The PYK2 tyrosine kinase is a negative regulator of bone formation, but aside from the requirement for PYK2 kinase activity there has been little progress toward understanding of the molecular mechanism involved in this function. To gain insight into the signaling pathways modulated by PYK2 we sought to identify PYK2 substrates. Challenges inherent to a quantitative phosphoproteomic analysis for non-receptor tyrosine kinases were overcome by employing an inducible PYK2 overexpression system in NIH3T3 cells in combination with a selective PYK2 inhibitor. The identification of a number of known PYK2 substrates and interacting partners validated the methodology. Results of the inducible cell system were extended to a cell model of osteogenesis, examining the effect of the PYK2 inhibitor on the phosphorylation state of targets identified in the phosphoproteomic study. Consistent with phosphoproteomic analysis, increased osteogenesis associated with a selective PYK2 inhibitor was accompanied by reduced phosphorylation of paxillin, Gab1 and p130(Cas), along with reduction of phosphorylation levels of the Met activation loop. These results further confirmed the utility of the methodology and point to a previously unknown bi-directional activation pathway between PYK2 and Met.
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