Activation of Ca²⁺/calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase in Jurkat T lymphocytes

Ca²⁺/calmodulin-dependent protein kinase IV (CaM-kinase IV), a member of the CaM-kinase family involved in transcriptional regulation, is stimulated by Ca²⁺/CaM but also requires phosphorylation by a CaM-kinase kinase for full activation. In this study we investigated the physiological role of a CaM-kinase cascade in Jurkat T human lymphocytes through antigen receptor (CD3) signaling. Total and Ca²⁺-independent CaM-kinase IV activities were increased 8-14-fold by anti-CD3 antibody. This CD3-mediated activation involved phosphorylation since the immunoprecipitated CaM-kinase IV from stimulated Jurkat cells could be subsequently inactivated in vitro by protein phosphatase 2A. CaM-kinase IV immunoprecipitated from unstimulated Jurkat cells or CD3-negative mutant Jurkat cells could be activated in vitro 10-40-fold by CaM-kinase kinase purified from rat brain or thymus, whereas CaM-kinase IV from CDS-stimulated wild-type Jurkat cells was only activated to 2-3-fold by exogenous CaM-kinase kinase. CaM-kinase IV activation was triggered by Ca²⁺ acting through calmodulin since activation could also be elicited by ionomycin treatment, and CD3-mediated activation was blocked by the calmodulin antagonist calmidazolium. These data are consistent with a CaM-kinase cascade in which CaM-kinase IV is activated by a CaM-kinase kinase cascade triggered by elevated intracellular calcium in Jurkat cells.