Proteomic screening for Rho-kinase substrates by combining kinase and phosphatase inhibitors with 14-3-3zeta affinity chromatography

DOI Web Site Web Site PubMed 被引用文献18件
  • Nishioka Tomoki
    Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University
  • Nakayama Masanori
    Department of Tissue Morphogenesis, Max-Planck-Institute for Molecular Biomedicine
  • Amano Mutsuki
    Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University
  • Kaibuchi Kozo
    Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University Japan Science and Technology Agency, CREST

書誌事項

タイトル別名
  • Proteomic Screening for Rho-kinase Substrates by Combining Kinase and Phosphatase Inhibitors with 14-3-3ζ Affinity Chromatography

この論文をさがす

抄録

The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.<br>

収録刊行物

被引用文献 (18)*注記

もっと見る

関連プロジェクト

もっと見る

キーワード

詳細情報 詳細情報について

問題の指摘

ページトップへ