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

• 久永 眞市

  首都大学東京大学院理工学研究科生命科学専攻神経分子機能研究室

書誌事項

タイトル別名

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

抄録

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>

収録刊行物

• 生物物理化学

  生物物理化学 56 (Suppl_1), S9-S13, 2012

  日本電気泳動学会

キーワード

• Phos-tag
• Cdk5
• p35
• phosphorylation
• quantification

詳細情報

• CRID: 1390282679179041024
• NII論文ID: 130004809458
• NII書誌ID: AN00129729
• DOI: 10.2198/sbk.56.s9
• ISSN: 13499785
• ISSN: 00319082
• NDL書誌ID: 023553691
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I023553691
• 本文言語コード: ja
• データソース種別:
  • JaLC
  • NDL
  • Crossref
  • CiNii Articles
  • KAKEN
• 抄録ライセンスフラグ: 使用不可