A Novel Serine/Threonine Kinase Binding the Ras-related RhoA GTPase Which Translocates the Kinase to Peripheral Membranes

doi:10.1074/jbc.270.49.29051

A Novel Serine/Threonine Kinase Binding the Ras-related RhoA GTPase Which Translocates the Kinase to Peripheral Membranes (*)

From the (1)Glaxo-IMCB Group, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511
(2)Institute of Neurology, 1 Wakefield St., London WC1N 1PJ, United Kingdom

() To whom correspondence should be addressed:
Glaxo-IMCB Group, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511.
Tel.:65-772-6167; Fax: 65-774-0742.

Abstract

We previously reported the cloning of a serine/threonine kinase, PAK (for p21 (Cdc42/Rac)-activated kinase), which binds to the Ras-related GTPases Cdc42Hs and Rac1 (Manser, E., Leung, T., Salihuddin, H., Zhao, Z-s., and Lim, L.(1994) Nature 367, 40-46). These p21 proteins together with RhoA comprise the Rho subfamily of proteins that are involved in morphological events. We now report the isolation of a rat cDNA encoding a 150-kDa protein, which specifically binds RhoA in its GTP form and contains an N-terminal serine/threonine kinase domain highly related to the human myotonic dystrophy kinase and a cysteine-rich domain toward the C terminus. The RhoA binding domain is unrelated to other p21 binding domains. Antibody raised against the kinase domain of the predicted protein, termed ROKα (for ROKα, RhoA-binding kinase), recognized a ubiquitous 150-kDa protein. The brain p150 purified by affinity chromatography with RhoA exhibited serine/threonine kinase activity. In cultured cells, immunoreactive p150 was recruited to membranes upon transfection with dominant positive RhoA mutant and was localized with actin microfilaments at the cell periphery. These results are consistent with a role for the kinase ROKα as an effector for RhoA.

Footnotes

↵(*) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38481[GenBank].
↵¹ The abbreviations used are:

ACK

activated Cdc42Hs-associated kinase

PAK

p21 (Cdc42/Rac)-activated kinase

ROKα

RhoA-binding kinase

GST

glutathione S-transferase

MES

4-morpholineethanesulfonic acid

HA

hemagglutinin

FBS

fetal bovine serum

DMEM

Dulbecco's modified Eagle's medium

GTP S

guanosine 5′-3-O-(thio)triphosphate

rbf

Rho-binding DNA fragment.
↵²T. Leung and L. Lim, unpublished observations.
- Received August 28, 1995.
- Revision received October 5, 1995.