Identification of basic residues involved in activation and calmodulin binding of rabbit smooth muscle myosin light chain kinase.
It is postulated that basic residues in the regulatory region of myosin light chain kinase are important for conferring autoinhibition by binding to the catalytic core. To investigate this proposal, 10 basic amino acids within the regulatory region of rabbit smooth muscle myosin light chain kinase (Lys961-Lys979) were replaced either singularly or in combination with acidic or nonpolar residues by site-directed mutagenesis. All active mutant kinases were dependent on Ca2+/calmodulin for catalytic activity. None of the mutants was active in the absence of Ca2+/calmodulin, suggesting that the autoinhibitory region has not been defined completely. Charge reversal mutants at Arg974, Arg975, and Lys976 resulted in loss of high affinity binding of calmodulin and increased the concentration of calmodulin required for half-maximal activation (KCaM). The charge reversal mutant at Lys979 also increased KCaM but to a lesser extent. Charge reversal mutants at Lys965 and Arg967 resulted in an inactive myosin light chain kinase that could not be proteolytically activated. When these residues were mutated to Ala, the expressed kinase was dependent upon Ca2+/calmodulin for activity and exhibited a decrease in KCaM. Charge reversal mutants in Lys961 and Lys962 also had decreased KCaM values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.
Fitzsimons
D P
DP
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235-9040.
Herring
B P
BP
Stull
J T
JT
Gallagher
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits
D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase; D002118:Calcium