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 stimulation of osteoblast proliferation; however, the mechanisms of fluoroaluminate 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. Biophys. 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 elusive. In this study, we show that in MC3T3-E1 cells, fluoroaluminate induces a 110-kDa tyrosine-phosphorylated protein that we identify as Pyk2, a cytoplasmic tyrosine kinase related to Fak (focal adhesion kinase), 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 treatment. Fluoroaluminate also induced activation of Src and the association of Pyk2 with Src, The phosphorylation of Src-associated Pyk2 increased >20-fold in in vitro kinase assays, suggesting that Pyk2 is phosphorylated by Src, Although MC3T3-E1 cells express much more Fak than Pyk2, Src preferentially associated with Pyk2. In vitro, Pyk2 bound to the Src SH2 domain, suggesting that this interaction mediates the Src-Pyk2 association in cells, These data indicate that osteoblastic cells express Pyk2, which is tyrosine-phosphorylated and activated in response to G protein activation by fluoroaluminate, and that the mechanism of Pyk2 activation most likely involves Src, Thus, Src and Pyk2 are tyrosine kinases involved in G protein-mediated tyrosine phosphorylation in osteoblastic cells and may be important for the osteogenic action of fluoroaluminate.