Investigating the regulation of calcium/calmodulin-dependent protein kinase IV

Calcium/calmodulin-dependent kinase IV (CaMKIV) is a multifunctional serine/threonine kinase that has been shown to potentiate Ca2+-dependent transcription. Although there had been previous studies examining various aspects of CaMKIV regulation, a comprehensive investigation into the regulation of CaMKIV transcriptional function was lacking; thus, I initiated an expansive study to address this deficiency. Previous studies had shown that CaMKIV is regulated by autoinhibitory interactions, which are relieved upon calcium/calmodulin-(Ca 2+/CaM-) binding, leading to basal CaMKIV activity. It had also been shown that Ca2+/CaM-bound CaMKIV could be phosphorylated on its T200 residue by the upstream kinase, calcium/calmodulin-dependent kinase kinase (CaMKK), leading to increased CaMKIV activity. I made the surprising discovery that phosphorylation of CaMKIV on T200 actually leads to its transformation from a Ca2+/CaM-dependent enzyme to a fully Ca2+/CaM-independent or autonomous enzyme. Through the analysis of wild-type and mutant CaMKIV proteins in biochemical experiments and transcriptional assays, I demonstrate that autonomous activity is both necessary and sufficient for CaMKIV transcriptional function. (CaMKIV mutants with constitutive autonomous activity can bypass the requirements for Ca2+/CaM-binding and T200 phosphorylation.); One of the major impediments in the study of CaM kinase functions in the cell has been the lack of specific inhibitors. Two compounds, KN62 and KN93, have been frequently used as specific CaMKII inhibitors. However, recent evidence indicated that KN62 and KN93 (and their purportedly inactive analogs KN04 and KN92, respectively) have effects on several unintended targets, including CaM kinases other than CaMKII. Thus, I performed a systematic analysis of the effects of these compounds on the multifunctional CaM kinase family members. I found that the water insolubility of KN62 and KN04 made these compounds unsuitable for study in our established kinase assay conditions. I demonstrate that, despite its frequent description as a specific CaMKII inhibitor, KN93 inhibits CaMKI, CaMKII, and CaMKIV similarly. In contrast, the purportedly inactive KN93 analog, KN92, has dissimilar effects on the CaM kinases; weakly inhibiting CaMKI and CaMKIV, while potently inhibiting CaMKII. These findings indicate that KN93 and KN92 may be used in conjunction to investigate the multifunctional CaM kinases, and to differentiate between CaMKI/IV- and CaMKII-dependent processes.