Biochemical characterization of four splice variants of mouse Ca2+/calmodulin-dependent protein kinase Iδ

Ca2+/calmodulin (CaM)-dependent protein kinase I delta (CaMKI delta) is a Ser/Thr kinase that plays pivotal roles in Ca2+ signalling. CaMKI delta is activated by Ca2+/CaM-binding and phosphorylation at Thr(180) by CaMK kinase (CaMKK). In this study, we characterized four splice variants of mouse CaMKI delta (mCaMKI delta s: a, b, c and d) found by in silico analysis. Recombinant mCaMKI delta s expressed in Escherichia coli were phosphorylated by CaMKK; however, only mCaMKI delta-a and c showed protein kinase activities towards myelin basic protein in vitro, with mCaMKI delta-b and mCaMKI delta-d being inactive. Although mCaMKI delta-a and mCaMKI delta-c underwent autophosphorylation in vitro, only mCaMKI delta-c underwent autophosphorylation in 293T cells. Site-directed mutagenesis showed that the autophosphorylation site is Ser(349), which is found in the C-terminal region of only variants c and b (Ser(324)). Furthermore, phosphorylation of these sites (Ser(324) and Ser(349)) in mCaMKI delta-b and c was more efficiently catalyzed by cAMP-dependent protein kinase in vitro and in cellulo as compared to the autophosphorylation of mCaMKI delta-c. Thus, variants of mCaMKI delta possess distinct properties in terms of kinase activities, autophosphorylation and phosphorylation by another kinase, suggesting that they play physiologically different roles in murine cells.