New insights into the structure-function relationships of Rho-associated kinase: a thermodynamic and hydrodynamic study of the dimer-to-monomer transition and its kinetic implications

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.

Figure 1. (A) Schematic representation of the predicted functional domains of ROCK I, and (B) SDS/PAGE of purified full-length ROCK I and II and the four truncated ROCK I constructs

(A) The limits of the four ROCK truncations are shown below the full-length sequence. Both full-length ROCK I (1354 amino acids) and ROCK II (1388 amino acids) and the four ROCK I constructs were cloned and expressed in insect cells. (B) Standard protein markers (lane 1) in the order of increasing molecular mass are: lysozyme (14.4 kDa), soybean trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), BSA (66.2 kDa), and phosphorylase b (97.4 kDa). The identities of the sample proteins are: lane 2, full-length ROCK II; lane 3, S6–L553; lane 4, S6–R415; lane 5, S6–E379; lane 6, M71–E379; lane 7, full–length ROCK I.

Figure 2. SEC chromatograms of full-length and truncated ROCK constructs

(A) Full-length ROCK II, (B) S6–L553, (C) S6–R415, (D) S6–E379, (E) M71–E379. The shaded portion in (D) represents the area integrated to give a value of 90000 Da for the dimeric portion of S6–E379.

Figure 3. Sedimentation velocity experiments with five ROCK constructs

Plots of the apparent sedimentation coefficient g(s*) against the sedimentation coefficient s_20,w corrected to the viscosimetric properties of water at 20 °C are shown. They were fitted using the DCDT+ program to models representing one or two sedimenting species. The circles represent the experimental data. The solid lines represent the best fit and are the sum of the contributions to the sedimenting boundary from individual species (dashed and dotted lines). (A) Full-length ROCK II, (B) S6–L553, (C) S6–R415, (D) S6–E379, (E) M71–E379.

Figure 4. Sedimentation equilibrium experiments with full-length ROCK II

The data are shown as absorbance against radial position at 20 °C. The raw data (○, 8000 rev./min; □, 10000 rev./min; ◇, 12000 rev./min) and the fit (lines) are shown. The distribution of the residuals for each of these fits is shown in the upper panels.

Figure 5. SDS/PAGE of cross-linking of ROCK with DFDNB

Standard protein markers were as in Figure 1(b). The three indicated constructs were incubated with either 0.1 or 0.5 mM DFDNB for 15 min, then the reaction was quenched with SDS sample buffer. Arrows indicate where the dimers of each construct are expected to appear.