Figure 4: Membrane binding and kinase activity of ROCK2.

(a) ROCK2 binds to membranes in vitro. Folch liposomes containing 0.5 mg ml⁻¹ Folch brain lipids were sufficient to bind >90% of ROCK2. (b) Recombinant ROCK2 is active against the substrate MLC2 (black curve), but activity is not influenced by membrane binding (red curve). Error bars are the s.d. of three independent measurements. (c) Fluorescence anisotropy binding assay. ROCK1 and ROCK2 Rho-binding domains do not bind to RhoA·GMPPNP, even at very high protein concentrations. The Dbs exchange factor for RhoA binds to nucleotide-free RhoA (1 mM EDTA) with a binding constant of 354 nM, compared with a 60-fold weaker binding affinity for nucleotide-loaded RhoA (2 mM Mg²⁺). Error bars are the s.d. of 20 measurements. (d) RhoA does not bind to ROCK2 in the context of membranes. RhoA was incubated with ROCK2 bound to Folch liposomes in a liposome pelleting assay. RhoA is exclusively in the supernatant, while ROCK2 is exclusively in the pellet, indicating that membrane binding by ROCK2 does not facilitate its interaction with RhoA. (e) Schematic illustrating ROCK2 constructs used in f–h. (f) Cellular phenotype of overexpression of an RBD mutant of ROCK2. Immunostaining of ΔROCK2 NIH3T3 fibroblasts expressing ROCK2^{N1027S K1028E E1031K}-EGFP shows normal stress fibres. (g) The RBD mutant has activity indistinguishable from ROCK2^WT. (h) Cells overexpressing the RBD of ROCK2 (ROCK2^929–1,053) also exhibit normal stress fibres.