ROLE OF SUBSTRATE IN IMPARTING CALCIUM AND PHOSPHOLIPID REQUIREMENTS TO PROTEIN-KINASE-C ACTIVATION
ROLE OF SUBSTRATE IN IMPARTING CALCIUM AND PHOSPHOLIPID REQUIREMENTS TO PROTEIN-KINASE-C ACTIVATION
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DOI：
10.1021/bi00381a029
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发表时间：
1987-04-07
期刊：
影响因子：
2.9
通讯作者：
NELSESTUEN, GL
中科院分区：
文献类型：
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作者：
BAZZI, MD;NELSESTUEN, GL
通过使用几种底物，研究了底物在影响磷脂和Ca2+依赖性蛋白激酶C （PKC）辅因子需求中的作用。所有测试的底物，包括组蛋白、肌钙蛋白I、肌球蛋白轻链、鱼精蛋白、聚精氨酸、丝氨酸（PAS）、聚赖氨酸、丝氨酸（PLS）和髓鞘碱性蛋白（MBP），都被发现与磷脂囊泡以及磷脂酰丝氨酸(PS)-Triton混合胶束相互作用并聚集。PKC对这些不同底物的磷酸化表明存在三种不同的底物类别：(A)底物，如鱼精蛋白，不需要辅助因子；(B)底物如PLS、PAS和MBP只需要磷脂的存在；(C)底物如组蛋白、肌球蛋白轻链和肌钙蛋白I需要Ca2+和磷脂的存在。二酰基甘油是C类底物的主要辅因子。这些不同的需求与底物与磷脂和/或酶的相互作用有关。A类底物与PKC相互作用强烈，并在二元混合物中聚集。在缺乏Ca2+的情况下，PKC直接与B类底物结合，而不与c类底物结合。因此，底物-酶结合消除了磷酸化对Ca2+的需求，底物-酶复合物的聚集也消除了磷脂的需求。增加盐浓度可抑制底物-磷脂相互作用和底物磷酸化，但所需的量取决于底物。PKC活性的丧失似乎与底物- ps聚集的丧失一致，而PKC从膜上解离需要更高的盐浓度。Poly（l -赖氨酸）和Poly（l -精氨酸）这两种有效的PKC抑制剂也表现出底物依赖的抑制特征。这两种聚合物与磷脂囊泡相互作用强烈，似乎通过干扰底物-磷脂结合发挥其抑制作用。这些研究表明，底物在向PKC的激活传递Ca2+、二酰基甘油和磷脂需求方面起着重要作用，PKC与磷脂活性的滴定实际上是由底物-磷脂相互作用主导的。底物传递到活性位点似乎是PKC磷酸化的关键事件，根据底物的选择，这一过程可能需要磷脂和/或Ca2+。
The role of substrate in influencing the cofactor requirements of the phospholipid- and Ca2+-dependent protein kinase C (PKC) was investigated by using several substrates. All of the substrates tested, including histone, troponin I, myosin light chain, protamine, poly(arginine, serine) (PAS), poly(lysine, serine) (PLS), and myelin basic protein (MBP), were found to interact with and aggregate phospholipid vesicles as well as phosphatidylserine (PS)-Triton mixed micelles. Phosphorylation of these different substrates by PKC indicated the presence of three distinct substrate categories: (A) substrates such as protamine requiring no cofactors; (B) substrates such as PLS, PAS, and MBP requiring only the presence of phospholipid; and (C) substrates such as histone, myosin light chain, and troponin I requiring the presence of Ca2+ and phospholipid. Diacylglycerol was a major cofactor only with category C substrates. These different requirements correlated with the interaction of the substrate with phospholipid and/or enzyme. The substrates in category A interacted strongly with and aggregated PKC in a binary mixture. In the absence of Ca2+, PKC bound to substrates of category B directly but not to substrates in category C. Thus, substrate-enzyme binding eliminated the Ca2+ requirement of phosphorylation, and aggregation of substrate-enzyme complex eliminated the phospholipid requirements as well. Substrate-phospholipid interaction and substrate phosphorylation were inhibited by increasing salt concentrations, but the amount needed depended upon the substrate. Loss of PKC activity appeared to coincide with loss of substrate-PS aggregation while dissociation of PKC from the membranes required much higher salt concentrations. Poly(L-lysine) and poly(L-arginine), two potent inhibitors of PKC, also showed substrate-dependent inhibition characteristics. Both of these polymers interaction strongly with phospholipid vesicles and appeared to exert their inhibition by interfering with substrate-phospholipid binding. These studies indicate that the substrate plays an important role in imparting Ca2+, diacyglycerol, and phospholipid requirements to the activation of PKC, and titrations of PKC activity with phospholipid were actually dominated by substrate-phospholipid interactions. Delivery of substrate to the active site appears to be a key event in PKC phosphorylation, and this process may exhibit a requirement for phospholipid and/or Ca2+ depending upon the choice of substrate.