Ono Y, Fujii T, Igarashi K, Kuno T, Tanaka C, Kikkawa U, Nishizuka Y
Central Research Division, Takeda Chemical Industries, Osaka, Japan.
Proc Natl Acad Sci U S A. 1989 Jul;86(13):4868-71. doi: 10.1073/pnas.86.13.4868.
Protein kinase C normally has a tandem repeat of a characteristic cysteine-rich sequence in C1, the conserved region of the regulatory domain. These sequences resemble the DNA-binding zinc finger domain. For the gamma subspecies of rat brain protein kinase C, various deletion and point mutants in this domain were constructed, and the mutated proteins were expressed in Escherichia coli by using the T7 expression system. Radioactive phorbol 12,13-dibutyrate binding analysis indicated that a cysteine-rich zinc-finger-like sequence was essential for protein kinase C to bind phorbol ester and that one of two sequences was sufficient for the phorbol ester binding. Conserved region C2, another region in the regulatory domain, was apparently needed for the enzyme to require Ca2+ for phorbol ester binding activity.
蛋白激酶C通常在其调节结构域的保守区域C1中具有一个富含半胱氨酸的特征性串联重复序列。这些序列类似于DNA结合锌指结构域。对于大鼠脑蛋白激酶C的γ亚型，构建了该结构域中的各种缺失和点突变体，并使用T7表达系统在大肠杆菌中表达突变蛋白。放射性佛波醇12,13 - 二丁酸结合分析表明，富含半胱氨酸的锌指样序列对于蛋白激酶C结合佛波酯至关重要，并且两个序列中的一个对于佛波酯结合就足够了。调节结构域中的另一个区域保守区域C2显然是该酶使佛波酯结合活性需要Ca2+所必需的。
