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In this report, we cloned a novel calmodulin-kinase (CaM-KIδ) from HeLa cells and characterized its activation mechanism. CaM-KIδ exhibits Ca2+/CaM-dependent activity that is enhanced (∼30-fold) in vitro by phosphorylation of its Thr180 by CaM-K kinase (CaM-KK)α, consistent with detection of CaM-KIδ-activating activity in HeLa cells. We also identified a novel CaM-KKβ isoform (CaM-KKβ-3) in HeLa cells whose activity was highly Ca2+/CaM-independent. Transiently expressed CaM-KIδ exhibited enhanced protein kinase activity in HeLa cells without ionomycin stimulation. This sustained activation of CaM-KIδ was completely abolished by Thr180Ala mutation and inhibited by CaM-KK inhibitor, STO-609, indicating a functional CaM-KK/CaM-KIδ cascade in HeLa cells. [Copyright &y& Elsevier]

Characterizes the inhibitory mechanism of STO-609, a selective inhibitor of Ca[sup 2+]/calmodulin-dependent protein kinase (CaM-KK), by using catalytic chimera and point mutants of CaM-KKalpha and CaM-KKbeta. Amino acid difference in the adenosine triphosphate-binding hydrophobic pocket of the catalytic domain of CaM-KK isoforms; Sensitivity of CaM-KK isoforms to STO-609.

Studies the differential regulatory mechanism of calcium(sup 2+)/calmodulin-dependent protein kinase kinase (CaM-KK) isoforms. Regulation of protein kinase kinase activity by an autoinhibitory mechanism; Identification of a region of 23 amino acids located at the N-terminus of the catalytic domain as an importance regulatory element; Calcium(sup 2+)/CaM dependency for the Cam-KK activity.

In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin.dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an ∼140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by actirated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Set residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simpie removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme. [ABSTRACT FROM AUTHOR]