REGULATION OF MITOGEN-ACTIVATED PROTEIN-KINASES BY A CALCIUM CALMODULIN-DEPENDENT PROTEIN-KINASE CASCADE/

Membrane depolarization of NG108 cells gives rapid (<5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK), To investigate whether th e Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have tr ansfected PC12 cells, which lack CaM-KIV, with constitutively active m utants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, resp ectively), In the absence of depolarization, CaM-KK, transfection had no effect on Elk-dependent transcription of a luciferase reporter gene , whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10- fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were cotransfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activa ted 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38), The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-depend ent transcriptional assays, We found that c-Jun/ATF2-dependent transcr iptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activat ed CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases, Mutation of the phosphorylation s ite (Thr(196)) in CaM-KIV, which mediates its activation by CaM-KIV ki nase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade, These results establish a new Ca2+-dependent mechanism for r egulating MAP kinase pathways and resultant transcription.