The novel role of the C-terminal region of SHP-2. Involvement of Gab1 and SHP-2 phosphatase activity in Elk-1 activation.
ABSTRACT SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, we generated six deletion mutants of SHP-2. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198-593 (SHP-2-(198-593)), but not the smaller SHP-2-(399-593), showed significantly higher SHP-2 phosphatase activity in vitro. In contrast, SHP-2-(198-593) mutant inhibited wild type SHP-2 phosphatase activity, whereas SHP-2-(399-593) mutant increased activity. To understand these functional changes, we focused on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggested that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associated with Gab1, but the SHP-2-(198-593) mutant did not associate with Gab1. SHP-2-(1-202) or SHP-2-(198-593) inhibited platelet-derived growth factorinduced Elk-1 activation, but SHP-2-(399-593) increased Elk-1 activation. Co-expression of SHP-2-(1-202) with SHP-2-(399-593) inhibited SHP-2-(399-593)/Gab1 interaction, and the SHP-2-(399-593) mutant induced SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, we identified a novel sequence for SHP-2/Gab1 interactions in the C-terminal region of SHP-2.
- SourceAvailable from: Masanori Yoshizumi[Show abstract] [Hide abstract]
ABSTRACT: Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-kappaB transcriptional activity. Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-alpha-induced c-Jun and NF-kappaB transcriptional activation. TNF-alpha-mediated JNK, c-Jun, and NF-kappaB activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-alpha-induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-alpha-induced c-Jun and NF-kappaB activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-kappaB activation. These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-alpha signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.Arteriosclerosis Thrombosis and Vascular Biology 11/2003; 23(10):1775-81. DOI:10.1161/01.ATV.0000094432.98445.36 · 5.53 Impact Factor
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ABSTRACT: Endothelial cell (EC) migration contributes to reendothelialization after angioplasty or rupture of atherosclerotic plaques. Extracellular signal-regulated kinase (ERK)1/2 translocates to the nucleus and activates transcription factors such as Ets-like transcription factor-1 and early growth response factor-1 (Egr-1) during reendothelialization. Because ERK1/2 does not possess a nuclear localization signal (NLS), its mechanism of translocation and accumulation in the nucleus remains unclear. Because Gab1 has a putative NLS in its N-terminal region, and Gab1 associates with phosphorylated ERK1/2, we hypothesized that Gab1 participates in ERK1/2 and Egr-1 nuclear accumulation. Using regenerating EC as a model system, we found that endogenous growth factor receptor-bound protein 2-associated binder-1 (Gab1) translocates into the nucleus in migrating EC. Wild-type red fluorescent protein-tagged Gab1 could be observed in both nucleus and cytoplasm, whereas the putative NLS deletion mutant (deltaNLS-Gab1) specifically localized in the cytoplasm. In addition, reduction of Gab1 expression by antisense Gab1 oligos or overexpression of deltaNLS-Gab1 inhibited serum-induced ERK1/2 and Egr-1 nuclear accumulation, suggesting a functional role for the NLS of Gab1 and a role for Gab1-ERK1/2 interactions in ERK1/2-Egr-1 nuclear accumulation. To investigate whether Gab1-ERK1/2 interaction is critical for ERK1/2 and Egr-1 nuclear accumulation, we created a dominant-negative Gab1 construct that consisted of the c-Met binding domain (amino acids 442-536) of Gab1. We found that overexpression of the c-Met binding domain of Gab1 disrupted serum-induced Gab1-ERK1 interaction and inhibited ERK1 and Egr-1 nuclear accumulation. These data suggest that Gab1-ERK1/2 binding and their nuclear translocation play a crucial role in Egr-1 nuclear accumulation.Journal of Biological Chemistry 08/2004; 279(28):29691-9. DOI:10.1074/jbc.M309371200 · 4.60 Impact Factor
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ABSTRACT: Insulin-like growth factor-I (IGF-I) plays a role in mutually exclusive processes such as proliferation and differentiation in a variety of cell types. IGF-I is a potent mitogen and motogen for dedifferentiated vascular smooth muscle cells (VSMCs) in vivo and in vitro. However, in differentiated VSMCs, IGF-I is only required for maintaining the differentiated phenotype. Here we investigated the VSMC phenotype-dependent signaling and biological processes triggered by IGF-I. In differentiated VSMCs, IGF-I activated a protein-tyrosine phosphatase, SHP-2, recruited by insulin receptor substrate-1 (IRS-1). The activated SHP-2 then dephosphorylated IRS-1 Tyr(P)-895, resulting in blockade of the pathways from IRS-1/Grb2/Sos to the ERK and p38 MAPK. Conversely, such negative regulation was silent in dedifferentiated VSMCs, where IGF-I activated both MAPKs via IRS-1/Grb2/Sos interaction-linked Ras activation, leading to proliferation and migration. Thus, our present results demonstrate that the IRS-1/SHP-2 interaction acts as a switch controlling VSMC phenotype-dependent IGF-I-induced signaling pathways and biological processes, and this mechanism is likely to be applicable to other cells.Journal of Biological Chemistry 10/2004; 279(39):40807-18. DOI:10.1074/jbc.M405100200 · 4.60 Impact Factor