Protein-tyrosine kinase CAKβ/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 α2 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.

Calcium Mobilization Triggered by the Chemokine CXCL12 Regulates Migration in Wounded Intestinal Epithelial Monolayers

“…The signaling intermediates linking G-protein-coupled receptors with calcium and Pyk2 activation have yet to be fully defined. Separate reports implicate calcium/calmodulin and the myosin light chain kinase as key Pyk2 activators (65,66). In addition to Pyk2, focal adhesion kinase (FAK) and paxillin have previously been shown to play roles in epithelial migration (15,67).…”

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

“…The functional differences between these isoforms are not known. Although the precise mechanism of Pyk2 activation remains unclear, increase in intracellular free Ca 2? can directly or indirectly induce Pyk2 dimerization and transautophosphorylation at Y402 creating a Src-homology-2 binding site that recruits Src family kinases and activates various signaling pathways [1,5,[13][14][15].…”

Traditional Chinese medicine in the prevention and treatment of cancer and cancer metastasis

“…Previous studies have revealed that the focal adhesion kinase (FAK) signalling pathway is important for cell matrix adhesion and cell-extracellular matrix adhesion (73)(74)(75). Following the connection of integrins and matrix, a series of cellular events are triggered and activated, particularly the FAK signal pathway, in the interaction between cells and matrix (76).…”