Phosphorylation of Grb2-associated binder 2 on serine 623 by ERK MAPK regulates its association with the phosphatase SHP-2 and decreases STAT5 activation.
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.
Arnaud
Mary
M
Institut National de la Santé et de la Recherche Médicale Unité 461, Faculté de Pharmacie Paris-XI, Chatenay-Malabry, France.
Crouin
Catherine
C
Deon
Catherine
C
Loyaux
Denis
D
Bertoglio
Jacques
J
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002455:Cell Division / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015536:Down-Regulation / Q000276:immunology; D006131:Growth Inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007376:Interleukin-2 / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System / Q000276:immunology; D008894:Milk Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D050799:STAT5 Transcription Factor; D012694:Serine / Q000378:metabolism*; D016176:T-Lymphocyte Subsets / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C490895:GAB2 protein, human; D006131:Growth Inhibitors; D007376:Interleukin-2; D047908:Intracellular Signaling Peptides and Proteins; D008894:Milk Proteins; D010750:Phosphoproteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D012694:Serine; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases