title: Protein-tyrosine kinase CAKbeta/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 alpha2 helix and thus forming its dimer.
abstract: CAKbeta (cell adhesion kinase beta)/PYK2 (proline-rich tyrosine kinase 2) is the second protein-tyrosine kinase of the FAK (focal adhesion kinase) subfamily. It is different from FAK in that it is activated following an increase in cytoplasmic free Ca2+. In the present study we have investigated how Ca2+ activates CAKbeta/PYK2. Calmodulin-agarose bound CAKbeta/PYK2, but not FAK, in the presence of CaCl2. An alpha-helix (F2-alpha2) present in the FERM (band four-point-one, ezrin, radixin, moesin homology) F2 subdomain of CAKbeta/PYK2 was the binding site of Ca2+/calmodulin; a mutant of this region, L176A/Q177A (LQ/AA) CAKbeta/PYK2, bound to Ca2+/calmodulin much less than the wild-type. CAKbeta/PYK2 is known to be prominently tyrosine phosphorylated when overexpressed from cDNA. The enhanced tyrosine phosphorylation was inhibited by W7, an inhibitor of calmodulin, and by a cell-permeable Ca2+ chelator and was almost defective in the LQ/AA-mutant CAKbeta/PYK2. CAKbeta/PYK2 formed a homodimer on binding of Ca2+/calmodulin, which might then induce a conformational change of the kinase, resulting in transphosphorylation within the dimer. The dimer was formed at a free-Ca2+ concentration of 8-12 muM and was stable at 500 nM Ca2+, but dissociated to a monomer in a Ca2+-free buffer. The dimer formation of CAKbeta/PYK2 FERM domain was partially defective in the LQ/AA-mutant FERM domain and was blocked by W7 and by a synthetic peptide with amino acids 168-188 of CAKbeta/PYK2, but not by a peptide with its LQ/AA-mutant sequence. It is known that the F2-alpha2 helix is found immediately adjacent to a hydrophobic pocket in the FERM F2 lobe, which locks, in the autoinhibited FAK, the C-lobe of the kinase domain. Our results indicate that Ca2+/calmodulin binding to the FERM F2-alpha2 helix of CAKbeta/PYK2 releases its kinase domain from autoinhibition by forming a dimer.
authors.affiliation: The Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060-8556, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kohno
authors.forename: Eiko
authors.initials: E
authors.lastname: Matsuda
authors.forename: Hiroko
authors.initials: H
authors.lastname: Sasaki
authors.forename: Terukatsu
authors.initials: T
authors.lastname: Sasaki
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D019281:Dimerization; D004789:Enzyme Activation; D051418:Focal Adhesion Kinase 2 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D014443:Tyrosine / Q000378:metabolism; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D017931:DNA Primers; D018076:DNA, Complementary; D014667:Vasopressins; D014443:Tyrosine; D051418:Focal Adhesion Kinase 2; D002118:Calcium