Simultaneous Degradation of αII- and βII-Spectrin by Caspase 3 (CPP32) in Apoptotic Cells
1998; Elsevier BV; Volume: 273; Issue: 35 Linguagem: Inglês
10.1074/jbc.273.35.22490
ISSN1083-351X
AutoresKevin Wang, Rand Posmantur, Rathna Nath, Kim McGinnis, Margaret M. Whitton, Robert V. Talanian, Susan B. Glantz, Jon S. Morrow,
Tópico(s)Cell death mechanisms and regulation
ResumoThe degradation of αII- and βII-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both αII- and βII-subunits. Major C-terminal αII-spectrin BDPs were detected at ≈150, 145, and 120 kDa (αII-BDP150, αII-BDP145, and αII-BDP120, respectively); major C-terminal βII-spectrin BDPs were at ≈110 and 85 kDa (βII-BDP110 and βII-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that αII-BDP150 and αII-BDP120 were generated by cleavages at DETD1185*S1186 and DSLD1478*S1479, respectively. For βII-spectrin, a major caspase site was detected at DEVD1457*S1458, and both βII-BDP110 and βII-BDP85 shared a common N-terminal sequence starting with Ser1458. An additional cleavage site near the C terminus, at ETVD2146*S2147, was found to account for βII-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both αII- and βII-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis. The degradation of αII- and βII-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both αII- and βII-subunits. Major C-terminal αII-spectrin BDPs were detected at ≈150, 145, and 120 kDa (αII-BDP150, αII-BDP145, and αII-BDP120, respectively); major C-terminal βII-spectrin BDPs were at ≈110 and 85 kDa (βII-BDP110 and βII-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that αII-BDP150 and αII-BDP120 were generated by cleavages at DETD1185*S1186 and DSLD1478*S1479, respectively. For βII-spectrin, a major caspase site was detected at DEVD1457*S1458, and both βII-BDP110 and βII-BDP85 shared a common N-terminal sequence starting with Ser1458. An additional cleavage site near the C terminus, at ETVD2146*S2147, was found to account for βII-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both αII- and βII-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis. The importance of proteases in the expression of mammalian apoptosis has been the subject of many recent studies. The mammalian interleukin-1β-converting enzyme (ICE) 1The abbreviations used are: ICEinterleukin-1β-converting enzymeMTXmaitotoxinZ-D-DCBbenzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzeneGSTglutathione S-transferase from Schistosoma japonicumPAGEpolyacrylamide gel electrophoresispAbpolyclonal antibodyCAPS3-(cyclohexylamino)propanesulfonic acidBDPbreakdown productαII-BDPαII-spectrin breakdown productβII-BDPβII-spectrin breakdown productPBSphosphate-buffered salineBSAbovine serum albumin.-like protease family (renamed caspase (1Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2158) Google Scholar)) is perhaps the best characterized. Overexpression of ICE in fibroblasts can lead to apoptosis (2Wang L. Miura M. Bergeron L. Zhu H. Yuan J. Cell. 1994; 78: 739-750Abstract Full Text PDF PubMed Scopus (817) Google Scholar). While at least eight other caspases have been identified (Ich-1 (Nedd2), ICE-LAP6, Ich-2, ICErelIII, Mch-2, Mch-3, Mch-4, and Mch-5/FLICE (for reviews, see Refs. 3Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar and 4Vaux D.L. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2239-2244Crossref PubMed Scopus (914) Google Scholar)), human caspase 3 (CPP32) is perhaps the most universal apoptosis mediator. It is present in most mammalian cells (5Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3839) Google Scholar), and its deletion by gene knockout blocks neuronal death during brain development with consequential lethality (6Kuida K. Zheng T.S. Na S. Kuan C. Yang D. Karasuyama H. Rakic P. Flavell R.A. Nature. 1996; 384: 368-372Crossref PubMed Scopus (1721) Google Scholar). Besides the caspases, a second family of proteases implicated in the initiation and control of apoptosis are the calpains (7Glantz S.B. Morrow J.S. Haddad G.G. Lister G. Tissue Oxygen Deprivation: Developmental, Molecular and Integrated Function. 95. Marcel Dekker, Inc., New York1996: 153-192Google Scholar, 8Murachi T. Biochem. Int. 1989; 18: 263-294PubMed Google Scholar), especially in several hematopoietic and neuronal cells (9Squier M.K. Miller A.C. Malkinson A.M. Cohen J.J. J. Cell. Physiol. 1994; 159: 229-237Crossref PubMed Scopus (416) Google Scholar, 10Sarin A. Adams D.H. Henkart P.A. J. Exp. Med. 1993; 178: 1693-1700Crossref PubMed Scopus (196) Google Scholar, 11Sarin A. Clerici M. Blatt S.P. Hendrix C.W. Shearer G.M. Henkart P.A. J. Immunol. 1994; 153: 862-872PubMed Google Scholar, 12Nath R. Raser K.J. Stafford D. Hajimohammadreza I. Posner A. Allen H. Talanian R.V. Yuen P. Gilbertsen R.B. Wang K.K.W. Biochem. J. 1996; 319: 683-690Crossref PubMed Scopus (398) Google Scholar). The relationship between these two protease families, the consequences of each on their respective substrates and on cellular physiology, or the conditions under which each is activated remain poorly understood. interleukin-1β-converting enzyme maitotoxin benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene glutathione S-transferase from Schistosoma japonicum polyacrylamide gel electrophoresis polyclonal antibody 3-(cyclohexylamino)propanesulfonic acid breakdown product αII-spectrin breakdown product βII-spectrin breakdown product phosphate-buffered saline bovine serum albumin. While many proteins are cleaved during apoptosis, a prominent target of both calpain and caspase action is αII-spectrin, the major component of the cortical membrane skeleton. In neurons, calcium-activated calpain cleavage of αII-spectrin (non-erythroid α-spectrin or α-fodrin) accompanies N-methyl-d-aspartic acid receptor activation (13del Cerro S. Arai A. Kessler M. Bahr B.A. Vanderklish P. Rivera S. Lynch G. Neurosci. Lett. 1994; 167: 149-152Crossref PubMed Scopus (67) Google Scholar), 2S. P. Glantz, C. D. Cianci, K. K. W. Wang, and J. S. Morrow, submitted for publication.does not directly cause neuronal toxicity (7Glantz S.B. Morrow J.S. Haddad G.G. Lister G. Tissue Oxygen Deprivation: Developmental, Molecular and Integrated Function. 95. Marcel Dekker, Inc., New York1996: 153-192Google Scholar, 15Di Stasi A.M. Gallo V. Ceccarini M. Petrucci T.C. Neuron. 1991; 6: 445-454Abstract Full Text PDF PubMed Scopus (46) Google Scholar), and is postulated to be necessary for synaptic and neuronal plasticity (16Lynch G. Kessler M. Arai A. Larson J. Prog. Brain Res. 1990; 83: 233-250Crossref PubMed Scopus (121) Google Scholar, 17Lynch G. Baudry M. Brain Res. Bull. 1987; 18: 809-815Crossref PubMed Scopus (118) Google Scholar, 18Siman R. Noszek J.C. Kegerise C. J. Neurosci. 1989; 9: 1579-1590Crossref PubMed Google Scholar). Indeed, αII-spectrin cleavage by calpain appears to be a molecular mechanism by which skeletal plasticity can be enhanced without complete dissolution of the spectrin skeleton since calpain-mediated cleavage of αII-spectrin bestows calmodulin regulation on oligomeric spectrin-actin complexes, but does not dissociate them (unless βII-spectrin is also cleaved) (19Harris A.S. Croall D.E. Morrow J.S. J. Biol. Chem. 1989; 264: 17401-17408Abstract Full Text PDF PubMed Google Scholar, 20Harris A.S. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3009-3013Crossref PubMed Scopus (106) Google Scholar). In addition to the action of calpain, αII-spectrin is also targeted by caspase 3 during apoptosis in lymphocytes, hematopoietic cells, and neurons (12Nath R. Raser K.J. Stafford D. Hajimohammadreza I. Posner A. Allen H. 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A central question is to understand the molecular consequences of each protease's action, both singly and in concert, on spectrin's many functions and on the integrity of the cortical spectrin skeleton, a structure required for the maintenance of membrane order and integrity (reviewed in Ref. 26Morrow J.S. Rimm D.L. Kennedy S.P. Cianci C.D. Sinard J.H. Weed S.A. Hoffman J. Jamieson J. Handbook of Physiology. 11. Oxford University Press, London1997: 485-540Google Scholar). In other work, we have demonstrated that beyond the specific and preferred site of calpain action at the Tyr1176–Gly1177 bond (VY*GMMPR) in αII-spectrin (27Harris A.S. Croall D. Morrow J.S. J. Biol. Chem. 1988; 263: 15754-15761Abstract Full Text PDF PubMed Google Scholar), calpain also targets several additional sites in both αII- and βII-spectrin (27Harris A.S. Croall D. Morrow J.S. J. Biol. Chem. 1988; 263: 15754-15761Abstract Full Text PDF PubMed Google Scholar).2 In the present report, we demonstrate the specific sites of caspase 3 cleavage within both αII- and βII-spectrin and show that during apoptotic induction in neuroblastoma SH-SY5Y cells, it is caspase 3 that most rapidly cleaves not only αII-spectrin, but also βII-spectrin, and that this process is accompanied by skeletal dissolution. Together, these results define the molecular targets of these two important protease systems on the spectrin skeleton and suggest a mechanism by which different proteases, acting at slightly different sites within spectrin, might alternatively induce either enhanced skeletal plasticity or membrane skeletal dissolution. Human neuroblastoma SH-SY5Y cells (SY5Y) were grown to confluence on 12-well plates (∼2 × 106 cells/well) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml Fungizone (amphotericin B). Cultures were washed three times with serum-free minimum essential medium. After 1 h of preincubation with calpain inhibitor I (acetyl-Leu-Leu-Nle-CHO, Calbiochem) or Z-D-DCB (a caspase inhibitor), cultures were challenged either with a 0.1 nm concentration of the calcium channel activator maitotoxin (28Wang K.K.W. Nath R. Raser K.J. Hajimohammadreza I. Arch. Biochem. Biophys. 1996; 331: 208-214Crossref PubMed Scopus (59) Google Scholar) or with 0.5 μm staurosporine, each for 1 h (29Bertrand R. Solary E. O'Connor P. Kohn K.W. Pommier Y. Exp. Cell Res. 1994; 211: 314-321Crossref PubMed Scopus (475) Google Scholar). Unless otherwise stated, cultures were washed and returned to regular non-serum medium for 24 h, when cell viability was monitored or protein or DNA extraction was performed. Purified heteromeric bovine brain spectrin2 was digested with purified recombinant caspases (30Kamens J. Paskind M. Hugunin M. Talanian R.V. Allen H. Banach D. Bump N. Hackett M. Johnston C.G. Li P. Mankovich J.A. Terranova M. Ghayur T. J. Biol. Chem. 1995; 270: 15250-15256Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 31Bump N.J. Hackett M. Hugunin M. Seshagiri S. Brady K. Chen P. Ferenz C. Franklin S. Ghayur T. Li P. Licari P. Mankovich J. Shi L. Greenberg A.H. Miller L.K. Wong W.W. Science. 1995; 269: 1885-1888Crossref PubMed Scopus (607) Google Scholar) or purified porcine μ-calpain or m-calpain in 100 mm Hepes (pH 7.2 at 25 °C), 10 mm dithiothreitol, 10% (v/v) glycerol, and 1 mm EGTA for 90 min. Digestion was halted by the addition of an equal volume of SDS-PAGE sample buffer. Triplicate samples were subjected to electrophoresis. One gel was stained with Coomassie Blue, whereas two were transferred to Immobilon® membranes and probed by Western blotting with anti-αII-spectrin (pAb RAF-A) and anti-βII-spectrin (pAb 10D) antibodies, respectively.2Similar experiments were also carried out on GST fusion peptides representing various regions of αII- and βII-spectrins, prepared and analyzed as described previously (32Lombardo C.R. Weed S.A. Kennedy S.P. Forget B.G. Morrow J.S. J. Biol. Chem. 1994; 269: 29212-29219Abstract Full Text PDF PubMed Google Scholar).2 GST-containing fusion peptides were also detected by Western blotting using anti-GST antibody (Amersham Pharmacia Biotech). After SDS-PAGE on either 8% polyacrylamide or 4–20% gradient gels, samples were transferred to polyvinylidene difluoride membranes using 20 mm CAPS and 10% methanol (pH 11.0) for 60–75 min. The membrane was rinsed with water, stained with 0.5% Coomassie Blue in 50% methanol, and destained briefly with 100% methanol. Upon drying, the bands of interest were cut from the blot and subjected to N-terminal determination using an Applied Biosystems protein sequencer. SY5Y cell death was assessed by measuring the cytosolic enzyme lactate dehydrogenase released into the medium (25-μl samples) (39Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO J. 1996; 15: 1012-1020Crossref PubMed Scopus (296) Google Scholar). Alternatively, 40 μg/ml propidium iodide was added directly to the cell culture wells at 24 h, and fluorescence of the dye-DNA complex (excitation at 530 nm and emission at 620 nm) was measured after 5 min with a Millipore Cytoflor 2300 fluorescence plate reader. At the end of an experiment, the medium was first removed, and the attached cells were washed twice with Tris-buffered saline and 1 mm EDTA. Protein extraction was accomplished by cell lysis with SDS, followed by protein precipitation with trichloroacetic acid and solubilization with Tris base (33Wang K.K. Posner A. Hajimohammadreza I. BioTechniques. 1996; 20: 662-668PubMed Google Scholar). Protein samples were analyzed for protein concentration with a modified Lowry assay (Bio-Rad). An equal amount of total protein (15 μg) was loaded onto each lane and run on SDS-polyacrylamide gel (4–20% acrylamide) with a Tris/glycine running buffer system and then transferred to polyvinylidene difluoride membrane (0.2 μm) using a Tris/glycine buffer system in a semidry electrotransfer unit (Bio-Rad) at 20 mA for 1.5–2 h. αII-Spectrin was detected with monoclonal antibody 1622 (Chemicon International, Inc.), pAb RAF-A, or pAb RAF-B (34Harris A.S. Anderson J.P. Yurchenco P.D. Green L.A.D. Ainger K.J. Morrow J.S. J. Cell. Biochem. 1986; 30: 51-70Crossref PubMed Scopus (35) Google Scholar). βII-Spectrin was detected by pAb 10D, raised against a recombinant βII-spectrin peptide representing repeat unit 13 to residue 2204 within domain III (βII13-CΔ) (32Lombardo C.R. Weed S.A. Kennedy S.P. Forget B.G. Morrow J.S. J. Biol. Chem. 1994; 269: 29212-29219Abstract Full Text PDF PubMed Google Scholar). Biotinylated second antibody and avidin conjugated to alkaline phosphatase were from Amersham Pharmacia Biotech. The blots were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Densitometric analysis of Western blots was performed using a color scanner (Umax UC630) and either the National Institutes of Health program Image 1.5 or the program ScanAnalysis (Biosoft) operating on an Apple Macintosh® computer. Nonlinear regression analysis was carried out using the Biosoft program Ultrafit (Version 3.0), also operating on a Macintosh®computer. Immunofluorescence studies employed SH-SY5Y cells cultured on German glass coverslips (Fisher) at medium density. At the desired time point upon staurosporine treatment (6 h, unless stated otherwise), cells from all experimental treatment groups were fixed in 4% paraformaldehyde for 2 h at room temperature. Following fixation, cells were washed twice with phosphate-buffered saline (136 mm NaCl, 81 mmKCl, 1.6 mm Na2HPO4, and 14 mm KH2PO4 (pH 7.4)). Cultures were first incubated in 5% nonfat dry milk at 4 °C for 2 h. Primary antibodies (anti-CPP32 and anti-poly(ADP-ribose)polymerase) in blocking solution (10 mm NaPO4 (pH 7.5), 0.9% NaCl, 0.1% Tween 20, and 5% nonfat dry milk) were incubated for 3 h at 25 °C. The cultures were then washed with blocking solution three times for 10 min. Secondary antibodies (anti-mouse IgG) linked to a specific fluorophore (fluorescein isothiocyanate) were applied for 2 h. The coverslips were then washed three times in phosphate-buffered saline solution, mounted on slides containing Elvanol (anti-fade agent, DuPont), and allowed to dry. Slides were stored in the dark prior to immunofluorescence microscopic examination. Control cultures without primary antibodies did not stain. Our previous studies have established that SY5Y cells subjected to staurosporine (0.5 μm) undergo apoptosis, with chromatin condensation and other stigmata of apoptosis appearing within 5–6 h. The number of cells dying in culture plateau within 16–24 h (12Nath R. Raser K.J. Stafford D. Hajimohammadreza I. Posner A. Allen H. Talanian R.V. Yuen P. Gilbertsen R.B. Wang K.K.W. Biochem. J. 1996; 319: 683-690Crossref PubMed Scopus (398) Google Scholar, 35Posmantur R. McGinnis K. Nadimpalli R. Gilbertsen R.B. Wang K.K.W. J. Neurochem. 1997; 68: 2328-2337Crossref PubMed Scopus (99) Google Scholar). In the current study, loss of intact αII- and βII-spectrin in staurosporine-treated cells was detected by 1 h and continued progressively for 24 h (Fig.1 A). This loss was mirrored by increased levels of αII-spectrin BDPs at ≈150, 145, and 120 kDa (αII-BDP150, αII-BDP145, and αII-BDP120, respectively). βII-Spectrin was also simultaneously degraded into two major immunoreactive fragments at ≈110 and 85 kDa (βII-BDP110 and βII-BDP85, respectively). A minor βII-BDP was also evident at ≈55 kDa (βII-BDP55) (Fig. 1 B). The time course of the breakdown of both αII- and βII-spectrins into their major cleavage fragments could be roughly modeled as a three-step kinetic process, in which each intact subunit (A) broke down to an intermediate product (B) and then was then further degraded to a smaller major product (C), with two corresponding rate constants, kAandkB (Equation 1).A→kAB→kBCEquation 1 Scaling data to unit maximum value for A (the starting value of αII- or βII-spectrin) and solving the resulting kinetic equation as a function of time (t) yield the relative amount of B or C (Equations 2 and 3).B=kAe−kAt−e−kBtkB−kAEquation 2 C=1−e−kAt−kAe−kAt−e−kBtkB−kAEquation 3 Fitting the observed breakdown patterns using Equations 2 and 3indicated that kA ≈ 0.17 h−1 (for generation of αII-BDP150) and that kB ≈ 0.12 h−1 (for generation of αII-BDP120) (Fig. 1 B). For βII-spectrin, the corresponding values were kA≈ 0.16 h−1 (for generation of βII-BDP110) andkB ≈ 0.03 h−1 (for generation of βII-BDP85). These rates correspond to half-lives (t½) for intact αII-spectrin of ≈2.9 h and for βII-spectrin of ≈3.1 h. The half-lives of the intermediates derived from these fits were ≈4.2 h for αII-BDP150 and ≈19.2 h for βII-BDP110. Although these t½ values tended to vary (±0.8 h) from experiment to experiment (data not shown), it was clear that unlike for calpain cleavage (19Harris A.S. Croall D.E. Morrow J.S. J. Biol. Chem. 1989; 264: 17401-17408Abstract Full Text PDF PubMed Google Scholar),2 the time course of βII-spectrin breakdown by caspase 3 essentially paralleled that of αII-spectrin. Except for the transient generation of αII-BDP150, which can arise from proteolysis by several different proteases at a hypersensitive region within αII-spectrin repeat unit 11 (27Harris A.S. Croall D. Morrow J.S. J. Biol. Chem. 1988; 263: 15754-15761Abstract Full Text PDF PubMed Google Scholar, 36Harris A.S. Morrow J.S. J. Neurosci. 1988; 8: 2640-2651Crossref PubMed Google Scholar),2 the pattern of BDPs generated by staurosporine action in the SY5Y cells appeared to be distinct from those produced by calpain.2 To further define the nature of the protease cascade operating in these experiments, the effects of specific calpain and caspase inhibitors on staurosporine-induced breakdown were compared (Fig.2). Calpain inhibitor I specifically blocked the generation of αII-BDP145, but did not affect the generation of the major product, αII-BDP120 (Fig. 2 A). Conversely, Z-D-DCB, a caspase inhibitor, blocked the appearance of αII-BDP120. Both inhibitors slowed the overall generation of αII-BDPs, and thus, it appeared that both proteases contributed in some measure to the generation of αII-spectrin BDPs. With respect to βII-spectrin, calpain inhibitor I had little effect on βII-spectrin degradation compared with Z-D-DCB, which blocked the cleavage of βII-spectrin almost completely (Fig. 2 B). These patterns of cleavage were distinct from those induced in SY5Y cells by treatment with MTX (0.01 nm), a highly potent marine toxin that activates both voltage-sensitive and receptor-operated calcium channels in the plasma membrane (Fig. 2). Presumably, the intracellular calcium load induced by such treatment activates cell death pathways similar to those operating during necrosis (7Glantz S.B. Morrow J.S. Haddad G.G. Lister G. Tissue Oxygen Deprivation: Developmental, Molecular and Integrated Function. 95. Marcel Dekker, Inc., New York1996: 153-192Google Scholar, 28Wang K.K.W. Nath R. Raser K.J. Hajimohammadreza I. Arch. Biochem. Biophys. 1996; 331: 208-214Crossref PubMed Scopus (59) Google Scholar). With MTX treatment, there was dramatic loss of both αII- and βII-spectrin coupled with the generation of αII-BDP150, βII-BDP110, and βII-BDP55. On the other hand, the αII-BDP120 and βII-BDP85 products, characteristic of caspase 3 activity, were not formed. Z-D-DCB provided minimal protection, whereas calpain inhibitor I almost completely blocked both αII- and βII-spectrin breakdown in MTX-treated cells (Fig. 2). A βII-spectrin cleavage product of ≈110 kDa observed with MTX is similar in size to the caspase-generated βII-BDP110; based on the lack of βII-BDP85 as well as the inhibition of this cleavage by calpain inhibitor I, it appears that this band is a calpain product. These data, together with our earlier studies (12Nath R. Raser K.J. Stafford D. Hajimohammadreza I. Posner A. Allen H. Talanian R.V. Yuen P. Gilbertsen R.B. Wang K.K.W. Biochem. J. 1996; 319: 683-690Crossref PubMed Scopus (398) Google Scholar, 28Wang K.K.W. Nath R. Raser K.J. Hajimohammadreza I. Arch. Biochem. Biophys. 1996; 331: 208-214Crossref PubMed Scopus (59) Google Scholar), indicate that staurosporine and MTX activate in large measure distinct pathways of spectrin proteolytic cleavage in SY5Y cells. These pathways appear to be characteristic of apoptotic and non-apoptotic (necrotic) cell death, respectively, and both involve cleavage of βII-spectrin as part of the cell death event. The pattern of spectrin degradation during staurosporine-induced apoptosis in SY5Y cells and the response of these cells to Z-D-DCB strongly implicated a caspase in the apoptotic breakdown of spectrin. Since several related caspases may be active during apoptosis (37Faleiro L. Kobayashi R. Fearnhead H. Lazebnik Y. EMBO J. 1997; 16: 2271-2281Crossref PubMed Scopus (342) Google Scholar), it was of interest to determine their relative activity against spectrin in the milieu of SY5Y cells. Cell lysates were thus digested for 1 h with comparable amounts of recombinant human caspases 1–4, 6, and 7, and the breakdown patterns were analyzed after Western blotting (Fig. 3). As expected, all caspases generated αII-BDP150, presumably due to cleavage within αII-spectrin's hypersensitive site. In contrast, only caspase 3 produced significant levels of αII-BDP120. All caspases also readily digested βII-spectrin, but differed significantly in the ratio of the major βII-spectrin fragments generated (Fig. 3). For example, the dominant products generated by caspases 2, 3, 6, and 7 were βII-BDP70, βII-BDP85, βII-BDP55, and βII-BDP110, respectively. Again, only caspase 3 produced significant levels of both βII-BDP110 and βII-BDP85. While additional βII-spectrin BDPs no doubt existed in these experiments that were not visualized by pAb 10D (which is directed to the C-terminal third of βII-spectrin), these findings establish that caspases display characteristic differences in their relative specificity and activityvis-á-vis spectrin. Variations in the amount of active enzyme or in the enzyme/substrate ratios are unlikely to be a factor in these experiments since even in the very same experiment (e.g. with caspase 2; Fig. 3), a protease that displayed minimal activity against αII-spectrin often showed the greatest activity against βII-spectrin. Interestingly, αII-BDP120, βII-BDP110, and βII-BDP85, the spectrin fragments most prominent in apoptotic SY5Y cells (cf. Fig. 1), are characteristic of caspase 3 action (Fig. 3). Conversely, neither μ-calpain nor m-calpain, even when added to the SY5Y lysates, generated these fragments. Taken together, these data indicate that caspase 3 is the dominant protease mediating spectrin cleavage in staurosporine-induced apoptosis in neuroblastoma SY5Y cells. The sites of calpain cleavage in both αII- and βII-spectrin have been identified (27Harris A.S. Croall D. Morrow J.S. J. Biol. Chem. 1988; 263: 15754-15761Abstract Full Text PDF PubMed Google Scholar).2 To identify the precise sites at which caspase 3 cleaves spectrin, purified bovine brain αII/βII-spectrin was digested in vitro, and the resulting digestion products were analyzed by Western blotting and N-terminal microsequencing. Caspase 3 generated multiple spectrin fragments ranging from ≈165 to ≈85 kDa on Coomassie Blue-stained gels (Fig. 4). Two of these products (αII-BDP150 and αII-BDP120) reacted with monoclonal antibody 1622, indicating their origin from the αII-subunit. Conversely, pAb 10D detected βII-fragments at ≈110, ≈100, ≈85, and ≈55 kDa. Only the Coomassie Blue-stained fragment at ≈165 kDa was unaccounted for on the Western blots; presumably this represents a βII-spectrin N-terminal fragment that was not detected by pAb 10D. Collectively, the in vitro caspase 3 digestion products were almost identical to those observed in staurosporine-induced SY5Y cells (except for the αII-BDP145 generated by calpain). N-terminal sequencing identified the origin of several major caspase 3-generated fragments. Due to the presence of multiple bands of very similar molecular mass, the identity of some fragments proved difficult to obtain. These results (in which at least six terminal residues could be determined with confidence) are summarized in TableI and in Fig. 8. The αII-BDP150 fragment mapped to the sequence DETD1185*S1186KTASP in repeat 11 (with * representing the site of cleavage and the beginning point of the determined sequences). This site is just distal to the major calpain cleavage site (VY1176*G1177MMP) and immediately proximal to the calmodulin-binding domain (residues 1187–1206) (27Harris A.S. Croall D. Morrow J.S. J. Biol. Chem. 1988; 263: 15754-15761Abstract Full Text PDF PubMed Google Scholar). It is also likely that the N-terminal half of αII-spectrin was present within the αII-BDP150 band, based on analogy with the cleavage of αII-spectrin by calpain (7Glantz S.B. Morrow J.S. Haddad G.G. Lister G. Tissue Oxygen Deprivation: Developmental, Molecular and Integrated Function. 95. Marcel Dekker, Inc., New York1996: 153-192Google Scholar, 36Harris A.S. Morrow J.S. J. Neurosci. 1988; 8: 2640-2651Crossref PubMed Google Scholar).2 However, given the blocked N terminus of αII-spectrin, this fragment did not appear in the microsequencing results. The αII-BDP120 fragment mapped to a second caspase 3 cleavage site (DSLD1478*S1479EALIKKHE) in repeat 14 of αII-spectrin. The fragment liberated from αII-BDP150 to yield αII-BDP120 appeared in αII-BDP35 (Table I). Both the βII-BDP110 and βII-BDP85 fragments shared a common N-terminal sequence, placing this site of cleavage in repeat 11 of βII-spectrin (DEVD1457*S1458KRLTVQT). Reliable sequence information was not obtained from βII-BDP55 due to its low abundance.Table IMajor caspase 3-generated spectrin fragmentsFragmentN-terminal sequencePredicted cleavage siteAssumed endCalculated MrNative protein αII-BDP150′ aIt is likely that a second cleavage product also is present in the αII-BDP150 band, representing αII-spectrin residues 1–1185; this product (αII-BDP150′) cannot be detected by end sequencing since the N terminus of spectrin is methylated.(MDPSGVKVLE)(Start of αII-spectrin, assumed)Asp1185
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