Barylko Barbara, Mao Yuntao S, Wlodarski Pawel, Jung Gwanghyun, Binns Derk D, Sun Hui-Qiao, Yin Helen L, Albanesi Joseph P
Departments of Pharmacology and Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
J Biol Chem. 2009 Apr 10;284(15):9994-10003. doi: 10.1074/jbc.M900724200. Epub 2009 Feb 11.
Phosphatidylinositol 4-kinases play essential roles in cell signaling and membrane trafficking. They are divided into type II and III families, which have distinct structural and enzymatic properties and are essentially unrelated in sequence. Mammalian cells express two type II isoforms, phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) and IIbeta (PI4KIIbeta). Nearly all of PI4KIIalpha, and about half of PI4KIIbeta, associates integrally with membranes, requiring detergent for solubilization. This tight membrane association is because of palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domains of both type II isoforms. Deletion of this motif from PI4KIIalpha converts the kinase from an integral to a tightly bound peripheral membrane protein and abrogates its catalytic activity ( Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708 ). Here we identify the first two cysteines in the CCPCC motif as the principal sites of palmitoylation under basal conditions, and we demonstrate the importance of the central proline for enzymatic activity, although not for membrane binding. We further show that palmitoylation is critical for targeting PI4KIIalpha to the trans-Golgi network and for enhancement of its association with low buoyant density membrane fractions, commonly termed lipid rafts. Replacement of the four cysteines in CCPCC with a hydrophobic residue, phenylalanine, substantially restores catalytic activity of PI4KIIalpha in vitro and in cells without restoring integral membrane binding. Although this FFPFF mutant displays a perinuclear distribution, it does not strongly co-localize with wild-type PI4KIIalpha and associates more weakly with lipid rafts.
磷脂酰肌醇4激酶在细胞信号传导和膜运输中发挥着重要作用。它们分为II型和III型家族，具有不同的结构和酶学特性，在序列上基本不相关。哺乳动物细胞表达两种II型异构体，即磷脂酰肌醇4激酶IIα（PI4KIIα）和IIβ（PI4KIIβ）。几乎所有的PI4KIIα以及大约一半的PI4KIIβ都与膜紧密结合，需要去污剂来溶解。这种紧密的膜结合是由于位于两种II型异构体催化结构域内的富含半胱氨酸基序CCPCC的棕榈酰化。从PI4KIIα中删除该基序会将激酶从整合膜蛋白转变为紧密结合的外周膜蛋白，并消除其催化活性（Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705 - 7708）。在这里，我们确定CCPCC基序中的前两个半胱氨酸是基础条件下棕榈酰化的主要位点，并且我们证明了中央脯氨酸对酶活性的重要性，尽管对膜结合不重要。我们进一步表明，棕榈酰化对于将PI4KIIα靶向反式高尔基体网络以及增强其与通常称为脂筏的低浮力密度膜组分的结合至关重要。用疏水性残基苯丙氨酸取代CCPCC中的四个半胱氨酸，在体外和细胞中基本上恢复了PI4KIIα的催化活性，但没有恢复整合膜结合。尽管这种FFPFF突变体显示出核周分布，但它与野生型PI4KIIα的共定位不强，并且与脂筏的结合较弱。
