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Identification of Grb10 as a direct substrate for members of the Src tyrosine kinase family

Oncogene volume 19, pages 2895–2903 (2000)Cite this article

Abstract

Treatment of cells with insulin and protein tyrosine phosphatase inhibitors such as vanadate and pervanadate resulted in the tyrosine phosphorylation of Grb10, a Src homology 2 (SH2) and pleckstrin homology domain-containing adaptor protein which binds to a number of receptor tyrosine kinases including the insulin receptor (IR). Although Grb10 binds directly to the kinase domain of the IR, our data show that Grb10 is not a direct substrate for the IR tyrosine kinase. Consistent with this finding, Grb10 tyrosine phosphorylation in cells was inhibited by herbimycin A, a relatively specific inhibitor for members of the Src tyrosine kinase family, and by the expression of dominant negative Src or Fyn. In addition, Grb10 tyrosine phosphorylation was stimulated by expression of constitutively active Src or Fyn in cells and by incubation with purified Src or Fyn in vitro. The insulin stimulated or Src/Fyn-mediated tyrosine phosphorylation in vivo was significantly reduced when Grb10 tyrosine 67 was changed to glycine. This mutant form of Grb10 bound with higher affinity to the IR in cells than that of the wild-type protein, suggesting that tyrosine phosphorylation of Grb10 may normally negatively regulate its binding to the IR. Our data show that Grb10 is a new substrate for members of the Src tyrosine kinase family and that the tyrosine phosphorylation of the protein may play a potential role in cell signaling processes mediated by these kinases.

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Abbreviations

CHO:

Chinese hamster ovary

GST:

glutathione S-transferase

IGF-I:

insulin-like growth factor-I

IR:

insulin receptor

IRS-1:

IR substrate-1

PAGE:

polyacrylamide gel electrophoresis

PH:

plekstrin-homology

PI 3-kinase:

phosphatidylinositol 3-kinase

PTPase:

protein tyrosine phosphatase

SH2:

Src homology 2

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Acknowledgements

We thank Dr SA Courtneidge for mammalian expression vectors encoding constitutively active and dominant negative Src and Fyn. This research was supported by National Institute of Health Grant DK52933 (to F Liu and LQ Dong) and a research grant from the American Heart Association, Texas Affiliate (To LQ Dong).

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  1. Department of Pharmacology and Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, 78229, Texas, TX, USA

    Paul Langlais, Lily Q Dong, Derong Hu & Feng Liu

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  1. Paul Langlais
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Langlais, P., Dong, L., Hu, D. et al. Identification of Grb10 as a direct substrate for members of the Src tyrosine kinase family. Oncogene 19, 2895–2903 (2000). https://doi.org/10.1038/sj.onc.1203616

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