ROCK (Rho-associated coiled-coil kinase) 2 is a member of the AGC kinase family that plays an essential role downstream of Rho in actin cytoskeleton assembly and contractility. The process of ROCK2 activation is complex and requires suppression of an autoinhibitory mechanism that is facilitated by Rho binding. ROCK2 harbours a C-terminal extension within the kinase domain that contains a hydrophobic cluster of phenylalanine and tyrosine residues surrounding a key threonine residue. In growth-factor-stimulated AGC kinases, the hydrophobic motif is important for the transition of the kinase from inactive to active complex and requires phosphorylation of the conserved serine/threonine residue. Less is understood about the contribution that the hydrophobic motif plays in the activation of ROCK, and the role of the hydrophobic motif threonine at position 405. In the present study, we show that this residue of ROCK is essential for substrate phosphorylation and kinase domain dimerization. However, in contrast with the growth-factor-activated AGC kinases, a phosphomimetic residue at position 405 was inhibitory for ROCK2 activity and dimerization. A soluble hydrophobic motif peptide allosterically activated ROCK2 In vitro, but not the equivalent peptide with Asp405 substitution. Mechanistically, both ROCK2 activity and dimerization were dependent upon the interaction between Thr405 of the hydrophobic motif and Asp39 of the N-terminal extension. The reciprocal exchange of these residues was permissive for kinase activity, but dimerization was lost. These results support the rationale for development of small-molecule inhibitors designed to block ROCK activation by selectively interfering with hydrophobic motif-mediated activation-state transition and dimer formation.
Skip Nav Destination
Article navigation
April 2009
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
March 13 2009
The hydrophobic motif of ROCK2 requires association with the N-terminal extension for kinase activity
Amber L. Couzens;
Amber L. Couzens
1Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada, M3J 1P3
Search for other works by this author on:
Vivian Saridakis;
Vivian Saridakis
1Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada, M3J 1P3
Search for other works by this author on:
Michael P. Scheid
Michael P. Scheid
1
1Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada, M3J 1P3
1To whom correspondence should be addressed (email mscheid@yorku.ca).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
July 08 2008
Revision Received:
December 09 2008
Accepted:
December 22 2008
Accepted Manuscript online:
December 22 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 419 (1): 141–148.
Article history
Received:
July 08 2008
Revision Received:
December 09 2008
Accepted:
December 22 2008
Accepted Manuscript online:
December 22 2008
Citation
Amber L. Couzens, Vivian Saridakis, Michael P. Scheid; The hydrophobic motif of ROCK2 requires association with the N-terminal extension for kinase activity. Biochem J 1 April 2009; 419 (1): 141–148. doi: https://doi.org/10.1042/BJ20081376
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.