TYROSINE PHOSPHORYLATION OF THE RELATED ADHESION FOCAL TYROSINE KINASE IN MEGAKARYOCYTES UPON STEM-CELL FACTOR AND PHORBOL-MYRISTATE ACETATE STIMULATION AND ITS ASSOCIATION WITH PAXILLIN

We have characterized signaling pathways involving the related adhesio n focal tyrosine kinase (RAFTK, also known as PYK2 or CAK-beta) in CMK human megakaryocytic cells, Stem cell factor, which potentiates the g rowth of megakaryocytes and their progenitors, and phorbol myristate a cetate, which causes differentiation of megakaryocytic cell lines, ind uced the tyrosine phosphorylation of RAFTK but not of focal adhesion k inase, Stimulation of CMK cells with stem cell factor resulted in an i ncrease in the autophosphorylation and kinase activity of RAFTK. Phosp horylation of RAFTK under these conditions was mediated by a protein k inase C-dependent pathway, Cytochalasin D, which disrupts the cytoskel eton, abolished the phosphorylation of RAFTK upon phorbol myristate ac etate and stem cell factor stimulation, indicating that RAFTK associat ion with the actin cytoskeleton appears to be critical for its phospho rylation. In addition, we observed an association of RAFTK with paxill in, a 68-kDa cytoskeleton protein. Using in vitro binding assays, RAFT K and paxillin were shown to bind directly through the C-terminal prol ine-rich domain, Transient overexpression of a dominant-negative mutan t of RAFTK inhibited significantly the tyrosine phosphorylation of pax illin upon phorbol myristate acetate stimulation. These observations i ndicate that RAFTK might play an important role in the phosphorylation of signaling pathways within the focal adhesions and that RAFTK parti cipates in signaling events that link signals from the cell surface to the cytoskeleton, Furthermore, this study suggests that RAFTK might b e involved in megakaryocyte proliferation and differentiation.