Figure 3: ROCK2 activity in vivo is coiled-coil sequence independent.
From: A molecular ruler regulates cytoskeletal remodelling by the Rho kinases
(a) Schematic illustrating the construction of a ROCK2 chimera. The coiled-coil region of human ROCK2 (amino acids 414–1,141) was replaced with the corresponding region of Drosophila melanogaster (amino acids 418–1,133). A kinase-dead chimera contains the same inactivating mutation (K112A) as the kinase-dead human ROCK2. (b) NIH3T3 ΔROCK2 cells expressing the ROCK2 chimera show stress fibres indistinguishable from untransfected cells. (c) Expression of the kinase-dead chimera results in complete loss of stress fibres. (d) Fluorescence size exclusion chromatography of human ROCK2 and chimeric ROCK2 shows them to elute with identical retention volumes, indicating that the chimera is soluble and correctly folded. (e) In vitro kinase assay confirms that the ROCK chimera isolated from NIH3T3 ΔROCK2 cells is active against MLC2, while its kinase-dead counterpart is not.
