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 (K(CaM)). The charge reversal mutant at Lys⁹⁷⁹ also increased K(CaM) 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 K(CaM). Charge reversal mutants in Lys⁹⁶¹ and Lys⁹⁶² also had decreased K(CaM) values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.