RhoE (Rho-related GTP Binding Protein, Rnd3)

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes. Phosphorylation has no effect on RhoE binding to ROCK I, but instead increases RhoE protein stability. Using phospho-specific antibodies, we show that ROCK phosphorylates endogenous RhoE at serine 11 upon cell stimulation with platelet-derived growth factor, and that this phosphorylation requires an active protein kinase C signalling pathway. In addition, we demonstrate that phosphorylation of RhoE correlates with its activity in inducing stress fibre disruption and inhibiting Ras-induced transformation. This is the first demonstration of an endogenous Rho family member being phosphorylated in vivo and indicates that phosphorylation is an important mechanism to control the stability and function of this GTPase-deficient Rho protein.\nApplications: \nSuitable for use in Western Blot and Immunocytochemistry. Other applications have not been tested. \n\nRecommended Dilution:\nWestern Blot: 1:500-1:2000. Detected RhoE/Rnd3 in RIPA lysates from 3T3/A31 cells.\nImmunocytochemistry: 1:50. Has been reported by an independent laboratory to show positive immunostaining for RhoE/Rnd3 in 3T3/A31 cells fixed with paraformaldehyde and permeabilized by Triton X-100.\nOptimal dilutions to be determined by the researcher. \n\nIncluded Positive Antigen Control:\nF4209: Fibroblast Cell, 3T3/A3. Add 2.5ul of 2-mercaptoethanol/100ul of lysate and boil for 5 minutes to reduce the preparation. Load 20ug of reduced lysate per lane for minigels.\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.\n

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SPECIFICATIONS

Catalog Number

R1998-13

Size

200ul

Applications

ICC, WB

Hosts

Mouse

Reactivities

Hum, Mouse

Form

Supplied as a liquid, 0.05% sodium azide, before the addition of glycerol to 30%.

P Type

Mab

Purity

Ascites

Isotype

IgG

References

1. Riento, K., et al., Mol. Cell. Biol. 23: 4219-4229, 2003. 2. Foster, R., et al., Mol. Cell. Biol. 16: 2689-2699,1996. 3. Guasch, R.M., et al., Mol. Cell. Biol. 18: 4761-4771,1998. 4. Nobes, C.D., et al., J. Cell. Biol. 141: 187-197, 1998. 5. Hansen, S.H., et al., Mol. Cell. Biol. 20: 9364-9375, 2000. 6. Katoh, H., et al., Mol. Cell. Biol. 22: 2952-2964, 2002. 7. Garavini, H.E., et al., Biochemistry 41: 6303-6310, 2002.

Additional Info

Recognizes mouse RhoE/Rnd3, Mr ~29kD. Weakly crossreacts with Rnd1, Mr ~27kD. Species Crossreactivity: human.