PYK2 Autophosphorylation, but Not Kinase Activity, Is Necessary for Adhesion-induced Association with c-Src, Osteoclast Spreading, and Bone Resorption

Abstract

Proline-rich tyrosine kinase 2 (PYK2) is the main adhesion-induced kinase in bone-resorbing osteoclasts. Previous studies have shown that ligation of ␣ v ␤ 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 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. 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. PYK21 is highly expressed in osteoclasts, terminally differentiated bone-resorbing cells of hematopoietic origin, and participates in the signaling initiated by osteoclast interaction with bone. Adhesion to bone matrix induces osteoclast differentiation, cytoskeletal reorganization, and cellular polarization, leading to formation of unique membrane areas for active bone resorption. These include the following: (i) the sealing zone, for tight adhesion to bone matrix; (ii) the ruffled border, for directional secretion of protons and proteases into the resorption lacuna; and (iii) functional secretory domain toward bone marrow space, for transcytosis and release of degraded bone matrix (1-3). ␣ v ␤ 3 integrin is highly expressed in osteoclasts (4) and was suggested to mediate osteoclast adhesion to various bone matrix proteins and to regulate cytoskeletal organization required for migration and formation of the sealing zone (5, 6). Interference with ␣ v ␤ 3 integrin function by blocking antibodies or by RGD-containing peptides or proteins leads to inhibition of osteoclast migration and of bone resorption in vitro and in vivo (6 -12). In additio...

The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration

“…Expression of Pyk2 Y402F mutant or antisense RNAs was reported to inhibit osteoclast migration in response to M-CSF and their spreading onto vitronectin (Duong et al, 2001, Lakkakorpi et al, 2003. Consistently, we found that Wrch1 interfered with Pyk2 Y402 phosphorylation and inhibited adhesion to vitronectin and migration in response to M-CSF.…”

“…It is also necessary for osteoclast bone resorbing activity (Zou et al, 2007). Integrin αvß3 interaction with extracellular matrix proteins activates multiple signaling pathways, in particular phosphorylation cascades triggered by the tyrosine kinases Src and Pyk2 (Lakkakorpi et al, 2003, Miyazaki et al, 2004, to regulate podosome dynamics and organization, sealing zone formation and bone resorption (Destaing et al, 2008, Gil-Henn et al, 2007, Shyu et al, 2007. Much less is known about the importance of integrin signaling during osteoclast differentiation.…”

“…This suggests that the two kinases are not essential for differentiation although both where shown to regulate prefusion osteoclast spreading and migration (Duong et al, 2001, Lakkakorpi et al, 2003). …”

“…Since Pyk2 Y402 phosphorylation was shown to be essential to stimulate preOC migration toward M-CSF (Lakkakorpi et al, 2003), we then tested if Wrch1 could regulate this process. We performed transwell assays using RAW264.7 cells expressing GFP and treated (preOCs) or not (undifferentiated) with RANKL for three days.…”

ErbB-2 via PYK2 upregulates the adhesive ability of androgen receptor-positive human prostate cancer cells

“…While elevated ERK/MAPK, p38/MAPK and c-Src activities are correlated with increased PYK2 activity and enhanced adhesion in C-81 as well as PYK2 cDNA-transfected C-33 cells (Supplementary Figure 1A and 2), only ERK/ MAPK activity was consistently reduced by expressing the Y402F or the K457A-PYK2 mutants in C-81 cells, correlated with a decreased adhesion (Figure 6, Supplementary Figure 1B). Interestingly, those K457A mutant proteins still exhibit functional Y402 which led to c-Src activation, as described in osteoclasts-like cells (Lakkakorpi et al, 2003). Thus, PYK2 activates ERK/MAPK and c-Src by differential mechanisms, in which ERK/ MAPK activation depends on both Y402 phosphorylation and kinase activity, while c-Src activation only requires Y402 phosphorylation.…”