title: Characterization of a modified ROCK2 protein that allows use of N6-ATP analogs for the identification of novel substrates.
abstract: BACKGROUND
  The Rho-associated coiled-coil kinase-2 (ROCK2) is an important signaling transducer in the transmission of extracellular signals effecting organization of the actin cytoskeleton. ROCK2 has been implicated in numerous pathologies and the current focus is on understanding the molecular events that couple ROCK2 activity to biological function. To aid in the search for new ROCK2 substrates, we have developed an analog-sensitive (AS) ROCK2 protein that allows the use of selective ATP analogs that are not efficiently utilized by other protein kinases.
  
  
  RESULTS
  The analog sensitive protein, M160A ROCK2, was highly active and could phosphorylate proteins from a cellular homogenate with γ32P-N6 (benzyl)ATP. We show the utility of this approach by identifying a putative ROCK2 substrate, elongation initiation factor-1-α1. We further show that the major site of ROCK2 phosphorylation of EIF1α1 is Thr432.
  
  
  CONCLUSIONS
  Our work demonstrates that AS-ROCK2 could be useful in a systematic proteomic approach for identifying novel ROCK2 substrates.
authors.forename: Amber L
authors.initials: AL
authors.lastname: Couzens
authors.forename: R Montgomery
authors.initials: RM
authors.lastname: Gill
authors.affiliation: Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J1P3, Canada. mscheid@yorku.ca.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Scheid
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D019943:Amino Acid Substitution; D015977:Eukaryotic Initiation Factor-1 / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D015977:Eukaryotic Initiation Factor-1; C026022:eukaryotic peptide initiation factor-1A; D000255:Adenosine Triphosphate; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases