FLUOROALUMINATE INDUCES ACTIVATION AND ASSOCIATION OF SRC AND PYK2 TYROSINE KINASES IN OSTEOBLASTIC MC3T3-E1 CELLS

Fluoride is known to increase bone mass in vivo, probably through stim ulation of osteoblast proliferation; however, the mechanisms of fluoro aluminate action in osteoblasts have not yet been elucidated. We have previously shown that in osteoblastic MC3T3-E1 cells, fluoroaluminate stimulates G protein-mediated protein tyrosine phosphorylation (SuSa, M., Standke, G, J, R,, Jeschke, R 1., and Rohner, D, (1997) Biochem. B iophys. Res. Commun. 235, 680-684). Although the Ser/Thr kinases Erk1, Erk2, and p70(S6K) were activated in response to fluoroaluminate, the identity of fluoroaluminate-activated tyrosine kinase(s) remained elu sive. In this study, we show that in MC3T3-E1 cells, fluoroaluminate i nduces a 110-kDa tyrosine-phosphorylated protein that we identify as P yk2, a cytoplasmic tyrosine kinase related to Fak (focal adhesion kina se), The tyrosine phosphorylation of Pyk2 increased in a dose-and time -dependent manner. The autophosphorylation activity of Pyk2 increased 3-fold and reached its maximum within 10 min of fluoroaluminate treatm ent. Fluoroaluminate also induced activation of Src and the associatio n of Pyk2 with Src, The phosphorylation of Src-associated Pyk2 increas ed >20-fold in in vitro kinase assays, suggesting that Pyk2 is phospho rylated by Src, Although MC3T3-E1 cells express much more Fak than Pyk 2, Src preferentially associated with Pyk2. In vitro, Pyk2 bound to th e Src SH2 domain, suggesting that this interaction mediates the Src-Py k2 association in cells, These data indicate that osteoblastic cells e xpress Pyk2, which is tyrosine-phosphorylated and activated in respons e to G protein activation by fluoroaluminate, and that the mechanism o f Pyk2 activation most likely involves Src, Thus, Src and Pyk2 are tyr osine kinases involved in G protein-mediated tyrosine phosphorylation in osteoblastic cells and may be important for the osteogenic action o f fluoroaluminate.