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1.
J Immunol ; 173(6): 3962-71, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15356145

ABSTRACT

IL-2 stimulation of T lymphocytes induces the tyrosine phosphorylation and adaptor function of the insulin receptor substrate/Grb2-associated binder (Gab) family member, Gab2. In addition, Gab2 undergoes a marked decrease in its mobility in SDS-PAGE, characteristic of migration shifts induced by serine/threonine phosphorylations in many proteins. This migration shift was strongly diminished by treating cells with the MEK inhibitor U0126, indicating a possible role for ERK in Gab2 phosphorylation. Indeed, ERK phosphorylated Gab2 on a consensus phosphorylation site at serine 623, a residue located between tyrosine 614 and tyrosine 643 that are responsible for Gab2/Src homology 2 domain-containing tyrosine phosphatase (SHP)-2 interaction. We report that pretreatment of Kit 225 cells with U0126 increased Gab2/SHP-2 association and tyrosine phosphorylation of SHP-2 in response to IL-2, suggesting that ERK phosphorylation of serine 623 regulates the interaction between Gab2 and SHP-2, and consequently the activity of SHP-2. This hypothesis was confirmed by biochemical analysis of cells expressing Gab2 WT, Gab2 serine 623A or Gab2 tyrosine 614F, a mutant that cannot interact with SHP-2 in response to IL-2. Activation of the ERK pathway was indeed blocked by Gab2 tyrosine 614F and slightly increased by Gab2 serine 623A. In contrast, STAT5 activation was strongly enhanced by Gab2 tyrosine 614F, slightly reduced by Gab2 WT and strongly inhibited by Gab2 serine 623A. Analysis of the rate of proliferation of cells expressing these mutants of Gab2 demonstrated that tyrosine 614F mutation enhanced proliferation whereas serine 623A diminished it. These results demonstrate that ERK-mediated phosphorylation of Gab2 serine 623 is involved in fine tuning the proliferative response of T lymphocytes to IL-2.


Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Down-Regulation , MAP Kinase Signaling System , Milk Proteins/antagonists & inhibitors , Milk Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Serine/metabolism , Trans-Activators/antagonists & inhibitors , Trans-Activators/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Cell Division/genetics , Cell Division/immunology , Cell Line, Tumor , Down-Regulation/immunology , Growth Inhibitors/genetics , Growth Inhibitors/metabolism , Growth Inhibitors/physiology , Humans , Interleukin-2/physiology , Intracellular Signaling Peptides and Proteins , MAP Kinase Signaling System/immunology , Mitogen-Activated Protein Kinase Kinases/physiology , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphoproteins/genetics , Phosphoproteins/physiology , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , STAT5 Transcription Factor , T-Lymphocyte Subsets/enzymology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Transfection
2.
Biochem J ; 382(Pt 2): 545-56, 2004 Sep 01.
Article in English | MEDLINE | ID: mdl-15170389

ABSTRACT

Gab2 (Grb2-associated binder-2), a member of the IRS (insulin receptor substrate)/Gab family of adapter proteins, undergoes tyrosine phosphorylation in response to cytokine or growth factor stimulation and serves as a docking platform for many signal transduction effectors, including the tyrosine phosphatase SHP-2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase]. Here, we report that, following IL-2 (interleukin-2) stimulation of human T lymphocytes, SHP-2 binds tyrosine residues 614 and 643 of human Gab2 through its N- and C-terminal SH2 domains respectively. However, the sole mutation of Tyr-614 into phenylalanine is sufficient to prevent Gab2 from recruiting SHP-2. Expression of the Gab2 Tyr-614-->Phe (Y614F) mutant, defective in SHP-2 association, prevents ERK (extracellular-signal-regulated kinase) activation and expression of a luciferase reporter plasmid driven by the c-fos SRE (serum response element), indicating that interaction of SHP-2 with Gab2 is required for ERK activation in response to IL-2. Further investigation of IL-2-dependent induction of SRE showed that expression of a constitutively active mutant of the RhoA GTPase synergizes with IL-2 for SRE-driven transcription, whereas a dominant-negative mutant reduces the IL-2 response. Thus, in response to IL-2, full induction of the SRE requires ERK-dependent as well as Rho-dependent signals that target the Ets-box and the CArG-box respectively. We also report that the synergy between Gab2/SHP-2 and RhoA for IL-2-dependent CArG-box-driven transcription depends upon MEK (mitogen-activated protein kinase/ERK kinase) activation, and is likely to involve regulation of the serum response factor co-activator MAL. Our studies thus provide new insights into the role of Gab2 and SHP-2 in IL-2 signal transduction.


Subject(s)
Acute-Phase Proteins/physiology , Genes, fos/physiology , Interleukin-2/physiology , Phosphoproteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Serum Response Element/physiology , Adaptor Proteins, Signal Transducing , CCAAT-Binding Factor/physiology , Cell Line, Tumor , Gene Expression Regulation/physiology , Gene Expression Regulation, Enzymologic/physiology , Glutathione Transferase , Humans , Intracellular Signaling Peptides and Proteins , Leukemia, Prolymphocytic, T-Cell/pathology , Mitogen-Activated Protein Kinase Kinases/physiology , Mitogen-Activated Protein Kinases/physiology , Peptides/metabolism , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatases/physiology , SH2 Domain-Containing Protein Tyrosine Phosphatases , Signal Transduction/physiology , T-Lymphocytes/enzymology , T-Lymphocytes/physiology , Tyrosine/metabolism , Tyrosine/physiology , src Homology Domains/physiology
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