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Cleavage and Nuclear Translocation of the Caspase 3 Substrate Rho GDP-Dissociation Inhibitor, D4-GDI, During Apoptosis

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Cleavage and Nuclear Translocation of the Caspase 3 Substrate Rho GDP-Dissociation Inhibitor, D4-GDI, During Apoptosis Cell Death and Differentiation (1999) 6, 412 ± 419 ã 1999 Stockton Press All rights reserved 13509047/99 $12.00 http://www.stockton-press.co.uk/cdd Cleavage and nuclear translocation of the caspase 3 substrate Rho GDP-dissociation inhibitor, D4-GDI, during apoptosis 1 ,1 Ronald J Krieser and Alan Eastman* Introduction 1 Department of Pharmacology and Toxicology, Dartmouth Medical School, Apoptosis plays a central role in such processes as Hanover, New Hampshire 03755, USA development, tissue homeostasis, and thymic selection, as * corresponding author: tel: (603)-650-1501; fax: (603)-650-1129; well as pathologies ranging from neurodegenerative disease, e-mail: [email protected] autoimmune disorders, and viral infection, to cancer. Much research on apoptosis has focused on determining proteins involved in decisions of cell fate, and regulation of the Received 17.11.98; revised 15.2.99; accepted 2.3.99 execution phase of cell death involving protease and Edited by G. Salvesen endonuclease activation. A great deal of this information has been gained from the study of small organisms such as Abstract the nematode C. elegans, which has a well-defined developmental program during which specific cells die. The While investigating endonucleases potentially involved in characterization of nematodes with mutations in the cell death apoptosis, an antisera was raised to bovine deoxyribonu- process has led to identification of genes which regulate clease II, but it recognized a smaller protein of 26 kDa protein apoptosis.1 The mammalian homologs have been identified in a variety of cell lines. The 26 kDa protein underwent for many of these regulatory genes. One gene which is central proteolytic cleavage to 22 kDa concomitantly with DNA to the execution phase of cell death is ced-3. This gene was digestion in cells induced to undergo apoptosis. Sequencing found to encode a homolog of the human cysteine protease 2 of the 26 kDa protein identified it as the Rho GDP- interleukin 1-b converting enzyme (ICE), and further studies dissociation inhibitor D4-GDI. Zinc, okadaic acid, calyculin led to the identification of a family of these cysteine proteases now termed caspases.3 A, cantharidin, and the caspase inhibitor z-VAD-fmk, all Caspases have a central role in the execution phase of prevented the cleavage of D4-GDI, DNA digestion, and apoptosis.2 Numerous substrates have been identified, apoptosis. The 26 kDa protein resided in the cytoplasm of including poly(ADP-ribose) polymerase,4 lamin A, lamin undamaged cells, whereas following cleavage, the 22 kDa B,5 DNA fragmentation factor (DFF/ICAD),6 70 kDa U1 form translocated to the nucleus. Human D4-GDI, and D4-GDI small ribonucleoprotein,7 and gelsolin.8 Although many mutated at the caspase 1 or caspase 3 sites, were expressed proteins are cleaved during apoptosis, a role has only in Chinese hamster ovary cells which show no detectable been identified for a few of them. ICAD has been found to endogenous D4-GDI. Mutation at the caspase 3 site prevented be cleaved by caspase 3 which leads to the release of an D4-GDI cleavage but did not inhibit apoptosis induced by endonuclease and subsequent DNA digestion observed 9,10 staurosporine. The cleavage of D4-GDI could lead to during apoptosis. Cleavage of gelsolin and lamin activation of Jun N-terminal kinase which has been reportedly cause some of the morphological changes observed during apoptosis.8,11 implicated as an upstream regulator of apoptosis in some In the process of investigating the potential role of systems. However, the results show that the cleavage of D4- deoxyribonuclease II (DNaseII) during apoptosis,12,13 the GDI and translocation to the nucleus do not impact on the 31 kDa protein was purified and used to immunize rabbits demise of the cell. for the production of a polyclonal antibody. The antiserum was used to probe Western blots of cell lysates. The major species recognized by the antiserum in undamaged cells Keywords: apoptosis; caspase; D4-GDI; deoxyribonuclease II; migrated at 26 kDa. Interestingly, it was observed that this protein phosphatase inhibitors; zinc 26 kDa protein was cleaved to a 22 kDa form as cells underwent apoptosis. Therefore this protein was purified and sequenced to confirm its identity. Amino acid sequence Abbreviations: AEBSF, 1-(aminoethyl)-benzenesulfonyl ¯uoride analysis of the protein identified it as D4-GDI rather than hydrochloride; CHO, Chinese hamster ovary; D4-GDI, Rho GDP- DNaseII. dissociation inhibitor protein; DFF, DNA fragmentation factor; D4-GDI is a Rho GDP-dissociation inhibitor protein. This DNase, deoxyribonuclease; EDC, 1-ethyl-3-(dimethylaminopro- protein functions to keep the Rho protein in its GDP-bound pyl)carbodiimide hydrochloride; EGFP, enhanced green fluores- inactive state. The Rho family of small GTPases including cent protein; ICE, interleukin 1-b converting enzyme; z-VAD-fmk, Rho, Rac, and Cdc42, have been shown to regulate many carbobenzoxy-valine-alanine-aspartate-¯uoromethylketone; SDS, cellular processes such as the assembly of the actin sodium dodecyl sulfate cytoskeleton and focal adhesions,14 oxidant production in Cleavage and nuclear translocation of D4-GDI in apoptosis RJ Krieser and A Eastman 413 leukocytes, and the stress response through activation of c- also detected in the human and murine cell lines, but was not Jun N-terminal kinase and p38mpk2.15 detected in CHO cells. In the human myelocytic leukemia cell Previously D4-GDI has been reported to be cleaved at line ML-1, the p26 was the most abundant species. When ML- aspartate 19 by caspase 3 during apoptosis in Jurkat T- 1 cells were induced to undergo apoptosis with staurosporine, cells induced with anti-Fas antibody, overexpression of the major species recognized migrated at 22 kDa (p22). To Bax, and staurosporine,16,17 and in BL60 Burkitt lymphoma investigate this further, ML-1 cells were incubated with 20 mg/ cells induced with anti-IgM.18 We add in this report the ml etoposide and assayed every 30 min thereafter. p22 observations that D4-GDI is cleaved in ML-1 cells and appeared concurrent with DNA fragmentation, while a many other cell types induced to undergo apoptosis by reduction of p26 was observed (Figure 2a). Hence, p22 incubation with etoposide, staurosporine, or anti-fas anti- appears to result from proteolytic cleavage of p26. Similar body. We also show that protease inhibitors, serine/ results were obtained during apoptosis induced by 1 mM threonine phosphatase inhibitors and zinc can inhibit this staurosporine (Figure 2b). The cleavage of p26 was also cleavage. In addition, this is the first report to our observed in these cells during apoptosis induced by anti-FAS knowledge of a caspase cleavage triggering translocation antibody (data not shown). We have also detected this of a substrate from the cytoplasm to the nucleus of a cell. cleavage during apoptosis in Jurkat cells, HL60 cells, MDA- 468 and MCF7 human breast cancer cells, murine 32D cells, as well as mouse and rat thymocytes (data not shown). Results Detection of a protein cleaved during apoptosis Determination of the identity of p26 The polyclonal antiserum raised against the 31 kDa bovine Due to the uncertainty of the identity of the protein recognized DNaseII detected a 30/31 kDa doublet in the commercial by the antiserum, a scheme was developed to purify and DNaseII protein (Figure 1). The antibody also detected the 30/ sequence it. Cytosolic fractions of cells were prepared as this 31 kDa doublet in bovine spleen lysates, but only a faint was determined to contain p26. The cytosol was then 30 kDa band was visible from the human breast cancer cell subjected to purification on a phenyl Sepharose column. lines MDA-468 and T47D, the murine leukemic L1210 cells, Fractions containing p26 were identified by Western analysis. and CHO cells. The most prominent band detected in the The protein was found to bind to the column in 3 M NaCl and bovine spleen lysate migrated at 26 kDa (p26). This band was was eluted at 1 M NaCl. The 1 M NaCl eluate was concentrated and subjected to reverse phase HPLC. The fractions containing p26 were electrophoresed, transferred to a membrane, excised from the blot, and subjected to N- terminal amino acid sequencing. p26 appeared to be blocked at the amino terminus and was therefore digested with endopeptidase Lys-C. p26 peptides were separated by reverse phase HPLC. Two peptides were subjected to amino terminal sequencing. The amino acid sequence obtained was compared to sequences in the Genbank database. Both peptides were found to be derived from Rho GDP-dissociation inhibitor protein D4-GDI (Figure 3). To further confirm the identity of p26, purified D4-GDI was used to immunodeplete the antisera. The undepleted antiserum detected p26 and p22 in ML-1 cells, recombinant D4-GDI, and the 31 kDa bovine DNaseII (Figure 4A). Recombinant D4-GDI was capable of depleting the antiserum of its ability to recognize p26, p22, and D4- GDI, while it still recognized the 31 kDa bovine DNaseII, suggesting the antiserum may contain an antibody that recognizes the originally intended antigen (Figure 4B). An antibody raised against D4-GDI also detected p26 and p22 in the normal and apoptotic ML-1 cells respectively, as well as purified D4-GDI. Interestingly, this antibody also detected a small amount of p26 in Sigma bovine DNaseII (Figure 4C). Therefore, a low level of D4-GDI presumably contaminated the DNaseII antigen leading to the production of the antiserum reported here. Figure 1 Expression of p26 in a variety of cell lines and bovine spleen. Western analysis was performed of bovine DNaseII, and lysates from bovine Analysis of p26 cleavage during apoptosis spleen and the indicated cell lines. Western blots were probed with serum after immunization with DNaseII (A)orwithpreimmuneserum(B).
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