Sugars J M, Cellek S, Manifava M, Coadwell J, Ktistakis N T
Department of Signalling, Babraham Institute, Cambridge CB2 4AT United Kingdom.
J Biol Chem. 1999 Oct 15;274(42):30023-7. doi: 10.1074/jbc.274.42.30023.
We have reported previously that phospholipase D1 (PLD1) is labeled specifically with [(3)H]palmitate following transient expression and immunoprecipitation and that this modification appeared important both for membrane localization and catalytic activity. In this work we identify by mutagenesis that the acylation sites on PLD1 are cysteine residues 240 and 241, with the cysteine at position 241 accounting for most but not all of the modification. Replacement of both cysteine residues with either serines or alanines resulted in a mutant protein that contained undetectable [(3)H]palmitate. In comparison with the wild type protein, the double mutant showed reduced catalytic activity in vivo, whereas its activity in vitro was unchanged. In addition, the localization of the double mutant was altered in comparison with the wild type protein, whereas wild type PLD1 is primarily on intracellular membranes and on punctate structures, the double mutant was on plasma membrane. Because cysteines 240 and 241 lie within a putative pleckstrin homology domain of PLD1, it is likely that fatty acylation on these residues modulates the function of the PLD1 pleckstrin homology domain.
我们之前报道过，磷脂酶D1（PLD1）在瞬时表达和免疫沉淀后会被[³H]棕榈酸特异性标记，并且这种修饰对于膜定位和催化活性似乎都很重要。在这项工作中，我们通过诱变确定PLD1上的酰化位点是半胱氨酸残基240和241，其中位置241的半胱氨酸占了大部分但不是全部的修饰。用丝氨酸或丙氨酸取代这两个半胱氨酸残基会产生一种突变蛋白，该蛋白含有无法检测到的[³H]棕榈酸。与野生型蛋白相比，双突变体在体内的催化活性降低，而其体外活性未改变。此外，与野生型蛋白相比，双突变体的定位发生了改变，野生型PLD1主要位于细胞内膜和点状结构上，而双突变体位于质膜上。由于半胱氨酸240和241位于PLD1假定的普列克底物蛋白同源结构域内，这些残基上的脂肪酰化很可能调节了PLD1普列克底物蛋白同源结构域的功能。
