Multiple in vivo phosphorylated tyrosine phosphatase SHP-2 engages binding to Grb2 via tyrosine 584.
SHP-2 (also named PTP1D, syp, or SH-PTP2) has been identified as a phosphotyrosine phosphatase comprising two src-homology-2 (SH2) domains. Upon growth factor stimulation, SHP-2 becomes tyrosine phosphorylated, thereby increasing its catalytic activity. Here, we identified SHP-2 to be phosphorylated on multiple tyrosine residues in response to different stimuli and unmasked the carboxyl-terminal tyrosine 584 as a major phosphorylation site in human cell lines. Tyrosine 584 shares, together with tyrosine 546, the consensus sequence pY-X-N-X, a characteristic of potential binding sites for the SH2 domain of growth factor receptor-bound protein 2 (Grb2). We show here that mutation of tyrosine 584, but not tyrosine 546, to phenylalanine totally abolished the binding of Grb2 to SHP-2. By using a systematic mutagenesis approach, phosphorylation of additional tyrosines in each of the SH2 domains of SHP-2 was detected after coexpression of epidermal growth factor receptor, but not after coexpression of platelet-derived growth factor receptor, whereas tyrosine 263 located in the interspace between SH2 and catalytic domain appears to be exclusively recognized by platelet-derived growth factor receptor. Immunoprecipitation of SHP-2 from a panel of mammary carcinoma cell lines copurifies several tyrosine phosphorylated proteins; the most prominent band has an apparent molecular weight of M(r) 115,000.
Vogel
W
W
Department of Molecular Biology, Max-Planck-Institut für Blochemie, Martinsried, Germany. vogel@mshri.on.ca
Ullrich
A
A
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line / Q000737:chemistry / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology; D016296:Mutagenesis / Q000502:physiology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D019000:Phosphotyrosine; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D066246:ErbB Receptors; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases