Paxillin Is Tyrosine-phosphorylated by and Preferentially Associates with the Calcium-dependent Tyrosine Kinase in Rat Liver Epithelial Cells*

Abstract

We and others have recently cloned a non-receptor, calcium-dependent tyrosine kinase (CADTK; also known as PYK2, CAKβ, and RAFTK) that shares both overall domain structure and 45% amino acid identity with p125^FAK. We have studied the signaling, activation, and potential function of these related enzymes in GN4 rat liver epithelial cells that express CADTK and p125^FAK at roughly similar levels. p125^FAK is nearly fully tyrosine-phosphorylated in resting GN4 cells. In contrast, while CADTK is not tyrosine-autophosphorylated in untreated cells, angiotensin II increases CADTK Tyr(P) by 5–10-fold. With regard to signaling, CADTK activation is correlated with stimulation of c-Jun N-terminal kinase and p70^S6Kpathways but not with the stimulation of mitogen-activated protein kinase or p90^RSK. In this report we assessed the contribution of CADTK and p125^FAK to tyrosine phosphorylation of focal contact proteins. In adherent GN4 cells, the constitutive activity of p125^FAK was correlated with basal paxillin, tensin, and p130^CAS tyrosine phosphorylation. A rapid increase in the tyrosine phosphorylation of each protein was detected after treatment with angiotensin II or other agonists that stimulate CADTK; the prolonged 3–4-fold increase in paxillin tyrosine phosphorylation was the most substantial change. In the WB cell line that expresses 3-fold less CADTK than GN4 cell line agonist-dependent paxillin tyrosine phosphorylation is similarly reduced. Immunoprecipitation of CADTK from GN4 cells revealed CADTK·paxillin complexes that persisted in 500 mm NaCl but not in 0.1% SDS cell lysis buffer. The complexes were largely independent of the tyrosine phosphorylation state of either protein. Surprisingly, we did not detect p125^FAK·paxillin complexes in immunoprecipitates using either of two p125^FAKantibodies. When CADTK and p125^FAK were transiently overexpressed in 293(T) cells, both enzymes associated with paxillin, but the avidity of CADTK appeared to be greater. In addition, in transfected 293(T) cells, complexes between CADTK and another potential substrate, p130^CAS, were detected. In summary, in GN4 rat liver epithelial cells stimulation of CADTK was highly correlated with paxillin tyrosine phosphorylation; in addition, CADTK but not p125^FAK was complexed to paxillin at detectable levels. This suggests that agonist-dependent cytoskeletal changes in epithelial cells might proceed, in part, by CADTK-dependent mechanisms.