Zhu J, Hansen H, Su L, Shieh H L, Riedel H
Section on Molecular Biology, Joslin Diabetes Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215.
J Biochem. 1994 May;115(5):1000-9. doi: 10.1093/oxfordjournals.jbchem.a124413.
Based on the finding by others that the conserved region C1 of conventional protein kinase C isoforms carries two independent, cysteine-rich phorbol ester binding sites, we have mapped the structural elements of the C1 region for their role in the phorbol ester- and phospholipid regulation of PKC alpha responses. We have prepared two amino terminal truncation mutants of bovine PKC alpha, ND91 lacking the first Cys-repeat of C1, and ND153 lacking both Cys-repeats of C1, as well as two internal deletion mutants, D162-245 lacking most of C2, and D109-263 lacking most of C2 and the second Cys-repeat of C1. The mutants were expressed in the yeast Saccharomyces cerevisiae which allows the rapid biochemical and physiological characterization of mammalian PKC isoforms. We found that all mutants displayed an elevated basal level of enzymatic activity in vitro but retained the basic catalytic PKC characteristics: regulation by Ca2+ and (except for ND153) by phospholipid or phorbol ester. In vivo we observed proportional physiological responses, the stimulation of Ca2+ uptake, and an increase in the cell doubling time for all mutants upon phorbol ester stimulation (constitutive for ND153) similar to the response of normal PKC alpha. Our findings indicate that after partial PKC activation by deletion mutagenesis, the presence of either Cys-repeat in C1 still allows phospholipid- and phorbol ester regulation of protein kinase C alpha responses.
基于其他人的发现，即传统蛋白激酶C亚型的保守区域C1带有两个独立的、富含半胱氨酸的佛波酯结合位点，我们绘制了C1区域的结构元件，以研究它们在佛波酯和磷脂对PKCα反应的调节中的作用。我们制备了牛PKCα的两个氨基末端截短突变体，ND91缺失C1的第一个半胱氨酸重复序列，ND153缺失C1的两个半胱氨酸重复序列，以及两个内部缺失突变体，D162 - 245缺失大部分C2，D109 - 263缺失大部分C2和C1的第二个半胱氨酸重复序列。这些突变体在酿酒酵母中表达，这使得能够对哺乳动物PKC亚型进行快速的生化和生理特性分析。我们发现所有突变体在体外均表现出升高的基础酶活性水平，但保留了基本的催化PKC特性：受Ca2+调节，以及（除ND153外）受磷脂或佛波酯调节。在体内，我们观察到所有突变体在佛波酯刺激后（ND153为组成型）呈现出成比例的生理反应、Ca2+摄取的刺激以及细胞倍增时间的增加，这与正常PKCα的反应相似。我们的研究结果表明，通过缺失诱变进行部分PKC激活后，C1中任一一个半胱氨酸重复序列的存在仍然允许对蛋白激酶Cα反应进行磷脂和佛波酯调节。
