Quantitative analysis of in vivo phosphorylation of Cyclin-dependent kinase activator p35 by Phos-tag SDS-PAGE/immunoblotting

  • Hisanaga Shin-ichi
    Laboratory of Molecular Neuroscience, Department of Biological Sciences, Faculty of Science and Engineering, Tokyo Metropolitan University

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  • Phos-tagを用いたCdk5制御サブユニットp35のin vivoリン酸化の定量的解析
  • Phos-tag オ モチイタ Cdk5 セイギョ サブユニット p35 ノ in vivo リン サンカ ノ テイリョウテキ カイセキ

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Abstract

Phosphorylation is a major post-translational modification widely used in 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, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes downregulation of Cdk5 whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser8 and Thr138 as major phosphorylation sites using metabolic labeling of cultured cells followed by 2D-phosphopeptide mapping and phospho-specific 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 in vivo phosphorylation states of p35. Using Phos-tag acrylamide, electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. 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 Ser8, Ser91 and Thr138 phosphorylation. This is 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.<br>

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