ROK alpha (Rho-associated, coiled-coil containing protein kinase 2, p150 serine/threonine kinase, ROCK-2, ROCK-II, Rho-associated kinase-alpha, Rho-kinase)

The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kD, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.\n\nApplications: \nSuitable for use in Western Blot and Immunoprecipitation. Other applications not tested.\n\nRecommended Dilution:\nWestern Blot: 1-2ug/ml of this lot detected ROKa/ROCK-II in RIPA lysates from PC-12 cells.\nImmunoprecipitation: 4ug of this lot immunoprecipitated ROKa/ROCK-II from 500ug of PC-12 RIPA lysate.\nOptimal dilutions to be determined by the researcher.\n\nRecommended Secondary Antibodies\nI1904-39: IgG, X-Adsorbed (HRP) Pab Gt xRb\nI1904-40A: IgG, H&L, X-Adsorbed (HRP) Pab Gt xRb\nI1904-46Q: IgG, H&L (HRP) Pab Gt xRb\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. For long-term storage, store at -20 degrees C. Aliquots are stable for at least 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

R2998-26

Size

200ug

Applications

IP, WB

Hosts

Rabbit

Reactivities

Rat

Form

Supplied as a liquid in 0.1M Tris-glycine, pH 7.4, 0.15M sodium chloride, 0.05% sodium azide before the addition of glycerol to 30%.

P Type

Pab

Purity

Purified by Protein A affinity chromatography.

Isotype

IgG

References

1. Leung,T., et al., Mol. Cell. Biol. 16: 5313-5327, 1996. 2. Vincent, S., et al., Mol. Cell. Biol. 17: 2247-2256, 1997. 3. Begum, N., et al., J. Biol. Chem. 277: 6214-6222, 2002. 4. Peck, J., et al., J. Biol. Chem. 277: 43924-43932, 2002.

Additional Info

Recognizes rat ROKa/ROCK-II, Mr ~150kD and an unknown protein, Mr ~51kD, both of which were inhibited by the fusion protein. Species sequence homology: mouse, bovine, and human.