Oh Jeong S, Manzerra Pasquale, Kennedy Mary B
Division of Biology 216-76, California Institute of Technology, Pasadena, California 91125, USA.
J Biol Chem. 2004 Apr 23;279(17):17980-8. doi: 10.1074/jbc.M314109200. Epub 2004 Feb 17.
synGAP is a neuron-specific Ras GTPase-activating protein found in high concentration in the postsynaptic density fraction from mammalian forebrain. Proteins in the postsynaptic density, including synGAP, are part of a signaling complex attached to the cytoplasmic tail of the N-methyl-d-aspartate-type glutamate receptor. synGAP can be phosphorylated by a second prominent component of the complex, Ca(2+)/calmodulin-dependent protein kinase II. Here we show that phosphorylation of synGAP by Ca(2+)/calmodulin-dependent protein kinase II increases its Ras GTPase-activating activity by 70-95%. We identify four major sites of phosphorylation, serines 1123, 1058, 750/751/756, and 764/765. These sites together with other minor phosphorylation sites in the carboxyl tail of synGAP control stimulation of GTPase-activating activity. When three of these sites and four other serines in the carboxyl tail are mutated, stimulation of GAP activity after phosphorylation is reduced to 21 +/- 5% compared with 70-95% for the wild type protein. We used phosphosite-specific antibodies to show that, as predicted, phosphorylation of serines 765 and 1123 is increased in cultured cortical neurons after exposure of the neurons to the agonist N-methyl-d-aspartate.
突触后致密蛋白（synGAP）是一种神经元特异性的Ras GTP酶激活蛋白，在哺乳动物前脑的突触后致密部分中含量很高。突触后致密区的蛋白质，包括synGAP，是附着于N-甲基-D-天冬氨酸型谷氨酸受体胞质尾部的信号复合物的一部分。synGAP可被该复合物的另一个主要成分钙/钙调蛋白依赖性蛋白激酶II磷酸化。在此我们表明，钙/钙调蛋白依赖性蛋白激酶II对synGAP的磷酸化使其Ras GTP酶激活活性提高了70 - 95%。我们确定了四个主要的磷酸化位点，即丝氨酸1123、1058、750/751/756和764/765。这些位点与synGAP羧基尾部的其他次要磷酸化位点共同控制GTP酶激活活性的刺激。当这些位点中的三个以及羧基尾部的其他四个丝氨酸发生突变时，磷酸化后GAP活性的刺激与野生型蛋白的70 - 95%相比降至21±5%。我们使用位点特异性磷酸化抗体表明，正如所预测的，在培养的皮质神经元暴露于激动剂N-甲基-D-天冬氨酸后，丝氨酸765和1123的磷酸化增加。
