Masahiro Tsuda

A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion.

Protein tyrosine phosphatases (PTPases), such as SHP-1 and SHP-2, that contain Src homology 2 (SH2) domains play important roles in growth factor and cytokine signal transduction pathways. A protein of approximately 115 to 120 kDa that interacts with SHP-1 and SHP-2 was purified from v-src-transformed rat fibroblasts (SR-3Y1 cells), and the corresponding cDNA was cloned. The predicted amino acid sequence of the encoded protein, termed SHPS-1 (SHP substrate 1), suggests that it is a glycosylated receptor-like protein with three immunoglobulin-like domains in its extracellular region and four YXX(L/V/I) motifs, potential tyrosine phosphorylation and SH2-domain binding sites, in its cytoplasmic region. Various mitogens, including serum, insulin, and lysophosphatidic acid, or cell adhesion induced tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2 in cultured cells. Thus, SHPS-1 may be a direct substrate for both tyrosine kinases, such as the insulin receptor kinase or Src, and a specific docking protein for SH2-domain-containing PTPases. In addition, we suggest that SHPS-1 may be a potential substrate for SHP-2 and may function in both growth factor- and cell adhesion-induced cell signaling.

PI 3-KINASE GAMMA AND PROTEIN KINASE C-ZETA MEDIATE RAS-INDEPENDENT ACTIVATION OF MAP KINASE BY A GI PROTEIN-COUPLED RECEPTOR

Receptors coupled to the inhibitory G protein Gi, such as that for lysophosphatidic acid (LPA), have been shown to activate MAP kinase through a RAS‐dependent pathway. However, LPA (but not insulin) has now been shown to activate MAP kinase in a RAS‐independent manner in CHO cells that overexpress a dominant‐negative mutant of the guanine nucleotide exchange protein SOS (CHO‐ΔSOS cells). LPA also induced the activation of MAP kinase kinase (MEK), but not that of RAF1, in CHO‐ΔSOS cells. The RAS‐independent activation of MAP kinase by LPA was blocked by inhibitors of phosphatidylinositol 3‐kinase (PI3K) or by overexpression of a dominant‐negative mutant of the γ isoform of PI3K. Furthermore, LPA induced the activation of the atypical ζ isoform of protein kinase C (PKC‐ζ) in CHO‐ΔSOS cells in a manner that was sensitive to wortmannin or to the dominant‐negative mutant of PI3Kγ, and overexpression of a dominant‐negative mutant of PKC‐ζ inhibited LPA‐induced activation of MAP kinase. These observations indicate that Gi protein‐coupled receptors induce activation of MEK and MAP kinase through a RAS‐independent pathway that involves PI3Kγ‐dependent activation of atypical PKC‐ζ.

Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Roles of Fak and Src family kinases

SHPS-1 is a receptor-like glycoprotein that undergoes tyrosine phosphorylation and binds SHP-2, an Src homology 2 domain containing protein tyrosine phosphatase, in response to various mitogens. Cell adhesion to extracellular matrix proteins such as fibronectin and laminin also induced the tyrosine phosphorylation of SHPS-1 and its association with SHP-2. These responses were markedly reduced in cells overexpressing the Csk kinase or in cells that lack focal adhesion kinase or the Src family kinases Src or Fyn. However, unlike Src, focal adhesion kinase did not catalyze phosphorylation of the cytoplasmic domain of SHPS-1 in vitro. Overexpression of a catalytically inactive SHP-2 markedly inhibited activation of mitogen-activated protein (MAP) kinase in response to fibronectin stimulation without affecting the extent of tyrosine phosphorylation of focal adhesion kinase or its interaction with the docking protein Grb2. Overexpression of wild-type SHPS-1 did not enhance fibronectin-induced activation of MAP kinase. These results indicate that the binding of integrins to the extracellular matrix induces tyrosine phosphorylation of SHPS-1 and its association with SHP-2, and that such phosphorylation of SHPS-1 requires both focal adhesion kinase and an Src family kinase. In addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, SHP-2 may play an important role, partly through its interaction with SHPS-1, in the activation of MAP kinase in response to the engagement of integrins by the extracellular matrix.

Tyrosine phosphorylation of p62 Dok induced by cell adhesion and insulin: possible role in cell migration

Dok, a 62‐kDa Ras GTPase‐activating protein (rasGAP)‐associated phosphotyrosyl protein, is thought to act as a multiple docking protein downstream of receptor or non‐receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induced marked tyrosine phosphorylation of Dok. This adhesion‐dependent phosphorylation of Dok was mediated, at least in part, by Src family tyrosine kinases. The maximal insulin‐induced tyrosine phosphorylation of Dok required a Src family kinase. A mutant Dok (DokΔPH) that lacked its pleckstrin homology domain failed to undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild‐type protein, DokΔPH did not localize to subcellular membrane components. Insulin promoted the association of tyrosine‐phosphorylated Dok with the adapter protein NCK and rasGAP. In contrast, a mutant Dok (DokY361F), in which Tyr361 was replaced by phenylalanine, failed to bind NCK but partially retained the ability to bind rasGAP in response to insulin. Overexpression of wild‐type Dok, but not that of DokΔPH or DokY361F, enhanced the cell migratory response to insulin without affecting insulin activation of mitogen‐activated protein kinase. These results identify Dok as a signal transducer that potentially links, through its interaction with NCK or rasGAP, cell adhesion and insulin receptors to the machinery that controls cell motility.

Roles of the Complex Formation of SHPS-1 with SHP-2 in Insulin-stimulated Mitogen-activated Protein Kinase Activation

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathioneS-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.

Roles for the protein tyrosine phosphatase SHP-2 in cytoskeletal organization, cell adhesion and cell migration revealed by overexpression of a dominant negative mutant.

SHP-2, a SRC homology 2 domain-containing protein tyrosine phosphatase, mediates activation of Ras and mitogen-activated protein kinase by various mitogens and cell adhesion. Inhibition of endogenous SHP-2 by overexpression of a catalytically inactive (dominant negative) mutant in Chinese hamster ovary cells or Rat-1 fibroblasts has now been shown to induce a marked change in cell morphology (from elongated to less polarized) that is accompanied by substantial increases in the numbers of actin stress fibers and focal adhesion contacts. Overexpression of the SHP-2 mutant also increased the strength of cell-substratum adhesion and resulted in hyperphosphorylation of SHPS-1, a substrate of SHP-2 that contributes to cell adhesion-induced signaling. Inhibition of SHP-2 also markedly increased the rate of cell attachment to and cell spreading on extracellular matrix proteins such as fibronectin and vitronectin, effects that were accompanied by enhancement of adhesion-induced tyrosine phosphorylation of paxillin and p130Cas. In addition, cell migration mediated by fibronectin or vitronectin, but not that induced by insulin, was impaired by overexpression of the SHP-2 mutant. These results suggest that SHP-2 plays an important role in the control of cell shape by contributing to cytoskeletal organization, and that it is an important regulator of integrin-mediated cell adhesion, spreading, and migration as well as of tyrosine phosphorylation of focal adhesion contact-associated proteins.

Characterization of a 115-kDa Protein That Binds to SH-PTP2, a Protein-tyrosine Phosphatase with Src Homology 2 Domains, in Chinese Hamster Ovary Cells

SH-PTP2, a non-transmembrane-type protein-tyrosine phosphatase with two Src homology 2 domains, was previously shown to play a positive signaling role in the insulin-induced activation of Ras and mitogen-activated protein kinase. SH-PTP2 was shown to associate with a 115-kDa tyrosine-phosphorylated protein (pp115), as well as with insulin receptor substrate 1, in insulin-stimulated Chinese hamster ovary cells that overexpress human insulin receptors (CHO-IR cells). In vivo and in vitro binding experiments revealed that SH-PTP2 bound to pp115 through one or both of its SH2 domains. The pp115 protein was partially purified from insulin-stimulated CHO-IR cells that overexpress a catalytically inactive SH-PTP2 by a combination of immunoaffinity and lectin-affinity chromatography. A monoclonal antibody to pp115 was then generated by injecting the partially purified protein into mice. Experiments with this monoclonal antibody revealed that pp115 is a transmembrane protein with a domain exposed on the cell surface and that it binds to SH-PTP2 in response to insulin. The insulin receptor kinase appeared to phosphorylate pp115 on tyrosine residues both in vivo and in vitro. The extent of tyrosine phosphorylation of pp115 associated with SH-PTP2 was greatly increased in CHO-IR cells that overexpress catalytically inactive SH-PTP2 compared with that observed in CHO-IR cells overexpressing wild-type SH-PTP2. Furthermore, recombinant SH-PTP2 preferentially dephosphorylated pp115 in vitro, indicating that SH-PTP2 may catalyze the dephosphorylation of phosphotyrosine residues in pp115 after it binds to this protein. These results suggest that pp115 may act as a transmembrane anchor to which SH-PTP2 binds in response to insulin. Furthermore, pp115 may be a physiological substrate for both the insulin receptor kinase and SH-PTP2.

Lysophosphatidic acid-induced association of SHP-2 with SHPS-1: Roles of RHO, FAK, and a SRC family kinase

SHPS-1 is an ∼120u2009kDa glycosylated receptor like protein that contains three immunoglobulin-like domains in its extracellular region as well as four potential tyrosine phosphorylation and SRC homology 2 (SH2) domain binding sites in its cytoplasmic region. Lysophosphatidic acid (LPA) stimulated the rapid tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2, a protein tyrosine phosphatase containing SH2 domains in Rat-1 fibroblasts. LAP-induced tyrosine phosphorylation of SHPS-1 was inhibited by Clostridium botulinum C3 exoenzyme (which inactivates RHO) but not by pertussis toxin. The protein kinase C activator phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) also stimulated tyrosine phosphorylation of SHPS-1; however, down-regulation of protein kinase C by prolonged exposure of cells to TPA did not affect LAP-induced tyrosine phosphorylation of SHPS-1. LPA-induced tyrosine phosphorylation of SHPS-1 was markedly reduced in either focal adhesion kinase (FAK)-deficient mouse cells or CHO cells overexpressing the tyrosine kinase CSK. Overexpression of a catalytically inactivate SHP-2 markedly inhibited MAP kinase activation in response to low concentrations of LPA in CHO cells, whereas overexpression of a wild-type SHPS-1 did enhance this effect of LPA. Furthermore, MAP kinase activation in response to a low concentration of LPA was inhibited by botulinum C3 exoenzyme. These results indicate that LPA-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2 may be mediated by a RHO-dependent pathway that includes FAK and a SRC family kinase. Thus, in addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, the formation of a complex between SHPS-1 and SHP-2 may, in part, play an important role in the activation of MAP kinase in response to low concentrations of LPA.