CARDIAC TROPONIN-I MUTANTS - PHOSPHORYLATION BY PROTEIN-KINASE-C AND PROTEIN-KINASE-A AND REGULATION OF CA2+-STIMULATED MGATPASE OF RECONSTITUTED ACTOMYOSIN S-1
CARDIAC TROPONIN-I MUTANTS - PHOSPHORYLATION BY PROTEIN-KINASE-C AND PROTEIN-KINASE-A AND REGULATION OF CA2+-STIMULATED MGATPASE OF RECONSTITUTED ACTOMYOSIN S-1
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DOI：
10.1074/jbc.270.43.25445
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发表时间：
1995-10-27
影响因子：
4.8
通讯作者：
KUO, JF
中科院分区：
文献类型：
--
作者：
NOLAND, TA;GUO, XD;KUO, JF
研究了蛋白激酶C和蛋白激酶A对心肌肌钙蛋白I（TnI）的位点特异性磷酸化在Ca ~（2+）刺激的重组肌动球蛋白S-1的MgATP酶调节中的意义。使用的TnI突变体是T144 A、S43 A/S45 A和S43 A/S45 A/T144 A（其中鉴定的蛋白激酶C磷酸化位点Thr-144和Ser-43/ Ser-45分别被Ala取代）和S23 A/S24 A和N32（其中蛋白激酶A磷酸化位点Ser-23/Ser-24被Ala取代或缺失）。突变引起蛋白激酶C磷酸化动力学的微妙变化，所有突变体最大磷酸化到不同程度（1.3-2.7摩尔磷酸/摩尔蛋白质）。蛋白激酶C可以交叉磷酸化蛋白激酶A位点，但基本上不能发生逆转。与野生型TnI和T144 A相比，未磷酸化的S43 A/S45 A、S43 A/S45 A/T144、S23 A/S24 A和N32导致重构肌动球蛋白S-1的Ca 2+刺激的MgATP酶的Ca 2+敏感性降低。磷酸化的野生型和所有的突变体，除了S43 A/S45 A和S43 A/S45 A/T144 A的蛋白激酶C引起显着减少的最大活性的Ca ~（2+）刺激的MgATPase和表观亲和力的肌球蛋白S-1重建（调节）肌动蛋白。蛋白激酶C与蛋白激酶A通过磷酸化Ser-23/Ser-24而使肌丝Ca ~（2+）敏感性降低，提示心肌肌钙蛋白I中的Ser-43/Ser-45和Ser-23/Ser-24对肌丝正常的Ca ~（2+）敏感性有重要作用。Ser-43/ Ser-45和Ser-23/Ser-24的磷酸化分别主要参与蛋白激酶C对肌动球蛋白S-1 MgATP酶活性和Ca（2+）敏感性的调节。
The significance of site-specific phosphorylation of cardiac troponin I (TnI) by protein kinase C and protein kinase A in the regulation of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1 was investigated. The TnI mutants used were T144A, S43A/S45A, and S43A/S45A/T144A (in which the identified protein kinase C phosphorylation sites, Thr-144 and Ser-43/ Ser-45, were, respectively, substituted by Ala) and S23A/S24A and N32 (in which the protein kinase A phosphorylation sites Ser-23/Ser-24 were either substituted by Ala or deleted). The mutations caused subtle changes in the kinetics of phosphorylation by protein kinase C, and all mutants were maximally phosphorylated to various extents (1.3-2.7 mol of phosphate/mol of protein). Protein kinase C could cross-phosphorylate protein kinase A sites but the reverse essentially could not occur. Compared to wild-type TnI and T144A, unphosphorylated S43A/S45A, S43A/S45A/T144, S23A/ S24A, and N32 caused a decreased Ca2+ sensitivity of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. Phosphorylation by protein kinase C of wildtype and all mutants except S43A/S45A and S43A/S45A/ T144A caused marked reductions in both the maximal activity of Ca2+-stimulated MgATPase and apparent affinity of myosin S-1 for reconstituted (regulated) actin. It was further noted that protein kinase C acted in an additive manner with protein kinase A by phosphorylating Ser-23/Ser-24 to bring about a decreased Ca2+ sensitivity of the myofilament.It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca2+ sensitivity of the myofilament, and that phosphorylation of Ser-43/ Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca(2+)sensitivity, respectively, of actomyosin S-1 MgATPase.