Thrombin stimulates association of src homology domain containing adaptor protein Nek with pp125FAK

Goutam Ghosh Choudhury, Fabio Marra, Hanna E. Abboud

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Abstract

Thrombin stimulates mitogenesis and tyrosine phosphorylation of several proteins in glomerular mesangial cells [T. Force, J. M. Kyriakis, J. Avruch, and J. V. Bonventre, J. Biol. Chem. 266: 6650-6656, 1991; and G. Grandaliano, G. Ghosh Choudhury, P. Biswas, and H. E. Abboud, Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36: F528-F536, 1994]. However, none of the tyrosine phosphorylated proteins have been identified. Here we show that thrombin stimulates phosphorylation of four major proteins of molecular masses 170, 125, 97, and 47 kDa in antiphosphotyrosine immunoprecipitates in vitro. Immunoblot analysis of antiphosphotyrosine immunoprecipitates from lysates of thrombin-treated cells with anti-Nek antibody revealed the presence of this src homology domain-containing adaptor molecule in the tyrosine-phosphorylated protein fraction. In addition, in thrombin-treated cells, direct immunoblotting of Nek immunoprecipitates with antiphosphotyrosine antibody showed no tyrosine phosphorylation of Nek. In these immunoprecipitates, we detected a 125-kDa tyrosine-phosphorylated protein. We identified this protein as pp125FAK (FAK, focal adhesion kinase) after analyzing Nek immunoprecipitates by anti-FAK immunoblotting. Treatment of mesangial cells with thrombin resulted in stimulation of the tyrosine kinase activity of pp125FAK in vitro. We conclude that activation of the cytoplasmic protein tyrosine kinase pp125FAK by thrombin stimulates its association with the src homology domaincontaining adaptor protein Nek. This indicates that Nek is a direct target for FAK in the thrombin-induced signal transduetion pathway. growth factors; signal transduction; mesangial cell; focal adhesion kinase

Original languageEnglish (US)
Pages (from-to)F295-F300
JournalAmerican Journal of Physiology
Volume270
Issue number2 PART 2
Publication statusPublished - Dec 1 1996

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ASJC Scopus subject areas

  • Physiology (medical)

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