Abstract
Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.
Highlights
From the University of Melbourne Department of Medicine, Repatriation General Hospital, Heidelberg, Victoria3081, Australia and the §Department of Cell Biology,Baylor College of Medicine, Houston, Texas 77030
Calmodulin-binding Peptide-Fig. 1 shows results of an experiment designed to provide preliminary evidence for calmodulin binding by the synthetic peptidesused in thisstudy
The 22-residuesynthetic peptidewas tested for its capacity to act asa calmodulin antagonist
Summary
Calmodulin-binding Peptide-Fig. 1 shows results of an experiment designed to provide preliminary evidence for calmodulin binding by the synthetic peptidesused in thisstudy. Calmodulin-binding Peptide as aSubstrate AntagonistThe MLC kinase 480-501 peptide was found to inhibit myosin light chain phosphorylation with an ICs0of 2.7 p~ when tested under conditions of saturating calmodulin concentration (15p ~ an) d limiting myosin light chain concentration (Fig. 3).This result suggested that the MLC kinase 480-501 peptide may be acting as a competitive inhibitor of myosin light chain phosphorylation (with respect to the substratea) t higher concentrations than those required to inhibit calmodulin-dependent activation of the myosin kinase The nature of this inhibition was investigated as a function of varying substrate concentration. Des-Valso4-smMLCkinase 493507, which had poor calmodulin antagonist and calmodulin
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