ANGIOTENSIN-II ACTIVATES AT LEAST 2 TYROSINE KINASES IN RAT-LIVER EPITHELIAL-CELLS - SEPARATION OF THE MAJOR CALCIUM-REGULATED TYROSINE KINASE FROM P125(FAK)

In rat liver epithelial cell lines (WB or GN4), angiotensin II (Ang II ) stimulates cytosolic tyrosine kinase activity, in part, through a ca lcium-dependent mechanism. In other cell types, selected hormones that activate G(i)- or G(q)-coupled receptors stimulate the soluble tyrosi ne kinase, p125(FAK). Immunoprecipitation of p125(FAK) from Ang II-act ivated GN4 cells demonstrated a doubling of p125(FAK) kinase activity. However, an additional Ang II-activated tyrosine kinase (or kinases) representing the majority of the total activity was detected when the remaining cell lysate, immunodepleted of p125(FAK), was reimmunoprecip itated with an anti phosphotyrosine antibody. Cytochalasin D pretreatm ent blocks G-protein receptor-dependent tyrosine phosphorylation in Sw iss 3T3 cells. While cytochalasin D decreased the Tyr(P) content of 65 -75-kDa substrates in Ang II-treated GN4 cells, it did not diminish ty rosine phosphorylation of 115-130-kDa substrates, again suggesting act ivation of at least two tyrosine kinase pathways in GN4 cells. To sear ch for additional Ang II-activated enzymes, we used molecular techniqu es to identify 20 tyrosine kinase sequences in these cell lines. None was the major cytosolic enzyme activated by Ang II. Specifically, JAK2 , which had been shown by others to be stimulated by Ang II in smooth muscle cells, was not activated by Ang II in GN4 cells. Finally, we pu rified Tyr(P)-containing tyrosine kinases from Ang II-treated cells, u sing anti-Tyr(P) and ATP affinity resins; 80% of the tyrosine kinase a ctivity migrated as a single 115-120-kDa tyrosine-phosphorylated prote in immunologically distinct from p125(FAK). In summary, Ang II activat es at least two separate tyrosine kinases in rat liver epithelial cell s; p125(FAK) and a presumably novel, cytosolic 115-120-kDa protein ref erred to as the calcium-dependent tyrosine kinase.