ROCK II in vitro and in vivo cleavage by purified grB. (a) ROCK II or ROCK I was immunoprecipitated from MCF-7 cells, and then incubated with the indicated concentrations of grB for 60 min at 37°C before performing Western blot analysis of ROCK II (left) and ROCK I (right). Western blot analysis of Jurkat cells, either left untreated (Jurkat) or treated with an agonistic anti-Fas antibody for 6 h (Jurkat Fas 6h) was used for controls. (b) Purified grB was introduced into MCF-7 cells treated with Chariot reagent. At the indicated times (minutes) after grB addition, cells were lysed and ROCK II was analyzed by Western blotting using clone 21 antibody.

Properties of cleaved ROCK II. (a) MCF-7 cells were either left untransfected (lanes A, B, and J) or transiently transfected with either wild-type (WT; lanes C, D, and K) or mutated D1131A (lanes E, F, and L) or truncated Δ1131 (lanes G, H, and M) ROCK II constructs tagged in NH₂ terminus with the AU1 peptide. 22 h after transfection, in vitro pull-down assay was performed: lysates were incubated with GDP-bound (A, C, E, and G) or GTP-bound (B, D, F, and H) GST-RhoA, and precipitated proteins were analyzed by Western blotting with anti-AU1 (top) or anti–ROCK II COOH terminus (bottom) antibody. Controls are Western blot analysis of GST Rho alone (I) and lysates of cells left untransfected (J) or transfected with either WT (K) or D1131A (L) or Δ1131 (M) AU-1–tagged constructs, indicating equal expression of the three transfected constructs in the tested cells. The blot was reprobed with the anti–ROCK II COOH terminus antibody to verify homogenous pull-down efficiency. (b) In vitro kinase assay. 293T cells were either left untransfected (lanes A and E) or transiently transfected with either WT (lanes B and F) or truncated Δ1131 (lanes C and G) AU1-tagged ROCK II constructs. 24 h later, cells were lysed, and exogenous ROCK II proteins were immunoprecipitated using the anti-AU1 antibody (IP AU1) and assayed for kinase activity in vitro with myelin basic protein as a substrate (bottom, IVK, in vitro kinase assay). Beads alone were used as a control (lane D, Ac beads). The efficiency of immunoprecipitation was determined by Western blotting using an anti-AU1 antibody (top, lanes A–D) with simultaneous analysis of constructs expression with an aliquot of cell lysates as controls (lanes E–G).

Granzyme B–deficient murine cytotoxic cells fail to induce ROCK II cleavage in their target. IL-2 activated splenocytes from WT or grB-deficient (gzmb−/−) mice were incubated with YAC-1 targets for the indicated times at a 2:1 ratio before cells lysis and analysis of ROCK I and ROCK II cleavage by Western blotting. Hsc70 was used as a loading control.

ROCK II cleavage during cytotoxic granule-mediated apoptosis. (a) K562 cells incubated with LAK cells for 5 h in the presence of 20 μM z-VAD-fmk, and then stained with 5 μg ml⁻¹ Hoechst 33342. Photomicrographs of the same field were obtained by phase-contrast microscopy (left) and by microscopy with ultraviolet irradiation (right). Target cells are much larger than LAK cells. (arrows) Blebbing target cells; no chromatin condensation is detected as caspases are inhibited. (b) Western blot analysis of the indicated proteins (MLC, myosin regulatory light chain; MLC-P, phosphorylated MLC; Casp-3, caspase-3 active fragments) in lysates of K562 cells exposed for the indicated times to LAK cells in the presence or absence of 20μM z-VAD-fmk. (c) Western blot analysis of the indicated proteins in lysates of K562 cells exposed to LAK cells in the presence or absence of 10 μM Y-27632 and in the presence or absence of 20 μM z-IETD-fmk. (b and c) Molecular mass is indicated in kilodaltons. The anti-MLC antibody was used also as the loading control. Antibodies used include H-85 anti–ROCK I and clone 21 anti–ROCK II. Results are representative of three independent experiments. (d) Quantification of the cleavage fragments. Images of the cleavage bands of ROCK I and ROCK II were acquired by densitometry scanning (Gel Doc; Bio-Rad Laboratories) and analyzed with Quantity One software. Band intensity relative to the baseline signal (set as 1) is shown in arbitrary units.

Identification of ROCK II cleavage site. (a) Schematic representation of the structure of ROCK I and ROCK II. BD, Rho-binding domain; PH, pleckstrin homology domain. DETD¹¹¹³ has been demonstrated to be a caspase-3–mediated cleavage site in ROCK I (reference 7). (b) MCF-7 cells were transiently transfected with either a wild-type (WT) or a mutated (D1131A) ROCK II (the two constructs were tagged in the NH₂ terminus with the AU1 peptide). 20 h after transfection, AU-1–tagged proteins were immunoprecipitated, exposed to 5 nM grB for 60 min, and analyzed by Western blotting using an anti-AU1 antibody. Western blot analysis of transiently transfected cell lysates was used as the control.

Functional activity of cleaved ROCK II. (a) 293T cells were transiently cotransfected with ROCK II and EGFP plasmids in the presence of 40μM z-VAD-fmk for 20 h. Images were obtained by phase-contrast (right) and fluorescence (left) confocal microscopy of identical fields for each population. (top) ΔROCK II; (bottom) WT ROCK II. (b) 293T cells were either left untransfected or transiently transfected with the indicated ROCK II constructs in the absence or presence of 10 μM Y-27632 or 50 μg/ml TAT-C3 for 20 h. Then, lysates were probed with antibodies against phosphorylated MLC (MLC-P), MLC (charge control), or AU1 (transfection efficiency control). (bottom) A gel shift assay performed with an anti-RhoA antibody that identifies non–ADP-ribosylated (Rho) and ADP-ribosylated (Rho*) Rho.