Roles of Calmodulin-dependent Protein Kinases and Phosphatase in Calcium-dependent Transcription of Immediate Early Genes

Recent studies indicate multiple mechanisms are involved in Ca²⁺ stimulation of gene expression. We have used cell-permeable, specific inhibitors of calmodulin-dependent protein kinases (CaM kinases) and phosphatase (calcineurin) to investigate the involvement of these enzymes in transcriptional regulation of three immediate early genes in PC12 cells stimulated with A₂₃₁₈₇ or KCl. Preincubation of PC12 cells with the CaM kinase inhibitor KN-62 blocked autophosphorylation of CaM kinase II in response to stimulation by the Ca²⁺ ionophore A₂₃₁₈₇. KN-62 treatment also resulted in a 60-70% inhibition of Ca²⁺-dependent transcription of c-fos, NGFI-A (zif 268), and NGFI-B (nur 77) as assessed by either Northern or nuclear run-on analyses. Preincubation with the calcineurin inhibitors FK-506 or cyclosporin A strongly enhanced expression of NGFI-A and blocked transcription of NGFI-B, but it had no significant effect on Ca²⁺-stimulated transcription of c-fos. Both FK-506 and KN-62 were specific for Ca²⁺-stimulated transcription as neither effected transcription in response to forskolin or phorbol ester (12-O-tetradecanoylphorbol-13-acetate) treatment. This is the first report of CaM kinase and calcineurin involvement in transcriptional regulation of NGFI-A and NGFI-B. Activation of CaM kinases and calcineurin, in response to elevated intracellular Ca²⁺, would exert antagonistic effects on transcription of NGFI-A- Since inhibition of either the kinase or phosphatase decreased transcription of NGFI-B by 60-90%, this suggests that each enzyme is necessary but not sufficient for Ca²⁺ stimulation. These results indicate that CaM kinases and calcineurin can mediate broad and complex regulation of Ca²⁺-stimulated gene expression.