title: Proteomic screening for Rho-kinase substrates by combining kinase and phosphatase inhibitors with 14-3-3ζ affinity chromatography.
abstract: 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.
authors.affiliation: Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University. tnishioka@med.nagoya-u.ac.jp
authors.forename: Tomoki
authors.initials: T
authors.lastname: Nishioka
authors.forename: Masanori
authors.initials: M
authors.lastname: Nakayama
authors.forename: Mutsuki
authors.initials: M
authors.lastname: Amano
authors.forename: Kozo
authors.initials: K
authors.lastname: Kaibuchi
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008863:Microspheres; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D010957:Plasmids; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D040901:Proteomics; D051381:Rats; D015398:Signal Transduction; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D008565:Membrane Proteins; C509689:PARD3 protein, human; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases; D020742:rhoA GTP-Binding Protein