Morabito Maria A, Sheng Morgan, Tsai Li-Huei
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Neurosci. 2004 Jan 28;24(4):865-76. doi: 10.1523/JNEUROSCI.4582-03.2004.
PSD-95 (postsynaptic density 95) is a postsynaptic scaffolding protein that links NMDA receptors to the cytoskeleton and signaling molecules. The N-terminal domain of PSD-95 is involved in the synaptic targeting and clustering of PSD-95 and in the clustering of NMDA receptors at synapses. The N-terminal domain of PSD-95 contains three consensus phosphorylation sites for cyclin-dependent kinase 5 (cdk5), a proline-directed serine-threonine kinase essential for brain development and implicated in synaptic plasticity, dopamine signaling, cocaine addiction, and neurodegenerative disorders. We report that PSD-95 is phosphorylated in the N-terminal domain by cdk5 in vitro and in vivo, and that this phosphorylation is not detectable in brain lysates of cdk5-/- mice. N-terminal phosphorylated PSD-95 is found in PSD fractions together with cdk5 and its activator, p35, suggesting a role for phosphorylated PSD-95 at synapses. In heterologous cells, coexpression of active cdk5 reduces the ability of PSD-95 to multimerize and to cluster neuronal ion channels, two functions attributed to the N-terminal domain of PSD-95. Consistent with these observations, the lack of cdk5 activity in cultured neurons results in larger clusters of PSD-95. In cdk5-/- cortical neurons, more prominent PSD-95 immunostained clusters are observed than in wild-type neurons. In hippocampal neurons, the expression of DNcdk5 (inactive form of cdk5) or of the triple alanine mutant (T19A, S25A, S35A) full-length PSD-95 results in increased PSD-95 cluster size. These results identify cdk5-dependent phosphorylation of the N-terminal domain of PSD-95 as a novel mechanism for regulating the clustering of PSD-95. Moreover, these observations support the possibility that cdk5-dependent phosphorylation of PSD-95 dynamically regulates the clustering of PSD-95/NMDA receptors at synapses, thus providing a possible mechanism for rapid changes in density and/or number of receptor at synapses.
突触后致密蛋白95（PSD - 95）是一种突触后支架蛋白，它将N - 甲基 - D - 天冬氨酸（NMDA）受体与细胞骨架及信号分子相连。PSD - 95的N端结构域参与PSD - 95的突触定位和聚集，以及NMDA受体在突触处的聚集。PSD - 95的N端结构域含有细胞周期蛋白依赖性激酶5（cdk5）的三个共有磷酸化位点，cdk5是一种脯氨酸定向的丝氨酸 - 苏氨酸激酶，对大脑发育至关重要，并且与突触可塑性、多巴胺信号传导、可卡因成瘾和神经退行性疾病有关。我们报告PSD - 95在体外和体内均被cdk5在N端结构域磷酸化，并且在cdk5基因敲除小鼠的脑裂解物中检测不到这种磷酸化。在突触后致密物（PSD）组分中发现N端磷酸化的PSD - 95与cdk5及其激活剂p35在一起，这表明磷酸化的PSD - 95在突触处发挥作用。在异源细胞中，共表达活性cdk5会降低PSD - 95多聚化以及聚集神经元离子通道的能力，这两种功能都归因于PSD - 95的N端结构域。与这些观察结果一致，培养的神经元中缺乏cdk5活性会导致PSD - 95形成更大的聚集体。在cdk5基因敲除的皮质神经元中，观察到比野生型神经元中更明显的PSD - 95免疫染色聚集体。在海马神经元中，表达失活形式的cdk5（DNcdk5）或全长PSD - 95的三重丙氨酸突变体（T19A、S25A、S35A）会导致PSD - 95聚集体尺寸增加。这些结果确定PSD - 95的N端结构域的cdk5依赖性磷酸化是调节PSD - 95聚集的一种新机制。此外，这些观察结果支持这样一种可能性，即PSD - 95的cdk5依赖性磷酸化动态调节突触处PSD - 95 / NMDA受体的聚集，从而为突触处受体密度和/或数量的快速变化提供一种可能的机制。
