Proteomic Screening for Rho-kinase Substrates by Combining Kinase and Phosphatase Inhibitors with 14-3-3ζ Affinity Chromatography

Cell Structure and Function

Masanori Nakayama²  Kozo Kaibuchi¹  Tomoki Nishioka¹  Mutsuki Amano¹ 

[1] Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University;Department of Tissue Morphogenesis, Max-Planck-Institute for Molecular Biomedicine

关键词: phosphorylation;    protein kinase;    signal transduction;    proteomics;    mass spectrometry;   

DOI  :  10.1247/csf.11044

学科分类：分子生物学，细胞生物学和基因

来源: Japan Society for Cell Biology

【 摘 要 】

References(28)Cited-By(11)Supplementary materials(3)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.

【 授权许可】

Unknown