Ras Activation In Response Research Articles

Functional involvement of mSos in interleukin-3 and thrombin stimulation of the Ras, mitogen-activated protein kinase pathway in BaF3 murine hematopoietic cells.

Stimulation of the interleukin (IL)-3 receptor provokes rapid activation of the Ras pathway in various hematopoietic cell lines. Also, a wide range of G-protein-coupled receptors induce Ras activation following ligand stimulation. In this report, we investigate the mechanism underlying Ras activation upon stimulation of these two types of receptors in hematopoietic cells. Thrombin, a G-protein-coupled receptor ligand, was found to stimulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) in IL-3-dependent BaF3 cells, suggesting a significant function of thrombin receptor-mediated signaling. We show that the Ras-guanine nucleotide exchange factor mSos is indispensable for activation of the Ras pathway in IL-3- or thrombin-stimulated BaF3 cells. The activation of Ras in response to IL-3 as defined by accumulation of the GTP-bound form was impaired by conditional overexpression of a dominant-negative mutant of mSos (DeltamSos1). Furthermore, following induction of DeltamSos1, IL-3 enhancement of the kinase activities of c-Raf-1, ERK2, and JNK1 downstream of Ras was almost completely blocked. Similarly, thrombin-induced Ras-dependent ERK2 activation was diminished by DeltamSos1. However, the tyrosine phosphorylation pattern of cellular substrates upon thrombin stimulation was entirely different from the pattern of IL-3-induced tyrosine phosphorylation. Collectively, these results provide evidence that mSos plays a crucial role in both IL-3 and thrombin activation of the Ras pathway in hematopoietic cells, although molecules (including tyrosine kinases) mediating the signal to mSos are likely to be different between the two types of receptors.

Functional Roles of the Shc Phosphotyrosine Binding and Src Homology 2 Domains in Insulin and Epidermal Growth Factor Signaling

Shc is involved in the activation of Ras in response to many growth factors. Shc contains two phosphotyrosine binding domains, an Src homology 2 (SH2) domain in the carboxyl terminus of the protein and a phosphotyrosine binding (PTB) domain in the amino terminus. Since functional roles for these two domains have not been established, we microinjected glutathione S-transferase fusion proteins of either the Shc PTB or SH2 domains into fibroblasts expressing insulin and epidermal growth factor receptors and measured their effects on DNA synthesis. We found that the Shc PTB was necessary for insulin-induced mitogenic signaling, whereas the SH2 domain was not. In contrast, for epidermal growth factor signaling, the Shc SH2 was functionally more important. These differential modes of signal transduction may be an important factor in determining the specificity of the response of a cell to external stimuli.

Analysis of the T-cell activation signaling pathway mediated by tyrosine kinases, protein kinase C, and Ras protein, which is modulated by intracellular cyclic AMP.

T-cell receptor (TCR) triggering by an anti-CD3 antibody or phytohemagglutinin (PHA) as well as the treatment with phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), induces activation of Ras in T-lymphocytes (Downward, J. et al. (1990)) Nature 364, 719-723). In this paper, we studied the role of Ras in the process of TCR-mediated T-cell activation using a human lymphomic Jurkat cell line. The stimulatory effect of TCR cross-linking on Ras activation was inhibited by herbimycin A, a specific inhibitor of protein tyrosine kinases (PTKs), whereas PMA-induced Ras activation was not affected. On the other hand, calphostin C, a specific inhibitor of PKC, blocked not only PMA-induced, but also TCR-mediated formation of Ras.GTP. Furthermore, down-regulation of PMA-sensitive PKC severely impaired the activation of Ras in response to TCR-stimulation. Tyrosine-phosphorylation and translocation to the particulate fraction of phospholipase C-gamma 1 (PLC-gamma 1) were observed upon T-cell activation. Subcellular localization of PKC was also changed when the cells were stimulated with an anti-CD3 antibody or PMA. While TCR-stimulated translocation of PKC was observed only transiently, PMA-induced translocation of PKC was more sustained. These results suggest that the activation of PLC-gamma 1 by PTK and subsequent activation of PKC are important for TCR-mediated Ras activation in Jurkat cells. An activated form of Ras enhanced the activation of interleukin 2 (IL-2) promoter by TCR stimulation or PMA treatment, although the activated Ras by itself was insufficient for IL-2 promoter activation. On the other hand, a dominant-inhibitory Ras diminished almost completely the activation of IL-2 promoter induced by PMA plus calcium ionophore, indicating that Ras is essential for transduction of T-cell activation signals. Cholera toxin (CTX), which directly activates Gs alpha, is shown to inhibit the activation of IL-2 promoter. TCR-mediated Ras activation, tyrosine phosphorylation and translocation of cellular proteins including ZAP-70, PLC-gamma 1 , and PKC. An activated Gs alpha mutant as well as dibutylyl cAMP (dBcAMP) also showed similar inhibitory effects.

Role of SH-PTP2, a protein-tyrosine phosphatase with Src homology 2 domains, in insulin-stimulated Ras activation.

SH-PTP2 is a nontransmembrane human protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains and binds to insulin receptor substrate 1 (IRS-1) via these domains in response to insulin. 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 attenuated insulin-stimulated Ras activation. Expression of mutant SH-PTP2 also inhibited MAP kinase activation in response to insulin but not in response to 12-O-tetradecanoyl phorbol-13-acetate. In contrast, the insulin-induced association of phosphoinositide 3-kinase activity with IRS-1 was 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 was greater than that observed in CHO-IR cells overexpressing wild-type SH-PTP2. Recombinant SH-PTP2 specifically dephosphorylated 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, inactivated SH-PTP2 in vitro and increased the insulin-induced association of SH-PTP2 with IRS-1. These results suggest 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. Furthermore, these results are 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.

Role of SH-PTP2, a protein-tyrosine phosphatase with Src homology 2 domains, in insulin-stimulated Ras activation.

SH-PTP2 is a nontransmembrane human protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains and binds to insulin receptor substrate 1 (IRS-1) via these domains in response to insulin. 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 attenuated insulin-stimulated Ras activation. Expression of mutant SH-PTP2 also inhibited MAP kinase activation in response to insulin but not in response to 12-O-tetradecanoyl phorbol-13-acetate. In contrast, the insulin-induced association of phosphoinositide 3-kinase activity with IRS-1 was 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 was greater than that observed in CHO-IR cells overexpressing wild-type SH-PTP2. Recombinant SH-PTP2 specifically dephosphorylated 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, inactivated SH-PTP2 in vitro and increased the insulin-induced association of SH-PTP2 with IRS-1. These results suggest 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. Furthermore, these results are 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.