Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site.
We previously proposed a molecular mechanism for the activation of smooth muscle myosin light chain kinase (smMLCK) by calmodulin (CaM). According to this model, smMLCK is autoinhibited in the absence of Ca2+/CaM due to the interaction of a pseudosubstrate prototope, contained within the CaM binding/regulatory region, with the active site of the enzyme. Binding of Ca2+/CaM releases the autoinhibition and allows access of the protein substrate to the active site of the enzyme, resulting in phosphorylation of the myosin light chains. We now provide direct experimental evidence that the pseudosubstrate prototope can associate with the active site. We constructed a smMLCK mutant in which the five-amino acid phosphorylation site of the myosin light chain substrate was inserted into the pseudosubstrate sequence of the CaM binding domain without disrupting the ability of the enzyme to bind Ca2+/CaM. We demonstrate that this mutant undergoes intramolecular autophosphorylation at the appropriate inserted serine residue in the absence of CaM and that this autophosphorylation activates the enzyme. Binding of Ca2+/CaM to the mutant enzyme stimulated myosin light chain substrate phosphorylation but strongly inhibited autophosphorylation, presumably by removing the pseudosubstrate from the active site. These results confirm that the pseudosubstrate sequence has access to the catalytic site and that the activation of the enzyme is accompanied by its removal from this position due to Ca2+/CaM binding as predicted by the model.
Bagchi
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Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Kemp
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002645:Chickens; D005895:Gizzard, Avian / Q000201:enzymology; D006706:Homeostasis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism
D002147:Calmodulin; D011994:Recombinant Proteins; D009219:Myosin-Light-Chain Kinase; D002118:Calcium