1,25-dihydroxyvitamin D₃ regulates pp60(c-src) activity and expression of a pp60(c-src) activating phosphatase

The nonreceptor tyrosine kinase, c-src, and the steroid hormone, 1,25- dihydroxyvitamin D₃(1,25(OH)₂D₃), are essential to development of the osteoclast phenotype. On the other hand, functional relationships between the activities of c-src and 1,25(OH)₂D₃ are as yet unknown. To determine if 1,25(OH)₂D₃ modulates c-src in osteoclastogenesis, we tested the steroid's effect on avian marrow-derived osteoclast precursors. We find c-src mRNA and immunoprecipitable c-src protein (pp60(c-src)) unaltered by 72 h exposure of these cells to 1,25(OH)₂D₃ (10^-11 to 10^-9 M). Despite no quantitative change in pp60(c-src), in vitro kinase assay of the immune complex reveals 1,25(OH)₂D₃ dose-dependently accelerates the catalytic activity of pp60(c- src), enhancing its autophosphorylation and phosphorylation of exogenous substrate. This observation represents the first documentation, in nontransformed cells, of humoral induction of pp60(c-src) kinase. Consistent with the fact pp60(c-src) is activated by dephosphorylation of tyrosine 527 (Y527), the phosphotyrosine content of the pp60(c-src) immunoprecipitate, measured by immunoblot, is decreased by 1,25(OH)₂D₃. Alternatively, mRNA and protein levels of c-src kinase (CSK), which inactivates pp60(c-src) by phosphorylating Y527, are not altered by the steroid. In contrast, 1,25(OH)₂D₃ enhances mRNA and especially protein levels of avian protein tyrosine phosphatase λ (PTPλ), an enzyme specifically activating pp60(c- src) by dephosphorylating Y527 [Fang et al. (1994): J Biol Chem 269:20194- 20200]. Thus, treatment of avian osteoclast precursors with 1,25(OH)₂D₃ accelerates the catalytic activity of pp60(c-src) independent of protein expression. Activation of the kinase may occur via the Y527 dephosphorylating enzyme PTP, expression of which, we show for the first time, is regulated.