Abstract
Proline-rich tyrosine kinase 2 (PYK2) is the main adhesion-induced kinase in bone-resorbing osteoclasts. Previous studies have shown that ligation of alpha(v)beta(3) integrin in osteoclasts induces c-Src-dependent tyrosine phosphorylation and PYK2 activation, leading to cytoskeletal rearrangement, migration, and polarization of these cells. In this study, we examined the role of PYK2 kinase activity and its major autophosphorylation site in adhesion-dependent signaling and cytoskeletal organization during osteoclast spreading and migration. By infecting pre-fusion osteoclasts using recombinant adenovirus expressing PYK2 and its mutants, we demonstrated that mutation at the autophosphorylation site (Y402F) abolishes PYK2 association with c-Src and reduces significantly phosphorylation at tyrosines 579/580 and 881 resulting in inhibition of osteoclast spreading and bone resorption. Overexpression of the kinase-dead PYK2(K475A) mutant had no effect on cell spreading, interaction with c-Src, or the phosphorylation level of Tyr-402, Tyr-579/580, and Tyr-881 relative to PYK2(wt)-expressing cells. Taken together these findings suggest that Tyr-402 is the major docking site for c-Src and can be phosphorylated by another tyrosine kinase in osteoclasts but not in HEK293 cells. Interestingly, both PYK2(Y402F) and PYK2(K457A) translocate normally to podosomes and have no effect on macrophage colony-stimulating factor-induced osteoclast migration. Whereas PYK2(Y402F) dominant negatively blocks osteoclast spreading and bone resorption, PYK2(K457A) may function in part as an adaptor by initially recruiting c-Src to the adhesion complex, which appears to activate PYK2 by phosphorylating additional tyrosines in its regulatory and C-terminal domains. We thus concluded that phosphorylation at Tyr-402 in PYK2 is essential in the regulation of adhesion-dependent cytoskeletal organization in osteoclasts.
Highlights
Proline-rich tyrosine kinase 2 (PYK2) is the main adhesion-induced kinase in bone-resorbing osteoclasts
By infecting pre-fusion osteoclasts using recombinant adenovirus expressing PYK2 and its mutants, we demonstrated that mutation at the autophosphorylation site (Y402F) abolishes PYK2 association with c-Src and reduces significantly phosphorylation at tyrosines 579/ 580 and 881 resulting in inhibition of osteoclast spreading and bone resorption
Reagents—Tissue culture media and cell dissociation buffer were purchased from Invitrogen; fetal bovine serum (FBS) was from JRH Bioscience (Lenexa, KS); collagenase was from Wako Chemicals (Richmond, VA); dispase was from Roche Molecular Biochemicals; and macrophage colony-stimulating factor (M-CSF) was from R & D Systems Inc. (Minneapolis, MN). 1␣,25-Dihydroxyvitamin D3 (1␣,25(OH)2D3) was a gift of Dr Milan R
Summary
Proline-rich tyrosine kinase 2 (PYK2) is the main adhesion-induced kinase in bone-resorbing osteoclasts. We examined the role of PYK2 kinase activity and its major autophosphorylation site in adhesion-dependent signaling and cytoskeletal organization during osteoclast spreading and migration. Overexpression of the kinasedead PYK2(K475A) mutant had no effect on cell spreading, interaction with c-Src, or the phosphorylation level of Tyr-402, Tyr-579/580, and Tyr-881 relative to PYK2(wt)-expressing cells Taken together these findings suggest that Tyr-402 is the major docking site for c-Src and can be phosphorylated by another tyrosine kinase in osteoclasts but not in HEK293 cells. Both PYK2(Y402F) and PYK2(K457A) translocate normally to podosomes and have no effect on macrophage colony-stimulating factor-induced osteoclast migration. Proline-rich regions in the PYK2 C terminus mediate interactions with yet other adaptor molecules, p130Cas and Graf [17, 23]
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