Parissenti A M, Kirwan A F, Kim S A, Colantonio C M, Schimmer B P
Department of Research, Northeastern Ontario Regional Cancer Center, Sudbury, Ontario P3E 5J1, Canada.
J Biol Chem. 1998 Apr 10;273(15):8940-5. doi: 10.1074/jbc.273.15.8940.
Two fusion proteins in which the regulatory domains of human protein kinase Calpha (Ralpha; amino acids 1-270) or mouse protein kinase Cepsilon (Repsilon; amino acids 1-385) were linked in frame with glutathione S-transferase (GST) were examined for their abilities to inhibit the catalytic activities of protein kinase Calpha (PKCalpha) and other protein kinases in vitro. Both GST-Ralpha and GST-Repsilon but not GST itself potently inhibited the activities of lipid-activated rat brain PKCalpha. In contrast, the fusion proteins had little or no inhibitory effect on the activities of the Ser/Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, myosin light chain kinase, and mitogen activated protein kinase or on the src Tyr kinase. GST-Ralpha and GST-Repsilon, on a molar basis, were 100-200-fold more potent inhibitors of PKCalpha activity than was the pseudosubstrate peptide PKC19-36. In addition, a GST-Ralpha fusion protein in which the first 32 amino acids of Ralpha were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKCalpha activity. The three GST-R fusion proteins also inhibited protamine-activated PKCalpha and proteolytically activated PKCalpha (PKM), two lipid-independent forms of PKCalpha; however, the IC50 values for inhibition were 1 order of magnitude greater than the IC50 values obtained in the presence of lipid. These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipid-activated PKCalpha may have resulted from sequestration of lipid activators. Nonetheless, as evidenced by their abilities to inhibit the lipid-independent forms of the enzyme, the GST-R fusion proteins also inhibited PKCalpha catalytic activity through direct interactions. These data indicate that the R domains of PKCalpha and PKCepsilon are specific inhibitors of protein kinase Calpha activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.
研究了两种融合蛋白，其中人蛋白激酶Cα的调节结构域（Rα；氨基酸1 - 270）或小鼠蛋白激酶Cε的调节结构域（Repsilon；氨基酸1 - 385）与谷胱甘肽S - 转移酶（GST）读框相连，检测它们在体外抑制蛋白激酶Cα（PKCα）和其他蛋白激酶催化活性的能力。GST - Rα和GST - Repsilon均能有效抑制脂质激活的大鼠脑PKCα的活性，而GST本身则无此作用。相反，这些融合蛋白对Ser/Thr蛋白激酶（如cAMP依赖性蛋白激酶、cGMP依赖性蛋白激酶、酪蛋白激酶II、肌球蛋白轻链激酶和丝裂原活化蛋白激酶）或src酪氨酸激酶的活性几乎没有抑制作用。以摩尔计，GST - Rα和GST - Repsilon对PKCα活性的抑制作用比假底物肽PKC19 - 36强100 - 200倍。此外，缺失Rα前32个氨基酸（包括19 - 31位氨基酸的假底物序列）的GST - Rα融合蛋白是PKCα活性的有效竞争性抑制剂。这三种GST - R融合蛋白还能抑制鱼精蛋白激活的PKCα和蛋白水解激活的PKCα（PKM），这两种是不依赖脂质的PKCα形式；然而，抑制的IC50值比在有脂质存在时获得的IC50值大1个数量级。这些结果表明，GST - R融合蛋白对脂质激活的PKCα的部分抑制作用可能是由于脂质激活剂的隔离。尽管如此，从它们抑制该酶不依赖脂质形式的能力可以证明，GST - R融合蛋白也通过直接相互作用抑制PKCα的催化活性。这些数据表明，PKCα和PKCε的R结构域是蛋白激酶Cα活性的特异性抑制剂，并表明假底物序列之外的R结构域区域有助于该酶的自身抑制。
