Novel mechanism of protein kinase C inhibition involving the pseudosubstrate region by secalonic acid D in vitro.
Novel mechanism of protein kinase C inhibition involving the pseudosubstrate region by secalonic acid D in vitro.
复制标题
体外癸二酸 D 抑制蛋白激酶 C 涉及假底物区域的新机制。
DOI：
10.1006/taap.1999.8850
复制
发表时间：
2000
影响因子：
3.8
通讯作者：
Reddy,CS
中科院分区：
文献类型：
--
作者：
Balasubramanian,G;Reddy,CS
来自小鼠的研究证据表明，在用真菌毒素Secalonic acid D(SAD)治疗的雌性小鼠中，蛋白激酶C(CPKC)的传统亚型(CPKC)在子代腭裂(CP)的发展中起到了机械作用。这些实验的目的是评估SAD是否抑制商业上可用的纯蛋白激酶C(PKCα，-β，-γ)，并在体外确定这种抑制的机制。对3种同工酶的抑制作用相似(直接外推法的IC_(50)为5~6.2μM，对数回归法的IC_(50)为2.7~4μM)。口中最主要的蛋白激酶CβII的调节结构域被去除后，SAD失去了抑制作用，这表明这种抑制是由调节亚基介导的。动力学分析表明，SAD与钙离子和磷脂酰丝氨酸(PS)的结合部位缺乏竞争性相互作用。然而，针对PKCβII假底物区19-32位残基的抗体竞争性地逆转了SAD对PKCβII的抑制作用，表明该假底物是SAD相互作用的部位。此外，SAD还可抑制内切酶Arg-C对PKCβII的假底物的切割。精氨酸脱氢酶本身的活性不受精氨酸脱氢酶抑制的事实表明，精氨酸脱氢酶干扰了辅因子诱导的假底物从PKCβII活性部位释放的前一步，这是一种新的机制。
Evidence from studies in mice suggests a mechanistic role for the inhibition of conventional isoforms of protein kinase C (cPKC) in the development of cleft palate (CP) in the offspring of female mice treated with the mycotoxin, secalonic acid D (SAD). These experiments were aimed at assessing whether SAD inhibits commercially available pure cPKC (PKCα, -β, -γ) and at identifying the mechanism of such an inhibition in vitro. Secalonic acid D inhibited the three isozymes similarly (IC50 of 5 to 6.2 μM by direct extrapolation and 2.7 to 4 μM by logarithmic regression). The loss of inhibitory effect of SAD upon removal of the regulatory domain of PKCβII, the most predominant cPKC in the palate, suggested that the inhibition was mediated by the regulatory subunit. Kinetic analysis suggested a lack of competitive interaction for SAD with the binding sites for Ca2+and phosphatidyl serine (PS). Antibody directed against residues 19–32 of the pseudosubstrate region of PKCβII, however, competitively reversed the inhibition of PKCβII by SAD, suggesting that the pseudosubstrate is the site of interaction of SAD. Further, SAD inhibited the cleavage of the pseudosubstrate from PKCβII by the endoproteinase Arg-C. The fact that the activity of Arg-C itself was not inhibited by SAD suggests that SAD interferes with the preceding step involving the cofactor-induced release of the pseudosubstrate from the active site of PKCβII, a novel mechanism.