Phosphorylation of Grb10 by mitogen-activated protein kinase: Identification of Ser150 and Ser476 of human Grb10ζ as major phosphorylation sites

Paul Langlais, Changhua Wang, Lily Q. Dong, Christopher A. Carroll, Susan T. Weintraub, Feng Liu

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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, Ser418, and Ser476 of human Grb10ζ as MAPK-mediated in vitro phosphorylation sites. In vivo labeling and two-dimensional phosphopeptide mapping studies revealed that Ser150 and Ser476 of human Grb10ζ are phosphorylated in intact cells. Replacing Ser150 and Ser476 with alanines reduced the inhibitory effect of human Grb10ζ 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.

Original languageEnglish (US)
Pages (from-to)8890-8897
Number of pages8
JournalBiochemistry
Volume44
Issue number24
DOIs
StatePublished - Jun 21 2005
Externally publishedYes

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Phosphorylation
Mitogen-Activated Protein Kinases
Phosphopeptides
Insulin
Mutagenesis
Mitogen-Activated Protein Kinase 1
Insulin Receptor
Receptor Protein-Tyrosine Kinases
Tandem Mass Spectrometry
Site-Directed Mutagenesis
Alanine
Labeling
Serine
Mass spectrometry
Tyrosine
Proteins
Phosphotransferases
High Pressure Liquid Chromatography
Feedback
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation of Grb10 by mitogen-activated protein kinase : Identification of Ser150 and Ser476 of human Grb10ζ as major phosphorylation sites. / Langlais, Paul; Wang, Changhua; Dong, Lily Q.; Carroll, Christopher A.; Weintraub, Susan T.; Liu, Feng.

In: Biochemistry, Vol. 44, No. 24, 21.06.2005, p. 8890-8897.

Research output: Contribution to journalArticle

Langlais, Paul ; Wang, Changhua ; Dong, Lily Q. ; Carroll, Christopher A. ; Weintraub, Susan T. ; Liu, Feng. / Phosphorylation of Grb10 by mitogen-activated protein kinase : Identification of Ser150 and Ser476 of human Grb10ζ as major phosphorylation sites. In: Biochemistry. 2005 ; Vol. 44, No. 24. pp. 8890-8897.
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