Activation and association of the Tec tyrosine kinase with the human prolactin receptor: Mapping of a Tec/Vav1-receptor binding site

Stimulation of the PRL receptor (PRLr) results in the activation of the guanine nucleotide exchange factor (GEF) p95(Vav1) with corresponding alterations in cytoarchitecture and cell motility. To better understand the mechanisms involved in the regulation of Vav1 activity, the role of the tyrosine kinase p70(Tec) was examined. Coimmunoprecipitation and in vitro kinase assays revealed that ligand stimulation of the PRLr resulted in the rapid activation of Tec and its concomitant association with the PRLr. When coexpressed in COS-1 cells, both Vav1 and Tec were found to associate with the PRLr in the presence of ligand. In the absence of receptor, a constitutive complex between Vav1 and Tec was noted. Both Vav1 and Tee, however, were capable of independent engagement of a bipartite intracellular domain of the PRLr. Deletion mapping studies confined this interaction to residues 323 to 527 of the intracellular domain of the PRLr. Furthermore, Tec enhanced the GEF activity of Vav1 as evidenced by an increase in GTP-bound Rad. These data would suggest a pivotal function for the formation of a Tec/Vav1/PRLr complex during PRL-driven signal transduction, given the role of Vav1 in the control of cell proliferation and the regulation of Rho family-mediated cytoskeletal alterations.