shp-2 tyrosine phosphatase in cell motility, which might guide the design of a new strategy for pharmaceutical interference of tumor metastasis.

Shp-2 Tyrosine Phosphatase Functions as a Negative Regulator of the Interferon-Stimulated Jak/STAT Pathway

ABSTRACT Shp-2 is an SH2 domain-containing protein tyrosine phosphatase. Although the mechanism remains to be defined, substantial experimental data suggest that Shp-2 is primarily a positive regulator in cell growth and development. We present evidence here that Shp-2, while acting to promote mitogenic signals, also functions as a negative effector in interferon (IFN)-induced growth-inhibitory and apoptotic pathways. Treatment of mouse fibroblast cells lacking a functional Shp-2 with IFN-α or IFN-γ resulted in an augmented suppression of cell viability compared to that of wild-type cells. To dissect the molecular mechanism, we examined IFN-induced activation of signal transducers and activators of transcription (STATs) by electrophoretic mobility shift assay, using a specific DNA probe (hSIE). The amounts of STAT proteins bound to hSIE upon IFN-α or IFN-γ stimulation were significantly increased in Shp-2−/− cells. Consistently, tyrosine phosphorylation levels of Stat1 upon IFN-γ treatment and, to a lesser extent, upon IFN-α stimulation were markedly elevated in mutant cells. Furthermore, IFN-γ induced a higher level of caspase 1 expression in Shp-2−/− cells than in wild-type cells. Reintroduction of wild-type Shp-2 protein reversed the hypersensitivity of Shp-2−/− fibroblasts to the cytotoxic effect of IFN-α and IFN-γ. Excessive activation of STATs by IFNs was also diminished in mutant cells in which Shp-2 had been reintroduced. Together, these results establish that Shp-2 functions as a negative regulator of the Jak/STAT pathway. We propose that Shp-2 acts to promote cell growth and survival through two mechanisms, i.e., the stimulation of growth factor-initiated mitogenic pathways and the suppression of cytotoxic effect elicited by cytokines, such as IFNs.

Molecular Mechanism for the Shp-2 Tyrosine Phosphatase Function in Promoting Growth Factor Stimulation of Erk Activity

ABSTRACT We have previously shown that activation of extracellular signal-regulated kinase (Erk) by epidermal growth factor (EGF) treatment was significantly decreased in mouse fibroblast cells expressing a mutant Shp-2 molecule lacking 65 amino acids in the SH2-N domain, Shp-2Δ46-110. To address the molecular mechanism for the positive role of Shp-2 in mediating Erk induction, we evaluated the activation of signaling components upstream of Erk in Shp-2 mutant cells. EGF-stimulated Ras, Raf, and Mek activation was significantly attenuated in Shp-2 mutant cells, suggesting that Shp-2 acts to promote Ras activation or to suppress the down-regulation of activated Ras. Biochemical analyses indicate that upon EGF stimulation, Shp-2 is recruited into a multiprotein complex assembled on the Gab1 docking molecule and that Shp-2 seems to exert its biological function by specifically dephosphorylating an unidentified molecule of 90 kDa in the complex. The mutant Shp-2Δ46-110 molecule failed to participate in the Gab1-organized complex for dephosphorylation of p90, correlating with a defective activation of the Ras-Raf-Mek-Erk cascade in EGF-treated Shp-2 mutant cells. Evidence is also presented that Shp-2 does not appear to modulate the signal relay from EGF receptor to Ras through the Shc, Grb2, and Sos proteins. These results begin to elucidate the mechanism of Shp-2 function downstream of a receptor tyrosine kinase to promote the activation of the Ras-Erk pathway, with potential therapeutic applications in cancer treatment.

A deletion mutation in the SH2-N domain of Shp-2 severely suppresses hematopoietic cell development.

Shp-1 and Shp-2 are cytoplasmic protein tyrosine phosphatases that contain two Src homology 2 (SH2) domains. A negative regulatory role of Shp-1 in hematopoiesis has been strongly implicated by the phenotype of motheaten mice with a mutation in the Shp-1 locus, which is characterized by leukocyte hypersensitivity, deregulated mast cell function, and excessive erythropoiesis. A targeted deletion of 65 amino acids in the N-terminal SH2 (SH2-N) domain of Shp-2 leads to an embryonic lethality at midgestation in homozygous mutant mice. To further dissect the Shp-2 function in hematopoietic development, we have isolated homozygous Shp-2 mutant embryonic stem (ES) cells. Significantly reduced hematopoietic activity was observed when the mutant ES cells were allowed to differentiate into embryoid bodies (EBs), compared to the wild-type and heterozygous ES cells. Further analysis of ES cell differentiation in vitro showed that mutation in the Shp-2 locus severely suppressed the development of primitive and definitive erythroid progenitors and completely blocked the production of progenitor cells for granulocytes-macrophages and mast cells. Reverse transcriptase PCR analysis of the mutant EBs revealed reduced expression of several specific marker genes that are induced during blood cell differentiation. Stem cell factor induction of mitogen-activated protein kinase activity was also blocked in Shp-2 mutant cells. Taken together, these results indicate that Shp-2 is an essential component and primarily plays a positive role in signaling pathways that mediate hematopoiesis in mammals. Furthermore, stimulation of its catalytic activity is not sufficient, while interaction via the SH2 domains with the targets or regulators is necessary for its biological functions in cells. The in vitro ES cell differentiation assay can be used as a biological tool in dissecting cytoplasmic signaling pathways.

Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling.

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-α-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485–620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.

Biased Suppression of Hematopoiesis and Multiple Developmental Defects in Chimeric Mice Containing Shp-2 Mutant Cells

ABSTRACT Shp-2 is a cytoplasmic tyrosine phosphatase that contains two Src homology 2 (SH2) domains at the N terminus. Biochemical data suggests that Shp-2 acts downstream of a variety of receptor and cytoplasmic tyrosine kinases. A targeted deletion mutation in the N-terminal SH2 (SH2-N) domain results in embryonic lethality of homozygous mutant mice at midgestation. In vitro embryonic stem (ES) cell differentiation assays suggest that Shp-2 might play an important role in hematopoiesis. By aggregating homozygous mutant (Shp-2−/−) ES cells and wild-type (WT) embryos, we created Shp-2−/−-WT chimeric animals. We report here an essential role of Shp-2 in the control of blood cell development. Despite the widespread contribution of mutant cells to various tissues, no Shp-2−/− progenitors for erythroid or myeloid cells were detected in the fetal liver and bone marrow of chimeric animals by using the in vitro CFU assay. Furthermore, hematopoiesis was defective in Shp-2−/− yolk sacs. In addition, the Shp-2 mutation caused multiple developmental defects in chimeric mice, characterized by short hind legs, aberrant limb features, split lumbar vertebrae, abnormal rib patterning, and pathological changes in the lungs, intestines, and skin. These results demonstrate a functional involvement of Shp-2 in the differentiation of multiple tissue-specific cells and in body organization. More importantly, the requirement for Shp-2 is more stringent in hematopoiesis than in other systems.

Molecular Characterization of Specific Interactions between SHP-2 Phosphatase and JAK Tyrosine Kinases

Interactions between SHP-2 phosphotyrosine phosphatase and JAK tyrosine kinases have recently been implicated in cytokine signal transduction. However, the molecular basis of these interactions is not well understood. In this study, we demonstrate that SHP-2 is tyrosine-phosphorylated by and associated with JAK1 and JAK2 but not JAK3 in COS-1 cell cotransfection experiments. SHP-2 phosphatase activity appears not to be required for JAK and SHP-2 interactions because SHP-2 with a mutation at amino acid 463 from Cys to Ser, which renders SHP-2 inactive, can still bind JAKs. We further demonstrate that SHP-2 SH2 domains (amino acids 1-209) are not essential for the association of JAKs with SHP-2, and the region between amino acids 232 and 272 in SHP-2 is important for the interactions. Furthermore, tyrosine residues 304 and 327 in SHP-2 are phosphorylated by JAKs, and phosphorylated SHP-2 can associate with the downstream adapter protein Grb2. Finally, deletion of the N terminus but not the kinase-like domain of JAK2 abolishes the association of JAK2 with SHP-2. Taken together, these studies identified novel sequences for SHP-2 and JAK interactions that suggest unique signaling mechanisms mediated by these two molecules.

GRAP IS A NOVEL SH3-SH2-SH3 ADAPTOR PROTEIN THAT COUPLES TYROSINE KINASES TO THE RAS PATHWAY

A human cytoplasmic signaling protein has been cloned that possesses the same structural arrangement of SH3-SH2-SH3 domains as Grb2. This protein is designated Grap for Grb2-related adaptor protein. The single 2.3-kilobase (kb) grap transcript was expressed predominantly in thymus and spleen, while the ubiquitously expressed grb2 gene produced two mRNA species of 3.8 and 1.5 kb. Grap and Grb2 consist of 217 amino acids and share 59% amino acid sequence identity, with highest homology in the N-terminal SH3 domain. The GrapSH2 domain interacts with ligand-activated receptors for stem cell factor (c-kit) and erythropoietin (EpoR). Grap also forms a stable complex with the Bcr-Abl oncoprotein via its SH2 domain in K562 cells. Furthermore, Grap is associated with a Ras guanine nucleotide exchange factor mSos1, primarily through its N-terminal SH3 domain. These results show that a family of Grb2-like proteins exist and couple signals from receptor and cytoplasmic tyrosine kinases to the Ras signaling pathway.

A coiled-coil tetramerization domain of BCR-ABL is essential for the interactions of SH2-containing signal transduction molecules.

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemia. We have previously shown SH2-containing phosphotyrosine phosphatase SHP-2 forms stable complexes with BCR-ABL and Grb2 in BCR-ABL-transformed cells (Tauchi, T., Feng, G. S., Shen, R., Song, H. Y., Donner, D., Pawson, T., and Broxmeyer, H. E. (1994) J. Biol. Chem. 269, 15381-15387). To elucidate the structural requirement of BCR-ABL for the interactions with SH2-containing signaling molecules, we examined a series of BCR-ABL mutants which include the Grb2 binding site-deleted BCR-ABL (1-63 BCR/ABL), the tetramerization domain-deleted BCR-ABL (64-509 BCR/ABL), and the SH2 domain-deleted BCR-ABL (BCR/ABL ΔSH2). These BCR-ABL mutants were previously shown to reduce the transforming activity in fibroblasts. We found that the tetramerization domain-deleted BCR-ABL did not induce the tyrosine phosphorylation of SHP-2 and the interactions of BCR-ABL, SHP-2, and Grb2. In vitro kinase assays have also shown that the tetramerization domain-deleted BCR-ABL mutant did not phosphorylate GST-SHP-2 in vitro. SHP-2 was co-immunoprecipitated with phosphatidylinositol 3-kinase in BCR/ABL p210-transformed cells; however, this interaction was not observed in the tetramerization domain-deleted BCR-ABL mutant. Therefore the tetramerization domain of BCR-ABL is essential for interactions of these downstream molecules.