Figure 2: ROCK2 coiled-coil length regulates activity in vivo but not in vitro.

(a) Schematic illustrating ROCK2 constructs used in b–e. (b) NIH3T3 ΔROCK2 cells expressing ROCK2^WT have stress fibres indistinguishable from untransfected cells. (c) ROCK2^K112A (kinase-dead)-expressing cells exhibit a dominant-negative phenotype characterized by the complete loss of stress fibres, while untransfected cells show normal stress fibres. (d) Cells expressing ROCK2^409–1,379, a construct comprising the entire coiled-coil and the membrane-binding C1 and PH domains, also exhibit the complete loss of stress fibres. (e) Cells overexpressing ROCK2^{1–409 K112A}, a kinase-dead construct of the catalytic domain alone, exhibit normal stress fibres. (f) Schematic illustrating truncation constructs of ROCK2 used in g–l. (g) NIH3T3 ΔROCK2 fibroblasts expressing ROCK2 truncated by just 2 nm show normal stress fibres. (h) NIH3T3 ΔROCK2 fibroblasts expressing ROCK2 truncated in its coiled-coil by 5–60 nm (h–l) exhibit a complete loss of stress fibres. (m) In vitro kinase assay of ROCK2 mutants. Immunoblot of pS¹⁹ MLC2 following incubation with EGFP-tagged ROCK2 immobilized in a GFP-Trap 96-well plate. ROCK2^WT is active against MLC2, while kinase-dead ROCK2^K112A is completely inactive. ROCK2^Δ2, ROCK2^Δ5, ROCK2^Δ10, ROCK2^Δ20, ROCK2^Δ30 and ROCK2^Δ60 are all active against MLC2, as is the isolated kinase domain (CAT). (n) In vitro kinase assay of ROCK2^wt, ROCK2^Δ60 and ROCK2^CAT. All three constructs show the same specific activity against MLC2. Error bars are the s.d. of three independent measurements. (o) Rotary shadowed electron microscopy of ROCK2^Δ60. ROCK2^Δ60 is an extended particle with a coiled-coil length of 46.5±1.5 nm (n=10). Asymmetry is illustrated by four particles with a single region of electron density at one end of the particle and two clear regions of electron density at the other.