Department of Anatomy and Cell Biology, University of Heidelberg, D-69120 Heidelberg, Germany.
J Biol Chem. 2012 Sep 7;287(37):30952-66. doi: 10.1074/jbc.M112.349597. Epub 2012 Jul 9.
Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABA(A) receptors. The cellular and molecular mechanisms involved in gephyrin-mediated receptor clustering are still not well understood. Here we provide evidence that the gephyrin-binding protein collybistin is involved in regulating the phosphorylation of gephyrin. We demonstrate that the widely used monoclonal antibody mAb7a is a phospho-specific antibody that allows the cellular and biochemical analysis of gephyrin phosphorylation at Ser-270. In addition, another neighbored epitope determinant was identified at position Thr-276. Analysis of the double mutant gephyrin(T276A,S277A) revealed significant reduction in gephyrin cluster formation and altered oligomerization behavior of gephyrin. Moreover, pharmacological inhibition of cyclin-dependent kinases in hippocampal neurons reduced postsynaptic gephyrin mAb7a immunoreactivities. In vitro phosphorylation assays and phosphopeptide competition experiments revealed a phosphorylation at Ser-270 depending on enzyme activities of cyclin-dependent kinases CDK1, -2, or -5. These data indicate that collybistin and cyclin-dependent kinases are involved in regulating the phosphorylation of gephyrin at postsynaptic membrane specializations.
网格蛋白是一种支架蛋白，对于抑制性甘氨酸和不同类型的 GABA(A) 受体的突触后聚集是必不可少的。网格蛋白介导的受体聚集的细胞和分子机制仍未得到很好的理解。在这里，我们提供的证据表明，网格蛋白结合蛋白 collybistin 参与调节网格蛋白的磷酸化。我们证明，广泛使用的单克隆抗体 mAb7a 是一种磷酸特异性抗体，允许对 Ser-270 处的网格蛋白磷酸化进行细胞和生化分析。此外，还确定了另一个相邻的表位决定簇位于 Thr-276 位置。双突变体 gephyrin(T276A,S277A)的分析显示，网格蛋白簇的形成明显减少，并且网格蛋白的寡聚化行为发生改变。此外，在海马神经元中抑制周期蛋白依赖性激酶会降低突触后网格蛋白 mAb7a 的免疫反应性。体外磷酸化实验和磷酸肽竞争实验表明，Ser-270 的磷酸化依赖于周期蛋白依赖性激酶 CDK1、-2 或 -5 的酶活性。这些数据表明，collybistin 和周期蛋白依赖性激酶参与调节突触后膜特化处网格蛋白的磷酸化。
