CHARACTERIZATION OF RAFTK, A NOVEL FOCAL ADHESION KINASE, AND ITS INTEGRIN-DEPENDENT PHOSPHORYLATION AND ACTIVATION IN MEGAKARYOCYTES

We have recently isolated a cDNA encoding a novel human intracellular tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine k inase). The RAFTK cDNA, which encodes a polypeptide of 1,009 amino aci ds, shares 65% homology to the focal adhesion kinase (FAK), including several consensus motifs. In this report, we describe the biochemical characterization and functional analysis of the RAFTK protein. Coexpre ssion of RAFTK and FAK proteins in megakaryocytic cells and blood plat elets was observed. Using a specific antibody to RAFTK and the monoclo nal antibody 2A7 to FAK, FAK and RAFTK could be distinguished antigeni cally. RAFTK had intrinsic tyrosine kinase and autokinase activities. It was phosphorylated on tyrosine in growing cultures of COS cells tra nsfected with the pCDNAIII/flag-RAFTK expression vector containing the RAFTK cDNA ligated with the 8 amino acid flag peptide sequence. Simil ar to FAK, dephosphorylation of RAFTK was observed when adherent trans fected COS cells were detached. Phosphorylation was regained upon repl ating of these cells on the fibronectin-coated dishes. Analysis of tyr osine-phosphorylated RAFTK from adherent transfected COS cells showed that the Src homology 2 (SH2) domains of the Src and Fyn protein kinas es as well as the Grb2 adaptor protein were able to specifically assoc iate with RAFTK. Tyrosine phosphorylation of endogenous RAFTK was obse rved upon fibronectin-induced activation of human megakaryocytic cells . Furthermore, colocalization of RAFTK protein with vinculin, a focal adhesion protein, was observed by confocal microscopy in focal adhesio n-like structures in adherent CMK cells and in transfected pCDNAIII/fl ag-RAFTK COS cells upon fibronectin activation. These data suggest tha t RAFTK is a novel member of the FAK family, that it localizes to foca l adhesion-like structures in CMK megakaryocytic cells, that it partic ipates in integrin-mediated signaling pathways in megakaryocytes, and that it is able to associate with the tyrosine kinases Src and Fyn as well as the adaptor protein Grb2 via SH2-phosphotyrosine interactions. (C) 1996 by The American Society of Hematology.