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Membrane Blebbing During Apoptosis Results from Caspase-Mediated Activation of ROCK I

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Membrane Blebbing During Apoptosis Results from Caspase-Mediated Activation of ROCK I articles Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I Mathew L. Coleman*†, Erik A. Sahai*†, Margaret Yeo*, Marta Bosch*, Ann Dewar‡ and Michael F. Olson*§ *CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK ‡National Heart and Lung Institute, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. †These authors contributed equally to this work §e-mail: [email protected] The execution phase of apoptosis is characterized by marked changes in cell morphology that include contrac- tion and membrane blebbing. The actin–myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin–myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phos- phorylation of myosin light-chains, myosin ATPase activity and coupling of actin–myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies. he evolutionarily conserved execution phase of apoptosis is Results characterized by events that occur during the final stages of ROCK activity is necessary for membrane blebbing. We created a Tdeath, including cell contraction, dynamic membrane bleb- cell-penetrating form of the Clostridium botulinum C3 toxin, which bing and DNA fragmentation. The distinct morphological trans- catalyses the specific ADP ribosylation and inactivation of Rho18,by formation is one of the earliest described and most obvious aspects fusing a portion of the human immunodeficiency virus Tat-coding of apoptosis, but is also perhaps the least well characterized. sequence to the N terminus of C3; we expressed and purified the Contractile force generated by actin–myosin cytoskeletal structures resulting Tat–C3 toxin in Escherichia coli. We pretreated NIH 3T3 is thought to drive the formation of membrane blebs and apoptot- mouse fibroblasts with this toxin for 18 h and then subjected them ic bodies1–3, however, the biochemical pathway that promotes the to the apoptotic stimulus of 0.5 µM recombinant mouse tumour- generation of this force during apoptosis has not been identified. necrosis factor-α (TNFα) plus 10 µgml–1 cycloheximide (CHX). The Rho GTPases (RhoA, B and C) are intracellular signalling Although pretreatment with Tat–C3 was sufficient to modify all molecules that regulate the actin cytoskeleton4. Upon activation, Rho detectable RhoA and to block serum-stimulated formation of stress proteins exchange GDP for GTP, transduce signals to downstream fibres (data not shown), Tat–C3 did not affect the generation of effector proteins and finally return to the inactive GDP-bound form membrane blebs after treatment with TNFα, compared with cells by hydrolysing the bound GTP. Two isoforms of a serine/threonine treated with TNFα alone (Fig. 1a). In contrast, inhibition of ROCK kinase (Rho-associated kinases ROCK I and ROCK II) have been activity using the small-molecule inhibitor Y-27632 (ref. 19) result- identified as effectors of Rho5–7. ROCK proteins bind to and are acti- ed in a marked reduction in formation of membrane blebs (Fig. vated by GTP-bound Rho. Deletion of the carboxy-terminal region 1a), although apoptotic cells still exhibited a rounded morphology of ROCK activates the amino-terminal kinase domain both in vitro compared with untreated cells. Expression of a dominant negative and in vivo6,8–10. Activation of ROCK proteins contributes positively form of ROCK I8 similarly reduced membrane blebbing during to the stabilization of filamentous actin, phosphorylation of myosin apoptosis (data not shown). Scanning electron microscopy of light chains, myosin ATPase activity and coupling of actin–myosin TNFα-treated cells revealed that, although the morphology of filaments to the plasma membrane, leading to increased membrane blebs was altered by pretreatment with Tat–C3, the actin–myosin force generation and cell contractility11–17. number of blebs was not significantly reduced (Fig. 1b). Cells treat- As Rho and ROCK have been shown to contribute both to the ed with Y-27632, however, had very few large blebs and exhibited formation of actin cytoskeletal structures and to consequent con- only small protrusions emanating from the cell surface (Fig. 1b). tractile force generation, we examined the role of this signalling Consistent with the inability of Tat–C3 to inhibit blebbing, only a pathway in the development of the apoptotic morphology. Here we small and transient activation of RhoA was observed after treat- show that the occurrence of apoptotic membrane blebbing is ment with TNFα plus CHX in a pull-down assay using the Rho- dependent on the function of ROCK but not on that of Rho. ROCK binding domain from the Rho effector Rhotekin20,21 (Fig. 1c). I, but not ROCK II, is cleaved during apoptosis by activated caspas- Activation of ROCK I by caspase-mediated cleavage. During apop- es, generating a truncated kinase with increased intrinsic activity. tosis, a class of cysteine proteases called caspases act as effectors of This cleaved form of the kinase is sufficient to drive cell contraction the cell-death programme22. One mechanism by which caspases and membrane blebbing without caspase activation, which is con- promote apoptosis is through cleavage and subsequent activation sistent with a direct effect of ROCK-induced cell contractility on of protein kinases23. Western blotting of whole-cell lysates prepared the generation of the apoptotic morphology. Finally, we show that from untreated or TNFα/CHX-treated NIH 3T3 cells showed no ROCK activity and consequent membrane blebbing are required detectable cleavage of ROCK II (Fig. 2a) but significant cleavage of for redistribution of fragmented DNA from the nuclear region into ROCK I, as well as cleavage of the well characterized caspase sub- membrane blebs and apoptotic bodies. strate poly-ADP ribose polymerase (PARP) after treatment with NATURE CELL BIOLOGY VOL 3 APRIL 2001 http://cellbio.nature.com 339 © 2001 Macmillan Magazines Ltd articles a Control Tat–C3 Y-27632 TNFα TNFα + Tat–C3 TNFα + Y-27632 b TNFα TNFα + Tat–C3 TNFα + Y-27632 c RhoA–GTP 1/20 total RhoA 0 2 10 30 60 90 Time after TNFα stimulation (min) Figure 1 Membrane blebbing during apoptosis is Rho-independent but as in a. Scale bars represent 2.5 µm. c, Activation of RhoA by TNFα. Upper panel, ROCK-dependent. a, Phase-contrast micrographs of NIH 3T3 mouse fibroblasts. RhoA–GTP pulled down by association with the Rho-binding domain of Rhotekin at Where indicated, cells were pretreated with 0.5 µM Tat–C3 for 18 h, treated with the indicated times after TNFα stimulation; lower panel, 1/20 fraction of the total 25 ng ml–1 TNFα and 10 µgml–1 cycloheximide for 2 h (TNFα), and/or treated with RhoA per lysate. 10 µM Y-27632 for 2 h. b, Scanning electron micrographs of NIH 3T3 cells treated TNFα (Fig. 2b). Similar patterns of ROCK I cleavage were observed DETA (ROCK(D1113A)) and DEQA (ROCK(D1158A)), respec- in Swiss 3T3 mouse fibroblasts and in MCF7 human breast carci- tively, by site-directed mutagenesis and analysed them for cleavage noma, MCF10A human breast epithelial and HA1 human embry- by transfecting them into NIH 3T3 cells and then treating the cells onic kidney cells after treatment with TNFα and CHX (data not with TNFα. Wild-type and D1158A ROCK I were cleaved to simi- shown). The time course of ROCK I cleavage, which preceeded the lar extents, whereas cleavage of ROCK(D1113A) was not detected generation of membrane blebs, paralleled that of PARP (Fig. 2b). (Fig. 3a). The cleaved forms of wild-type and D1158A ROCK I had Cleavage of both ROCK I and PARP was inhibited by pretreatment the same apparent relative molecular weight as a mutant with an with the caspase inhibitor z-VAD-fmk (Fig. 2c). opal stop codon substituted for the glycine that is situated after the Treatment with TNFα of NIH 3T3 cells transfected with a plas- DETD (1110–1113) caspase-cleavage site (ROCK I(G1114opa)). As mid encoding human ROCK I with an N-terminal Myc tag, fol- the C-terminal region of ROCK I has been reported to act as an lowed by western blotting with the anti-Myc monoclonal antibody auto-inhibitory domain6,8–10, we compared the in vitro kinase activ- 9E10 revealed that cleavage occurred ~200 amino acids from the C ities of full-length wild-type ROCK I and the truncated G1,114opa terminus of ROCK I (Fig. 3a and data not shown). Two potential mutant. Removal of the C-terminal 241 amino acids increased the sites of caspase cleavage, resembling the prototypical caspase-3 phosphorylation of histone H1 by a factor of 7.8 ± 0.5 relative to cleavage sequence DEVD, are situated in this region in both human the activity of full-length ROCK I (Fig. 3b). In addition, there was and mouse ROCK I, at positions 1110–1113 (DETD) and no detectable autophosphorylation of ROCK I(G1114opa), such as 1155–1158 (DEQD), respectively. We changed these motifs to was observed for full-length ROCK I (Fig. 3b). Mutation to alanine 340 NATURE CELL BIOLOGY VOL 3 APRIL 2001 http://cellbio.nature.com © 2001 Macmillan Magazines Ltd articles a a Untransfected Wild-type Myc–ROCK I Myc–ROCK I ROCK I ROCK I ROCK II Myc–ROCK I (D1,113A) (D1,158A) (G1,114opa) PARP TNFα – + – + TNFα – + – + – + ––+ b b Immunoprecipitation In vitro kinase assay western blot ROCK I Myc– ROCK I – ROCK I (G1,114opa) PARP Myc– ROCK I – ROCK I (G1,114opa) ROCK I 0 15 30 60 90 120 150 180 Time after TNFα stimulation (min) c ROCK I IP: 9E10 Histone H1 WB: 9E10 PARP Relative kinase activity: 1.0 7.8 ± 0.5 Figure 3 Caspase-cleavage of ROCK I generates a truncated form with higher intrinsic kinase activity.
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