Phosphorylation of Grb10 by mitogen-activated protein kinase: identification of Ser150 and Ser476 of human Grb10zeta as major phosphorylation sites.
Grb10 is a Src-homology 2 (SH2) and Pleckstrin-homology (PH) domain-containing protein that binds to several autophosphorylated receptor tyrosine kinases including the insulin receptor (IR). Our previous studies showed that Grb10 underwent insulin-stimulated serine phosphorylation, yet the kinase(s) responsible for phosphorylation and the sites of the phosphorylation remain unknown. In this report, we show that Grb10 is a direct substrate of the p42/44 mitogen-activated protein kinase (MAPK). In addition, we found that inhibition of the MAPK signaling pathway reduced Grb10 phosphorylation in cells. Using site-directed mutagenesis, phosphopeptide mapping, and capillary HPLC-electrospray-tandem mass spectrometry analysis, we identified Ser(150), Ser(418), and Ser(476) of human Grb10zeta as MAPK-mediated in vitro phosphorylation sites. In vivo labeling and two-dimensional phosphopeptide mapping studies revealed that Ser(150) and Ser(476) of human Grb10zeta are phosphorylated in intact cells. Replacing Ser(150) and Ser(476) with alanines reduced the inhibitory effect of human Grb10zeta on insulin-stimulated IRS1 tyrosine phosphorylation. Taken together, our findings suggest that phosphorylation of the adaptor protein may provide a feedback inhibitory mechanism by which Grb10 regulates insulin signaling.
Langlais
Paul
P
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Wang
Changhua
C
Dong
Lily Q
LQ
Carroll
Christopher A
CA
Weintraub
Susan T
ST
Liu
Feng
F
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051383:GRB10 Adaptor Protein; D006801:Humans; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D021241:Spectrometry, Mass, Electrospray Ionization; D014162:Transfection
D017931:DNA Primers; C096358:GRB10 protein, human; D011506:Proteins; D011994:Recombinant Proteins; D051383:GRB10 Adaptor Protein; D010768:Phosphoserine; D012694:Serine; D011972:Receptor, Insulin; D048049:Extracellular Signal-Regulated MAP Kinases