Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag SDS-PAGE
Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag SDS-PAGE
复制标题
DOI：
10.1074/mcp.m900578-mcp200
复制
发表时间：
2010-06-01
影响因子：
7
通讯作者：
Hisanaga, Shin-ichi
中科院分区：
文献类型：
--
作者：
Hosokawa, Tomohisa;Saito, Taro;Hisanaga, Shin-ichi
磷酸化是一种主要的翻译后修饰，广泛用于调节许多细胞过程。细胞周期蛋白依赖性激酶5（Cdk 5）是由激活亚基p35激活的脯氨酸导向的丝氨酸/苏氨酸激酶。cdk 5-p35调节多种神经元活动，如神经元迁移、棘形成、突触活动和细胞死亡。Cdk 5的激酶活性受p35的蛋白水解调节：蛋白酶体降解导致Cdk 5下调，而钙蛋白酶对p35的裂解导致Cdk 5过度活化。p35的磷酸化决定蛋白水解途径。我们以前已经确定了Ser（8）和Thr（138）作为主要的磷酸化位点，使用代谢标记的培养细胞，然后通过二维磷酸肽图谱和磷酸特异性抗体。然而，这些方法不能确定体内p35磷酸化的程度。在这里，我们报告使用Phos标记的SDS-PAGE来揭示p35在神经元培养和脑中的磷酸化状态。使用Phos标签丙烯酰胺，磷酸化的p35的电泳迁移率被延迟，因为它被困在Phos标签网站。我们在Ser（91）处发现了一个新的磷酸化位点，该位点在体外被Ca（2+）-钙调素依赖性蛋白激酶II磷酸化。我们在COS-7细胞中构建了与Cdk 5共表达的磷酸化位点的p35和Ala取代突变体的磷酸化依赖性带型。使用标准带谱，我们将内源性p35的各个带与磷酸化状态的组合和定量的Ser（8）、Ser（91）和Thr（138）磷酸化进行了分配。在胚胎脑中观察到最高水平的p35磷酸化; Ser（8）在所有p35分子中被磷酸化，而Ser（91）在60%中被磷酸化，Thr（138）在12%的p35分子中被磷酸化。这是第一次定量和位点特异性测量p35的磷酸化，证明了Phos标记的SDS-PAGE用于分析体内蛋白质磷酸化状态的有用性。Molecular & Cellular Proteomics 9：1133-1143，2010.
Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, spine formation, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes down-regulation of Cdk5, whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser(8) and Thr(138) as major phosphorylation sites using metabolic labeling of cultured cells followed by two-dimensional phosphopeptide mapping and phosphospecific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the phosphorylation states of p35 in neuronal culture and brain. Using Phos-tag acrylamide, the electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We found a novel phosphorylation site at Ser(91), which was phosphorylated by Ca(2+)-calmodulin-dependent protein kinase II in vitro. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states and quantified Ser(8), Ser(91), and Thr(138) phosphorylation. The highest level of p35 phosphorylation was observed in embryonic brain; Ser(8) was phosphorylated in all p35 molecules, whereas Ser(91) was phosphorylated in 60% and Thr(138) was phosphorylated in similar to 12% of p35 molecules. These are the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins. Molecular & Cellular Proteomics 9:1133-1143, 2010.