Biochem. J. (2009) 419
(141–148) (Printed in Great Britain)
The hydrophobic motif of ROCK2 requires association with the N-terminal extension for kinase activity
Amber L. COUZENS, Vivian SARIDAKIS and Michael P. SCHEID1
Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada, M3J 1P3
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.
Key words: cytoskeleton, protein kinase, Rho, Rho-associated coiled-coil kinase (ROCK), signal transduction.
Abbreviations used: CRIK, citron Rho-interacting kinase; DMEM, Dulbecco's modified Eagle's medium; DMPK, dystrophia myotonica protein kinase; HA, haemagglutinin; HEK, human embryonic kidney; HM, hydrophobic motif; LDS, lithium dodecyl sulfate; LIMK, LIM kinase; MBP, myelin basic protein; MLC, myosin light chain; MRCK, myotonic dystrophy kinase-related Cdc42-binding kinase; PH, pleckstrin homology; PKB, protein kinase B; RBD, Rho-binding domain; ROCK, Rho-associated coiled-coil kinase; ROK, Rho-associated kinase.
1To whom correspondence should be addressed (email mscheid@yorku.ca).
Received 8 July 2008/9 December 2008; accepted 22 December 2008
Published as BJ Immediate Publication 22 December 2008, doi:10.1042/BJ20081376
© The Authors Journal compilation © 2009 Biochemical Society
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