Thrombin stimulates association of the src homology domain
containing adaptor protein nck with pp125fak (focal adhesion
kinase).
Choudhury, Goutam Ghosh, Fabio Marra, and Hanna E. Abboud.
Department of Medicine, University of Texas Health Science Center
at San Antonio, 7703 Floyd Curl Drive, San Antonio TX 78284-7882,
USA
APStracts 2:0154F, 1995.
Thrombin stimulates mitogenesis and tyrosine phosphorylation of
several proteins in glomerular mesangial cells (Force et. al. (1991)
J. Biol. Chem. 266, 6650-6656; Grandaliano et. al. (1994) Am. J.
Physiol. 267 (Renal Fluid and Electrolyte Physiol. 36, F528-F536).
However, none of the tyrosine phosphorylated proteins have been
identified. Here we show that thrombin stimulates phosphorylation of
four major proteins of molecular weights 170, 125, 97 and 47 kDs 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 kD tyrosine
-phosphorylated protein. We identified this protein as pp125FAK (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 focal adhesion kinase in the thrombin
-induced signal transduction pathway.
Received 3 May 1995; accepted in final form 16 August 1995.
APS Manuscript Number F146-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 September 1995.