Jideama N M, Noland T A, Raynor R L, Blobe G C, Fabbro D, Kazanietz M G, Blumberg P M, Hannun Y A, Kuo J F
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 1996 Sep 20;271(38):23277-83. doi: 10.1074/jbc.271.38.23277.
Protein kinase C (PKC) isozymes alpha, delta, epsilon, and zeta, shown to be expressed in adult rat cardiomyocytes, displayed distinct substrate specificities in phosphorylating troponin I and troponin T subunits in the bovine cardiac troponin complex. Thus, because they have different substrate affinities, PKC-alpha, -delta, and -epsilon phosphorylated troponin I more than troponin T, but PKC-zeta conversely phosphorylated the latter more than the former. Furthermore, PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in these proteins as free subunits or in the troponin complex. Unlike other isozymes, PKC-delta was uniquely able to phosphorylate Ser-23/Ser-24 in troponin I, the bona fide phosphorylation sites for protein kinase A (PKA); and consequently, like PKA, it reduced Ca2+ sensitivity of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. In addition, PKC-delta, like PKC-alpha, readily phosphorylated Ser-43/Ser-45 (sites common for all PKC isozymes) and reduced maximal activity of MgATPase. In this respect, PKC-delta functioned as a hybrid of PKC-alpha and PKA. In contrast to PKC-alpha, -delta, and -epsilon, PKC-zeta exclusively phosphorylated two previously unknown sites in troponin T. Phosphorylation of troponin T by PKC-alpha resulted in decreases in both Ca2+ sensitivity and maximal activity, whereas phosphorylation by PKC-zeta resulted in a slight increase of the Ca2+ sensitivity without affecting the maximal activity of MgATPase. Most of the in vitro phosphorylation sites in troponin I and troponin T were confirmed in situ in adult rat cardiomyocytes. The present study has demonstrated for the first time distinct specificities of PKC isozymes for phosphorylation of two physiological substrates in the myocardium, with functional consequences.
蛋白激酶C（PKC）的α、δ、ε和ζ同工酶在成年大鼠心肌细胞中表达，它们在磷酸化牛心肌肌钙蛋白复合物中的肌钙蛋白I和肌钙蛋白T亚基时表现出不同的底物特异性。因此，由于它们具有不同的底物亲和力，PKC-α、-δ和-ε对肌钙蛋白I的磷酸化作用比对肌钙蛋白T的更强，但PKC-ζ则相反，对后者的磷酸化作用比对前者更强。此外，PKC同工酶在将这些蛋白质中的不同位点作为游离亚基或在肌钙蛋白复合物中进行磷酸化时表现出不同的特异性。与其他同工酶不同，PKC-δ能够独特地磷酸化肌钙蛋白I中的Ser-23/Ser-24，这是蛋白激酶A（PKA）的真正磷酸化位点；因此，与PKA一样，它降低了重组肌动球蛋白S-1的Ca2+刺激的MgATPase的Ca2+敏感性。此外，PKC-δ与PKC-α一样，容易磷酸化Ser-43/Ser-45（所有PKC同工酶共有的位点）并降低MgATPase的最大活性。在这方面，PKC-δ起到了PKC-α和PKA的混合体的作用。与PKC-α、-δ和-ε相反，PKC-ζ专门磷酸化肌钙蛋白T中两个以前未知的位点。PKC-α对肌钙蛋白T的磷酸化导致Ca2+敏感性和最大活性都降低，而PKC-ζ的磷酸化导致Ca2+敏感性略有增加，而不影响MgATPase的最大活性。肌钙蛋白I和肌钙蛋白T中的大多数体外磷酸化位点在成年大鼠心肌细胞中得到了原位证实。本研究首次证明了PKC同工酶对心肌中两种生理底物磷酸化的不同特异性及其功能后果。
