Rybin Vitalyi O, Sabri Abdelkarim, Short Jacob, Braz Julian C, Molkentin Jeffery D, Steinberg Susan F
Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
J Biol Chem. 2003 Apr 18;278(16):14555-64. doi: 10.1074/jbc.M212644200. Epub 2003 Jan 31.
Recent studies identify conventional protein kinase C (PKC) isoform phosphorylations at conserved residues in the activation loop and C terminus as maturational events that influence enzyme activity and targeting but are not dynamically regulated by second messengers. In contrast, this study identifies phorbol 12-myristoyl 13-acetate (PMA)- and norepinephrine-induced phosphorylations of PKC epsilon (at the C-terminal hydrophobic motif) and PKC delta (at the activation loop) as events that accompany endogenous novel PKC (nPKC) isoform activation in neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are prevented (and the kinetics of PMA-dependent PKC down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase activity. PKC delta is recovered from PMA-treated cultures with increased in vitro lipid-independent kinase activity (and altered substrate specificity); the PMA-dependent increase in PKC delta kinase activity is attenuated when PKC delta activation loop phosphorylation is prevented. To distinguish roles of individual nPKC isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKC delta and PKC epsilon mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKC delta and WT-PKC epsilon are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKC delta is phosphorylated at the activation loop but not the hydrophobic motif; DN-PKC epsilon is phosphorylated at the hydrophobic motif but not the activation loop. Collectively, these results identify a role for PKC epsilon in nPKC activation loop phosphorylations and PKC delta in nPKC hydrophobic motif phosphorylations. Agonist-induced nPKC isoform phosphorylations that accompany activation/translocation of the enzyme contribute to the regulation of PKC delta kinase activity, may influence nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes.
最近的研究表明，传统蛋白激酶C（PKC）亚型在激活环和C末端保守残基处的磷酸化是成熟事件，这些事件影响酶活性和靶向性，但不受第二信使的动态调节。相比之下，本研究确定佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和去甲肾上腺素诱导的PKCε（在C末端疏水基序处）和PKCδ（在激活环处）的磷酸化是新生大鼠心肌细胞内源性新型PKC（nPKC）亚型激活时伴随的事件。nPKC激酶活性的药理抑制剂可阻止激动剂诱导的nPKC磷酸化（并减缓PMA依赖性PKC下调的动力学）。从PMA处理的培养物中回收的PKCδ具有增加的体外脂质非依赖性激酶活性（以及改变的底物特异性）；当PKCδ激活环磷酸化被阻止时，PKCδ激酶活性的PMA依赖性增加会减弱。为了区分单个nPKC亚型在nPKC磷酸化中的作用，使用腺病毒介导的基因转移将野生型（WT）和显性负性（DN）PKCδ和PKCε突变体引入心肌细胞培养物中。即使在没有变构激活剂的情况下，WT-PKCδ和WT-PKCε在激活环和疏水基序位点也高度磷酸化。DN-PKCδ在激活环处磷酸化，但在疏水基序处不磷酸化；DN-PKCε在疏水基序处磷酸化，但在激活环处不磷酸化。总体而言这些结果确定了PKCε在nPKC激活环磷酸化中的作用以及PKCδ在nPKC疏水基序磷酸化中的作用。伴随酶激活/易位的激动剂诱导的nPKC亚型磷酸化有助于调节PKCδ激酶活性，可能影响nPKC亚型的转运/下调，并为心肌细胞中的nPKC信号通路引入功能上重要的相互作用。
