Product Pathways - Kinases
ROCK1 Kinase #7532
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
Description
Purified recombinant human ROCK1 (Leu17-Leu535) kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system using sf9 cells and a recombinant virus encoding human ROCK1 (Leu17-Leu535) (GenBank Accession No. NM_005406) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using GSH-agarose.
Gel Staining
Figure 1. The purity of the GST-ROCK1 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. ROCK1 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: S6K substrate peptide (KRRRLASLR) 400 ng/μL, and recombinant ROCK1: variable.
Quality Control
The theoretical molecular weight of the GST-ROCK1 fusion protein is 85 kDa. The purity of the kinase was assessed using SDS-PAGE followed by Coomassie stain [Fig.1]. ROCK1 kinase activity was determined using a radiometric assay [Fig.2].
Background
ROCK (Rho-associated kinase), a family of serine/threonine kinases, is an important downstream target of GTPase Rho and plays an important role in Rho-mediated signaling. Two isoforms of ROCK have been identified (ROCK1 and ROCK2). ROCK is composed of N-terminal catalytic, coiled-coil and C-terminal PH (pleckstrin homology) domains. The C-terminus of ROCK negatively regulates its kinase activity (1,2). Caspase-3-induced cleavage of ROCK1 and direct cleavage of ROCK2 by granzyme B (grB) activates ROCK and leads to phosphorylation of myosin light chain and inhibition of myosin phosphatase (3). This phosphorylation may account for the mechanism by which Rho regulates cytokinesis, cell motility, cell membrane blebbing during apoptosis and smooth muscle contraction (4-6).
- Nakagawa, O. et al. (1996) FEBS Lett. 392, 189-193.
- Lee, J.H. et al. (2004) J. Cell. Biol. 167, 327-337.
- Sebbagh, M. et al. (2005) J. Exp. Med. 201, 465-471.
- Amano, M. et al. (1996) J. Biol. Chem. 271, 20246-20249.
- Kureishi, Y. et al. (1997) J. Biol. Chem. 272, 12257-12260.
- Totsukawa, G. et al. (2000) J. Cell Biol. 150, 797-806.
Application References
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Companion Products
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
