PKC and PTPα participate in Src activation by 1α,25(OH)2 vitamin D3 in C2C12 skeletal muscle cells

We previously demonstrated that 1 alpha,25(OH)(2)-vitamin D-3 [1 alpha,25(OH)(2)D-3] induces Src activation, which mediates the hormone-dependent ERK1/2 and p38 MAPK phosphorylation in skeletal muscle cells. In the present study, we have investigated upstream steps whereby 1 alpha,25(OH)(2)D-3 may act to transmit its signal to Src. Preincubation with the PKC inhibitor Ro318220 demonstrated the participation of PKC in 1 alpha,25(OH)(2)D-3-dependent Src activation. Of interest, the hormone promoted the activation of delta the isoform of PKC. We also explored the role of PTP alpha in PKC-mediated Src stimulation. Silencing of PTPa with a specific siRNA suppressed Src activation induced by 1 alpha,25(OH)(2)D-3. Hormone treatment increased PTP alpha (Tyr789) phosphorylation and PKC-dependent phosphatase activity. Accordingly, 1 alpha,25(OH)(2)D-3 promoted serine phosphorylation of PTPa in a PKC-dependent manner. Confocal immunocytochemistry and co-immunoprecipitation assays revealed that the hormone induces the co-localization of Src and PTPa with PKC participation. Computational analysis revealed that the electrostatic interaction between Src and PTPa is favored when PTPa is phosphorylated in Tyr789. These data suggest that 1 alpha,25(OH)(2)D-3 acts in skeletal muscle upstream on MAPK cascades sequentially activating PKC. PTP alpha and Src. (C) 2011 Elsevier Ireland Ltd. All rights reserved.