Laboratory of Cellular and Neuronal Dynamics, Department of Biology, Faculty of Sciences, Universidad de Chile, Santiago, Chile.
Functional Genomics Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda MD, USA.
PLoS One. 2014 Mar 21;9(3):e90363. doi: 10.1371/journal.pone.0090363. eCollection 2014.
Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.
蛋白质磷酸化是最常见的翻译后修饰之一，它调节细胞中的几个关键功能。周期蛋白依赖性激酶 5（Cdk5）是一种脯氨酸导向的丝氨酸/苏氨酸激酶，主要在神经系统中活跃。它调节几个生物过程，如神经元迁移、细胞骨架动力学、轴突导向和突触可塑性等。为了在大脑中寻找 Cdk5 的新底物，我们进行了定量磷酸蛋白质组学分析，使用固定化金属离子亲和色谱法（IMAC）从 E18.5 Cdk5+/+和 Cdk5-/-胚胎的全脑中分离磷酸蛋白质，该方法特异性结合磷酸化蛋白质。分离的磷酸蛋白质被洗脱并进行同位素标记相对和绝对定量（iTRAQ）和质谱鉴定。我们发现 40 种蛋白质在 Cdk5-/-脑中的磷酸化程度降低。此外，在这 40 种低磷酸化蛋白质中，我们鉴定了两种蛋白质：MARCKS（蛋白激酶 C 底物的豆蔻酰化丙氨酸丰富蛋白）和 Grin1（G 蛋白调节的神经突生长诱导物 1）。MARCKS 已知在鸡神经细胞中被 Cdk5 磷酸化，而 Grin1 尚未被报道被 Cdk5 磷酸化。当这些蛋白质与 p35 一起在 N2A 神经母细胞瘤细胞系中过表达时，发现它们的 Cdk5 模体中的丝氨酸磷酸化增加。相比之下，用 Cdk5 抑制剂罗昔考汀处理会导致 N2A 细胞和转染 MARCKS 的原代海马神经元中的丝氨酸磷酸化产生相反的效果。总之，这里呈现的结果确定了 Grin1 为新的 Cdk5 底物，并证实了先前鉴定的 MARCKS 为真正的 Cdk5 底物。
