A subset of protein kinase C phosphorylation sites on the myosin II regulatory light chain inhibits phosphorylation by myosin light chain kinase.
Protein kinase C (PKC) phosphorylates the regulatory light chains of smooth muscle and cytoplasmic myosin II at three known sites: S1, S2, and T9 [Ikebe, M., Hartshorne, D. J., & Elzinga, M. (1987) J. Biol. Chem. 262, 9569-9573]. Phosphorylation at these sites inhibits the actomyosin ATPase and inhibits phosphorylation of S19 on the regulatory light chain by myosin light chain kinase (MLCK) [Nishikawa, M., Sellers, J. R., Adelstein, R. S., & Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814]. To compare the effects of phosphorylation at a subset of PKC sites on the rate of MLCK phosphorylation, we substituted alanines for the known PKC phosphorylation sites in the Xenopus regulatory light chain (XRLC). PKC phosphorylation of S1A/S2A/T9A revealed secondary phosphorylation sites at T7 and T10, which are accessible both on isolated S1A/S2A/T9A and S1A/S2A/T9A-myosin hybrids. Apparent kinetic constants were determined for MLCK phosphorylation of WT XRLC and XRLC mutants: T9A, S1A/S2A, S1A/S2A/T9A, and T7A/T9A/T10A. PKC prephosphorylation of S1/2 had no effect on the rate of MLCK phosphorylation, while PKC prephosphorylation of T7/9/10 inhibited MLCK phosphorylation due to a 6-fold increase in Km. Our results suggest that phosphorylation of RLC S1/2 as observed in vivo may not be responsible for an inhibition of MLCK phosphorylation.
Turbedsky
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Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Pollard
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TD
Bresnick
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D016415:Sequence Alignment; D014981:Xenopus
D018994:Myosin Light Chains; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase