AJP - Renal Physiology, Vol 270, Issue 2 295-F300, Copyright © 1996 by American Physiological Society

ARTICLES

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

G. G. Choudhury, F. Marra and H. E. Abboud
Department of Medicine, University of Texas Health Science Center at San Antonio 78284-7882, USA.

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-Nck 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 Nck immunoprecipitates with antiphosphotyrosine antibody showed no tyrosine phosphorylation of Nck. In these immunoprecipitates, we detected a 125-kDa tyrosine-phosphorylated protein. We identified this protein as pp125FAK (FAK, focal adhesion kinase) after analyzing Nck 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 domain-containing adaptor protein Nck. This indicates that Nck is a direct target for FAK in the thrombin-induced signal transduction pathway.

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