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 (Scaronuscarona, M., Standke, G. J. R., Jeschke, M., 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.
Jeschke
M
M
Research Bone Metabolism, Novartis Pharma AG, CH-4002 Basel, Switzerland.
Standke
G J
GJ
Scaronuscarona
M
M
D016475:3T3 Cells; D000535:Aluminum / Q000494:pharmacology*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005461:Fluorine / Q000494:pharmacology*; D051418:Focal Adhesion Kinase 2; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects* / Q000201:enzymology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
C053629:fluoroaluminum; D005461:Fluorine; D014443:Tyrosine; D000535:Aluminum; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse; D019061:src-Family Kinases