Abstract
The effect of the length of ROCK (Rho-associated kinase) on its oligomerization state has been investigated by analysing full-length protein and four truncated constructs using light-scattering and analytical ultracentrifugation methods. Changes in size correlate with the kinetic properties of the kinase. Sedimentation velocity, sedimentation equilibrium and light-scattering data analyses revealed that protein constructs of size Ser6-Arg415 and larger exist predominantly as dimers, while smaller constructs are predominantly monomeric. The amino acid segments comprising residues 379-415 and 47-78 are shown to be necessary to maintain the dimeric ROCK structure. kcat values ranged from 0.7 to 2.1 s(-1) and from 1.0 to 5.9 s(-1) using ROCK peptide (KKRNRTLSV) and the 20000 Da subunit of myosin light chain respectively as substrate, indicating that the effect of the ROCK oligomerization state on the kcat is minor. Values of ATP K(m) for monomeric constructs were increased by 50-80-fold relative to the dimeric constructs, and K(i) comparisons using the specific competitive ROCK inhibitor Y-27632 also showed increases of at least 120-fold, demonstrating significant perturbations in the ATP binding site. The corresponding K(m) values for the ROCK peptide and myosin light chain substrates increased in the range 1.4-16-fold, demonstrating that substrate binding is less sensitive to the ROCK oligomerization state. These results show that the oligomerization state of ROCK may influence both its kinase activity and its interactions with inhibitors, and suggest that the dimeric structure is essential for normal in vivo function.