Turbedsky K, Pollard T D, Bresnick A R
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry. 1997 Feb 25;36(8):2063-7. doi: 10.1021/bi9624651.
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.
蛋白激酶C（PKC）可在三个已知位点对平滑肌和细胞质肌球蛋白II的调节轻链进行磷酸化：S1、S2和T9 [池部，M.，哈茨霍恩，D. J.，& 埃尔津加，M.（1987年）《生物化学杂志》262卷，9569 - 9573页]。这些位点的磷酸化会抑制肌动球蛋白ATP酶，并抑制肌球蛋白轻链激酶（MLCK）对调节轻链上S19位点的磷酸化 [西川，M.，塞勒斯，J. R.，阿德尔斯坦，R. S.，& 日高，H.（1984年）《生物化学杂志》259卷，8808 - 8814页]。为了比较PKC位点亚组的磷酸化对MLCK磷酸化速率的影响，我们将非洲爪蟾调节轻链（XRLC）中已知的PKC磷酸化位点替换为丙氨酸。S1A/S2A/T9A的PKC磷酸化揭示了T7和T10处的二级磷酸化位点，这些位点在分离的S1A/S2A/T9A和S1A/S2A/T9A - 肌球蛋白杂交体上均可被磷酸化。测定了野生型XRLC和XRLC突变体（T9A、S1A/S2A、S1A/S2A/T9A和T7A/T9A/T10A）的MLCK磷酸化的表观动力学常数。S1/2的PKC预磷酸化对MLCK磷酸化速率没有影响，而T7/9/10的PKC预磷酸化由于Km增加6倍而抑制了MLCK磷酸化。我们的结果表明，体内观察到的RLC S1/2磷酸化可能不是抑制MLCK磷酸化的原因。
