INHIBITORY CROSS-TALK BY CAMP KINASE ON THE CALMODULIN-DEPENDENT PROTEIN-KINASE CASCADE

The calmodulin-dependent kinase (CaM-K) cascade, a Ca2+-triggered syst em involving phosphorylation and activation of CaM-KI and CaM-KIV by C aM kinase kinase (CaM-KK), regulates transcription through direct phos phorylation of transcription factors such as cAMP response element-bin ding protein, We have shown previously that activated CaM-KIV can acti vate the mitogen-activated protein kinases (Enslen, H., Tokumitsu, H., Stork, P. J. S., Davis, R. J., and Soderling, T. R. (1996) Proc. Natl ., Acad. Sci. U. S. A. 93, 10803-10808), and the present paper describ es a novel regulatory cross-talk between cAMP kinase (PKA) and CaM-KK. PKA gave rapid phosphorylation in vitro and in cells of recombinant C aM-KK, resulting in 50-75% inhibition of CaM-KK activity, part of whic h was due to suppression of CaM-binding by phosphorylation of Ser(458) in the CaM-binding domain, However, the Ser(458) --> Ala mutant, or a truncation mutant in which the CaM-binding and autoinhibitory domains were deleted, was still partially suppressed by PKA-mediated phosphor ylation, The second inhibitory site was identified as Thr(108) by site -specific mutagenesis, Treatments of COS-7, PC12, hippocampal, or Jurk at cells with the PKA activators forskolin or isoproterenol gave 30-90 % inhibition of either endogenous or transfected CaM-KK and/or CaM-KIV activities. These results demonstrate that the CaM kinase cascade is negatively regulated in cells by the cAMP/PKA pathway.