Jeong Jaehoon, Li Yan, Roche Katherine W
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
eNeuro. 2021 May 25;8(3). doi: 10.1523/ENEURO.0481-20.2021. Print 2021 May-Jun.
SHANK3 is a large scaffolding protein in the postsynaptic density (PSD) that organizes protein networks, which are critical for synaptic structure and function. The strong genetic association of with autism spectrum disorder (ASD) emphasizes the importance of SHANK3 in neuronal development. SHANK3 has a critical role in organizing excitatory synapses and is tightly regulated by alternative splicing and posttranslational modifications. In this study, we examined basal and activity-dependent phosphorylation of Shank3 using mass spectrometry (MS) analysis from phosphorylation assays, experiments, and studies with cultured neurons. We found that Shank3 is highly phosphorylated, and we identified serine 782 (S782) as a potent CaMKII phosphorylation site. Using a phosphorylation state-specific antibody, we demonstrate that CaMKII can phosphorylate Shank3 S782 and in heterologous cells on cotransfection with CaMKII. We also observed an effect of a nearby ASD-associated variant (Shank3 S685I), which increased S782 phosphorylation. Notably, eliminating phosphorylation of Shank3 with a S782A mutation increased Shank3 and PSD-95 synaptic puncta size without affecting Shank3 colocalization with PSD-95 in cultured hippocampal neurons. Taken together, our study revealed that CaMKII phosphorylates Shank3 S782 and that the phosphorylation affects Shank3 synaptic properties.
SHANK3是突触后致密区（PSD）中的一种大型支架蛋白，它组织对突触结构和功能至关重要的蛋白质网络。SHANK3与自闭症谱系障碍（ASD）的强烈遗传关联强调了其在神经元发育中的重要性。SHANK3在组织兴奋性突触方面起着关键作用，并受到可变剪接和翻译后修饰的严格调控。在本研究中，我们通过磷酸化分析、实验以及对培养神经元的研究，利用质谱（MS）分析检测了Shank3的基础磷酸化和活性依赖性磷酸化。我们发现Shank3高度磷酸化，并确定丝氨酸782（S782）是一个有效的CaMKII磷酸化位点。使用磷酸化状态特异性抗体，我们证明CaMKII可以磷酸化Shank3 S782，并且在与CaMKII共转染的异源细胞中也能磷酸化。我们还观察到一个附近的与ASD相关的变体（Shank3 S685I）的作用，它增加了S782的磷酸化。值得注意的是，用S782A突变消除Shank3的磷酸化增加了Shank3和PSD - 95突触小体的大小，而不影响培养海马神经元中Shank3与PSD - 95的共定位。综上所述，我们的研究表明CaMKII磷酸化Shank3 S782，并且这种磷酸化影响Shank3的突触特性。
