Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
J Neurochem. 2010 Mar;112(6):1562-73. doi: 10.1111/j.1471-4159.2010.06569.x. Epub 2010 Jan 7.
Calcium/calmodulin-dependent serine kinase (CASK), a causative gene in X-linked mental retardation, acts as a multi-domain scaffold protein and interacts with more than 20 cellular proteins in different subcellular regions of neurons. It is of interest, therefore, to explore whether post-translational modification regulates CASK's protein-protein interactions. Here, we provide evidence that CASK is phosphorylated by protein kinase A (PKA), identifying residue S562 in the PSD-95-Dlg-ZO-1 domain and residue T724 in the guanylate kinase domain as PKA sites by an in vitro PKA kinase reaction and site-directed mutagenesis. Although the role of S562 phosphorylation is not clear, T724 phosphorylation up-regulates the interaction between CASK and T-box transcription factor T-brain-1 (Tbr-1). NMDAR2b, a downstream target of the CASK-Tbr-1 complex, was then used to explore the significance of CASK phosphorylation by PKA. In cultured cortical neurons, the PKA pathway stimulates both the protein expression and the promoter activity of NMDAR2b. Deletion of the Tbr-1-binding sites greatly reduces the 3'-5'-cyclic AMP responsiveness of the NMDAR2b promoter, and the CASK T724A mutation does not promote the 3'-5'-cyclic AMP responsiveness of NMDAR2b. In conclusion, our data provide evidence that PKA phosphorylates CASK, regulates the nuclear function of CASK, and consequently modulates NMDAR2b expression.
钙/钙调蛋白依赖性丝氨酸激酶(CASK)是 X 连锁智力低下的致病基因,作为一种多结构域支架蛋白,与神经元不同亚细胞区域的 20 多种细胞蛋白相互作用。因此,研究是否存在翻译后修饰来调节 CASK 的蛋白-蛋白相互作用非常有趣。在这里,我们提供了证据表明蛋白激酶 A(PKA)可使 CASK 磷酸化,通过体外 PKA 激酶反应和定点突变鉴定 PSD-95-Dlg-ZO-1 结构域中的残基 S562 和鸟苷酸激酶结构域中的残基 T724 为 PKA 位点。虽然 S562 磷酸化的作用尚不清楚,但 T724 磷酸化可上调 CASK 与 T 脑-1(Tbr-1)转录因子的相互作用。然后使用 NMDAR2b(CASK-Tbr-1 复合物的下游靶标)来研究 PKA 对 CASK 磷酸化的意义。在培养的皮质神经元中,PKA 通路可刺激 NMDAR2b 的蛋白表达和启动子活性。Tbr-1 结合位点的缺失大大降低了 NMDAR2b 启动子对 3'-5'-环磷酸腺苷的反应性,而 CASK T724A 突变不会促进 NMDAR2b 对 3'-5'-环磷酸腺苷的反应性。总之,我们的数据提供了证据表明 PKA 可使 CASK 磷酸化,调节 CASK 的核功能,从而调节 NMDAR2b 的表达。