Hu Tianhui, Exton John H
Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Biol Chem. 2003 Jan 24;278(4):2348-55. doi: 10.1074/jbc.M210093200. Epub 2002 Nov 12.
It has been suggested that protein-protein interaction is important for protein kinase C (PKC) alpha to activate phospholipase D1 (PLD1). To determine the one or more sites on PKCalpha that are involved in binding to PLD1, fragments containing the regulatory domain, catalytic domain, and C1-C3 domain of PKCalpha were constructed and shown to be functional, but they all failed to bind and activate PLD1 in vivo and in vitro. A C-terminal 23-amino acid (aa) deletion mutant of PKCalpha was also found to be inactive. To define the binding/activation site(s) in the C terminus of PKCalpha, 1- to 11-aa deletion mutants were made in this terminus. Deletion of up to 9 aa did not alter the ability of PKCalpha to bind and activate PLDl, whereas a 10-aa deletion was inactive. The residue at position 10 was Phe(663). Mutations of this residue (F663D and F663A) caused loss of binding, activation, and phosphorylation of PLD1, indicating that Phe(663) is essential for these activities. Time course experiments showed that the activation of PLD1 by PMA was much faster than its phosphorylation, and its activity decreased as phosphorylation increased with time. Staurosporine, a PKC inhibitor, completely inhibited PLD1 phosphorylation in response to 4beta-phorbol 12-myristate 13-acetate PMA and blocked the later decrease in PLD activity. The same results were found with the D481E mutant of PKCalpha, which is unable to phosphorylate PLD1. These results indicate that neither the regulatory nor catalytic domains of PKCalpha alone can bind to or activate PLD1 and that a residue in the C terminus of PKCalpha (Phe(663)) is required for these effects. The initial activation of PLD1 by PMA is highly correlated with the binding of PKCalpha. Although PKCalpha can phosphorylate PLD1, this is a relatively slow process and is associated with inactivation of the enzyme.
有人提出，蛋白质 - 蛋白质相互作用对于蛋白激酶C（PKC）α激活磷脂酶D1（PLD1）很重要。为了确定PKCα上参与与PLD1结合的一个或多个位点，构建了包含PKCα调节结构域、催化结构域和C1 - C3结构域的片段，并证明其具有功能，但它们在体内和体外均未能结合并激活PLD1。还发现PKCα的C末端23个氨基酸（aa）缺失突变体无活性。为了确定PKCα C末端的结合/激活位点，在此末端制作了1至11个氨基酸的缺失突变体。缺失多达9个氨基酸不会改变PKCα结合并激活PLD1的能力，而10个氨基酸的缺失则无活性。第10位的残基是苯丙氨酸（Phe(663)）。该残基的突变（F663D和F663A）导致PLD1的结合、激活和磷酸化丧失，表明苯丙氨酸（Phe(663)）对于这些活性至关重要。时间进程实验表明，佛波酯（PMA）对PLD1的激活比对其磷酸化快得多，并且随着时间的推移，其活性随着磷酸化增加而降低。蛋白激酶C抑制剂星形孢菌素完全抑制了4β - 佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）诱导的PLD1磷酸化，并阻止了随后PLD活性的降低。PKCα的D481E突变体也得到了相同的结果，该突变体无法使PLD1磷酸化。这些结果表明，PKCα的调节结构域或催化结构域单独都不能结合或激活PLD1，并且PKCα C末端的一个残基（苯丙氨酸（Phe(663)））是这些效应所必需的。PMA对PLD1的初始激活与PKCα的结合高度相关。虽然PKCα可以使PLD1磷酸化，但这是一个相对缓慢的过程，并且与该酶的失活有关。
