PMID- 24405853
OWN - NLM
STAT- MEDLINE
DCOM- 20140730
LR  - 20211021
IS  - 1472-6750 (Electronic)
IS  - 1472-6750 (Linking)
VI  - 14
DP  - 2014 Jan 9
TI  - Characterization of a modified ROCK2 protein that allows use of N6-ATP analogs 
      for the identification of novel substrates.
PG  - 2
LID - 10.1186/1472-6750-14-2 [doi]
AB  - 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 gamma32P-N6 
      (benzyl)ATP. We show the utility of this approach by identifying a putative ROCK2 
      substrate, elongation initiation factor-1-alpha1. We further show that the major site 
      of ROCK2 phosphorylation of EIF1alpha1 is Thr432. CONCLUSIONS: Our work demonstrates 
      that AS-ROCK2 could be useful in a systematic proteomic approach for identifying 
      novel ROCK2 substrates.
FAU - Couzens, Amber L
AU  - Couzens AL
FAU - Gill, R Montgomery
AU  - Gill RM
FAU - Scheid, Michael P
AU  - Scheid MP
AD  - Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J1P3, 
      Canada. mscheid@yorku.ca.
LA  - eng
GR  - Canadian Institutes of Health Research/Canada
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20140109
PL  - England
TA  - BMC Biotechnol
JT  - BMC biotechnology
JID - 101088663
RN  - 0 (Eukaryotic Initiation Factor-1)
RN  - 0 (eukaryotic peptide initiation factor-1A)
RN  - 8L70Q75FXE (Adenosine Triphosphate)
RN  - EC 2.7.11.1 (ROCK2 protein, human)
RN  - EC 2.7.11.1 (rho-Associated Kinases)
SB  - IM
MH  - Adenosine Triphosphate/*analogs & derivatives
MH  - Amino Acid Substitution
MH  - Eukaryotic Initiation Factor-1/metabolism
MH  - HEK293 Cells
MH  - Humans
MH  - Mutagenesis, Site-Directed
MH  - Phosphorylation
MH  - Signal Transduction
MH  - rho-Associated Kinases/genetics/*metabolism
PMC - PMC3913329
EDAT- 2014/01/11 06:00
MHDA- 2014/07/31 06:00
PMCR- 2014/01/09
CRDT- 2014/01/11 06:00
PHST- 2013/09/04 00:00 [received]
PHST- 2014/01/07 00:00 [accepted]
PHST- 2014/01/11 06:00 [entrez]
PHST- 2014/01/11 06:00 [pubmed]
PHST- 2014/07/31 06:00 [medline]
PHST- 2014/01/09 00:00 [pmc-release]
AID - 1472-6750-14-2 [pii]
AID - 10.1186/1472-6750-14-2 [doi]
PST - epublish
SO  - BMC Biotechnol. 2014 Jan 9;14:2. doi: 10.1186/1472-6750-14-2.