The actions of prolactin (PRL), a hormone utilized in normal and malignant mammary tissues, are mediated through its receptor (PRLr), a member of the cytokine receptor superfamily. PRL acts to stimulate the survival, motility, and the progression of human breast cancer. PRL-induced dimerization of the PRLr induces the association, tyrosine phosphorylation, and activation of members of the Vav family (Vav1-3) of guanine nucleotide exchange factors (GEF), an event necessary for PRL-driven cellular signaling and function. We have discovered two novel protein kinases in association with Vav1 and Vav2, namely the tyrosine kinase Tec, and the serine/threonine kinase Nek3. Both kinases are rapidly activated during PRL-stimulated signaling and associate with the PRLr-Vav2 complex in human breast cancer, forming a macromolecular signaling assembly. It is the central hypothesis of this proposal that the structures and interactions in the PRLr-Tec/Vav2/Nek3 complex coordinately activate the downstream effectors and functions associated with PRLr action. This hypothesis will be tested by three specific aims using breast cancer and PRL-responsive lines. First, the functional role of Tec and Nek3 during PRL-driven proliferation, survival, and cytoskeletal change will be assessed at the cellular and molecular levels, through the transfection of wild-type, activated, and kinase-dead forms of Tec and Nek3. Second, the location of Vav2 tyrosine phosphorylation will be determined by mass spectroscopy and the functional significance of these residues evaluated by replacement mutagenesis. Third, mutagenic and biophysical approaches will identify and characterize the interaction motifs within the PRLr-Tec/Vav2 complex, while the role of such motifs during PRLr-mediated signaling and action will be evaluated through the transfection of interaction defective mutants of the PRLr, Tec and Vav2. The structure/function studies proposed here will provide insight into the assembly of a multimeric PRLr-associated signaling complex and relate these findings to cellular processes of relevance to the pathophysiology of human breast cancer. These analyses of PRLr-associated transduction may ultimately provide the basis for novel therapeutic strategies aimed at modulating the function of the PRL/PRLr complex.
