Department of Molecular Physiology and Biophysics.
Vanderbilt Brain Institute.
J Neurosci. 2020 Mar 4;40(10):2000-2014. doi: 10.1523/JNEUROSCI.0893-19.2020. Epub 2020 Feb 4.
The activation of neuronal plasma membrane Ca channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKIIα also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues Arg-Arg-Lys to three alanines disrupts CaMKII binding and CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory. Precise neuronal expression of genes is essential for normal brain function. Proteins involved in signaling pathways that underlie activity-dependent gene expression, such as CaMKII, Shank3, and L-type calcium channels, are often mutated in multiple neuropsychiatric disorders. Shank3 and CaMKII were previously shown to bind L-type calcium channels, and we show here that Shank3 also binds to CaMKII. Our data show that each of these interactions is required for depolarization-induced phosphorylation of the CREB nuclear transcription factor, which stimulates the expression of c-Fos, a neuronal immediate early gene with key roles in synaptic plasticity, brain development, and behavior.
神经元质膜钙通道的激活可刺激多种细胞内反应。支架蛋白可优先将特定的钙通道与不同的下游输出偶联，例如增加基因表达，但这些信号通路的高度特异性的分子机制尚不完全清楚。在这里，我们显示包含 CaMKII 和 Shank3 的复合物可从鼠大脑前脑提取物中特异性共免疫沉淀，Shank3 是一种已知与 L 型钙通道 (LTCC) 相互作用的突触后支架蛋白。激活的纯化 CaMKIIα 还可在残基 829 和 1130 之间直接结合 Shank3。将 Shank3 的精氨酸-精氨酸-赖氨酸突变为三个丙氨酸会破坏 CaMKII 结合和 CaMKII 与异源细胞中的 Shank3 之间的关联。我们的 shRNA/拯救研究表明，Shank3 与 CaMKII 和 LTCC 的结合对于由培养的海马神经元去极化诱导的核 CREB 转录因子的磷酸化和 c-Fos 的表达增加是重要的。因此，这种新型的 CaMKII-Shank3 相互作用对于从质膜中的 LTCC 起始特定的长程信号是必不可少的，该信号对于学习和记忆过程中神经元基因表达的活性依赖性变化是必需的。精确的神经元基因表达对于正常的大脑功能至关重要。参与活动依赖性基因表达的信号通路中的蛋白质，例如 CaMKII、Shank3 和 L 型钙通道，在多种神经精神疾病中经常发生突变。Shank3 和 CaMKII 先前被显示与 L 型钙通道结合，我们在这里显示 Shank3 也与 CaMKII 结合。我们的数据表明，这些相互作用中的每一种都需要去极化诱导的 CREB 核转录因子的磷酸化，这刺激了 c-Fos 的表达，c-Fos 是一种具有关键作用的神经元即刻早期基因，在突触可塑性、大脑发育和行为中。
