Calmodulin kinase II chimeras used to investigate the structural requirements for smooth muscle myosin light chain kinase autoinhibition and calmodulin-dependent activation.
Segments of the autoregulatory domain of MK, a catalytically active fragment of the monomeric smooth muscle myosin light chain kinase (smMLCK) (residues 472-972), were replaced with their counterparts from a homologous but multimeric enzyme, calmodulin-dependent protein kinase II (CaM KII). Chimeric proteins in which both the autoregulatory and oligomerization domains of CaM KII (residues 281-478) were substituted for residues 781-972 of smMLCK, MK(CK281-478), or only the autoregulatory domain of CaM KII (residues 281-315) was exchanged for residues 781-813 of smMLCK, MK(CK281-315), exhibited significant enzymatic activity in the absence of Ca(2+)/CaM. In contrast, both MK and a chimeric protein in which the C-terminal half of the autoregulatory domain of smMLCK was replaced with CaM KII residues 301-315, MK(CK301-315), were inactive in the absence of Ca(2+)/CaM. These results indicate that the sequence of the N-terminal half of the autoregulatory domain of smMLCK is important for complete autoinhibition of its enzymatic activity. All proteins bound to Ca(2+)/CaM, and the chimeric proteins MK(CK281-478) and MK(CK281-315) were activated by Ca(2+)/CaM with activation constants (K(CaM)) and maximal enzymatic activities comparable to those of the wild-type MK enzyme. This demonstrates that the entire autoregulatory domain of CaM KII can replace that of smMLCK in its ability to promote efficient CaM-dependent activation of the smMLCK enzyme. However, the inability of the chimeric protein MK(CK301-315) to be activated by Ca(2+)/CaM suggests that replacement of only the C-terminal half of the autoregulatory domain of smMLCK, while still retaining the ability to bind Ca(2+)/CaM, also substitutes residues that prevent activation of the enzyme by Ca(2+)/CaM.
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Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Schreiber
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JL
Means
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AR
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002147:Calmodulin / Q000502:physiology*; D002645:Chickens; D004789:Enzyme Activation / Q000235:genetics; D009130:Muscle, Smooth / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D014422:Turkeys
D002147:Calmodulin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase