Cytoskeleton-dependent tyrosine phosphorylation of the p130(Cas) family member HEF1 downstream of the G protein-coupled calcitonin receptor - Calcitonin induces the association of HEF1, paxillin, and focal adhesion kinase

HEF1 is a recently described p130(Cas)-like docking protein that contains o ne SH3 domain and multiple SH2 binding motifs, In B cells, HEF1 is phosphor ylated by a cytoskeleton-dependent mechanism that is triggered by integrin ligation. However, the induction of HEF1 phosphorylation by G protein-coupl ed receptors has not been reported. We found that HEF1, but not p130(Cas), is tyrosine-phosphorylated following stimulation of the rabbit C1a calciton in receptor stably expressed in HEK-293 cells. The calcitonin-induced tyros ine phosphorylation of HEF1 increased in a time- and dose-dependent manner. Dibutyryl cAMP and forskolin had little or no effect on HEF1 phosphorylati on, and the protein kinase A inhibitor H89 failed to detectably inhibit the response to calcitonin, indicating that the G(s)/cAMP/protein kinase A pat hway does not mediate the calcitonin effect. Pertussis toxin, which selecti vely blocks G(i/o) signaling, also had no effect. Increasing cytosolic Ca2 with ionomycin stimulated HEF1 phosphorylation and preventing any calciton in-induced change in cytosolic calcium by a combination of BAPTA and extrac ellular EGTA completely blocked the calcitonin induced tyrosine phosphoryla tion of HEF1. Phorbol 12-myristate 13-acetate also induced HEF1 tyrosine ph osphorylation, and the protein kinase C inhibitor calphostin C completely i nhibited both calcitonin- and phorbol 12-myristate 13-acetate-stimulated HE F1 phosphorylation. Calcitonin also induced the tyrosine phosphorylation of paxillin and focal adhesion kinase, and the association of these two prote ins with HEF1. Pretreatment with cytochalasin D, which disrupts actin micro filaments, prevented the calcitonin-induced HEF1 and paxillin phosphorylati on. In conclusion, the calcitonin-stimulated tyrosine phosphorylation of HE F1 is mediated by calcium- and protein kinase C-dependent mechanisms and re quires the integrity of the actin cytoskeleton.