©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proteolysis of Fodrin (Non-erythroid Spectrin) during Apoptosis (*)

(Received for publication, January 10, 1995)

Seamus J. Martin (1)(§) Geraldine A. O'Brien (1) Walter K. Nishioka (1) Anne J. McGahon Artin Mahboubi (1) Takaomi C. Saido (2) Douglas R. Green (1)

From the  (1)Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037 and the (2)Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Several recent studies have implicated proteases as important triggers of apoptosis. Thus far, substrates that are cleaved during apoptosis have been elusive. In this report we demonstrate that cleavage of alpha-fodrin (non-erythroid spectrin) accompanies apoptosis, induced by activation via the CD3/T cell receptor complex in a murine T cell hybridoma, ligation of the Fas (CD95) molecule on a human T cell lymphoma line and other Fas-expressing cells, or treatment of cells with staurosporine, dexamethasone, or synthetic ceramide. Furthermore, inhibition of activation-induced apoptosis by pretreatment of T hybridoma cells with antisense oligonucleotides directed against c-myc also inhibited fodrin proteolysis, confirming that this cleavage process is tightly coupled to apoptosis. Fodrin cleavage during apoptosis may have implications for the membrane blebbing seen during this process.


INTRODUCTION

Apoptosis(1) , a mode of cell death characterized by a series of distinct morphological changes, largely involving the nucleus and the plasma membrane, is currently the subject of intense study. The majority of programmed cell deaths from insects to mammals exhibit strikingly consistent morphological features, typical of apoptosis, suggesting that a molecular machinery for death has been well conserved through evolution(2) . This machinery, which is still largely uncharacterized, executes a series of changes in the cell resulting in condensation of the nucleus(1) , cleavage of chromatin(3) , disruption of the nuclear lamin network(4, 5) , and plasma membrane changes that result in recognition and engulfment of apoptotic cells by neighboring phagocytes(6) .

Recent studies have revealed important physiological triggers for apoptosis. These include the Fas (CD95/Apo-1) cell surface molecule, which is a member of the tumor necrosis factor/nerve growth factor receptor family(7, 8) . Some of the intracellular signals generated distal to engagement of these ``death receptors'' have now been partly characterized(9) . (^1)Much attention has also focused on gene products found to act as intracellular regulators of the cell death machinery; these include Bcl-2, Abl, Myc, Ras, and p53 (see (10) and (11) for recent reviews). It is notable also that mutated or dysregulated forms of all of these genes are found in association with many cancers, underscoring the importance of the cell death machinery as a regulator of cell growth in multicellular organisms(12) . However, although we are rapidly uncovering information about the triggers and regulators of this machinery, the death machinery itself still remains a largely unknown quantity.

Several recent studies have implicated proteases as important players in apoptosis(13, 14, 15, 16, 17, 18, 19, 20, 21, 22) . It has also been suggested that activation of a protease may be a central control point in apoptosis, much like activation of the p34/cyclin B complex is essential for mitosis(23) . In this scenario, upon activation, this putative cytoplasmic regulator acts on several substrates simultaneously, rather than initiating a linear cascade of events. However, although several proteases have been reported to become activated during apoptosis, the only proteins to date that have been reported to become cleaved during this process are poly(A)DP-ribose polymerase (15) and the protein component of the U1 small nuclear ribonucleoprotein(22) , although there is also a possibility that nuclear lamins become cleaved(5) , rather than depolymerized(4) , during this process.

In this report, we present evidence that fodrin (non-erythroid spectrin), a major component of the cortical cytoskeleton that has binding sites for several proteins, becomes cleaved during apoptosis induced by ligation of the CD3/T cell receptor complex, Fas ligation, or treatment of cells with staurosporine, glucocorticoid, or synthetic ceramide. Fodrin cleavage was detected during apoptosis of a variety of cell lines of murine and human origin and was inhibited under conditions where apoptosis was inhibited. These data provide further support for protease activation as a general feature of apoptosis and, given the localization of fodrin to the cortical cytoskeleton, suggest that membrane blebbing in apoptosis may be at least partly due to disruption of the fodrin network in these cells.


MATERIALS AND METHODS

Cell Lines and Reagents

All cell lines were cultured under standard conditions. Murine mastocytoma P815 cells were retrovirally infected with a human Fas cDNA construct (pLXSN-APO-1, designated pLRcASN) by coculture with the amphotrophic retrovirus packaging line, PA317, which had been infected previously by coculture with the ecotropic retrovirus packaging line, 2, as described previously.^1 After selection in G418 for 7 days, P815 cells were found to express human Fas and to be susceptible to anti-Fas mAb-induced apoptosis.

A rabbit polyclonal antibody against the peptide GMMPR, which matches the N-terminal sequence of the calpain-catalyzed proteolytic fragment of the fodrin alpha subunit, was generated as described previously(24) . Anti-fodrin monoclonal antibody (mAb) (^2)1622 was purchased from Chemicon International Inc. (Temecula, CA), anti-Fas mAb CH-11 was purchased from Kamiya Biomedical Co. (Thousand Oaks, CA), anti-CD3 mAb (145-2C11) was purified as described(25) , N-acetylsphingosine (C(2)-ceramide) was purchased from Biomol (Plymouth Meeting, PA), and phosphothioate-derivatized antisense oligonucleotides directed against murine c-myc (CACGTTGAGGGGCAT), as well as a same base composition scrambled control (AGTGGCGGAGACTCT), were purchased from Quality Controlled Biochemicals Inc. (Hopkinton, MA). Staurosporine, dexamethasone, and all other chemicals were purchased from Sigma, unless otherwise indicated.

Cell Death Assays

Since apoptosis is accompanied by cell shrinkage and plasma membrane blebbing (events that change the granularity and size of the cell), death was routinely assessed by measuring these changes by flow cytometry, reflected in changes to the light scattering properties of the cells, as described previously(26) . Cell death was also assessed by evaluating the uptake of propidium iodide (PI) dye by flow cytometry, as described(27) .

Preparation of Cell Lysates

Briefly, cells were pelleted at 200 times g, followed by several washes in ice-cold phosphate-buffered saline, pH 7.2, containing 1 µM phenylmethylsulfonyl fluoride, and were then resuspended in running buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol) at 10^7 cells/ml, followed by immersion in a boiling water bath for 10 min to solubilize proteins. Lysates were cleared of particulate matter by further centrifugation at 13,000 times g for 10 min at 4 °C and were then stored at -70 °C until required.

Electrophoresis and Western Blotting of Proteins

Cell lysates were assayed for protein content using the Bio-Rad microassay. Prior to loading on the gel, bromphenol blue dye was added to each sample (0.002% final concentration), equal amounts (25 µg) of total protein were loaded per lane, and proteins were separated under reducing conditions for 2 h at 70 V in 6-8% SDS-polyacrylamide gels and then Western blotted at 150 mA overnight. Blots were blocked for 1 h in TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat dried milk, then probed for 2 h with the appropriate primary antibody diluted in the same buffer, washed for 1 h in several changes of TBST, followed by probing for another 1 h with a peroxidase-coupled secondary antibody of the appropriate specificity (Amersham) and detection of bound antibody by enhanced chemiluminescence (Amersham). In some experiments, blots were then stripped for reprobing by incubating for 30 min at 70 °C in 62.5 mM Tris, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol and, after reblocking in TBST, 5% nonfat dried milk for 1 h, were reprobed with different primary mAb, as described above.


RESULTS AND DISCUSSION

Previous studies have shown that activation of A1.1 cells, a T cell hybridoma line, via the T cell receptor/CD3 complex results in apoptosis of these cells(25) . This form of apoptosis requires c-myc expression since it can be inhibited by antisense oligonucleotides targeted against c-myc mRNA(28) , as well as a dominant negative inhibitor of Myc/Max heterodimerization, MaxRx(29) . Recent studies also indicate that apoptosis in this context is due to activation-induced up-regulation of Fas and Fas ligand on these cells, which interact in a cell-autonomous manner to generate a death signal(30) . Protease inhibitors, particularly inhibitors of calpain activity, have been demonstrated to have inhibitory effects on activation-induced cell death of T cell hybridomas(14) , as well as death of mature T cells(31) , suggesting the involvement of a protease in this process. However, protein substrates that are cleaved during this type of apoptosis have yet to be reported.

Since there is indirect evidence that calpain becomes activated during apoptosis, we wished to determine whether a known intracellular substrate for calpain, the fodrin alpha subunit, became cleaved during activation-induced cell death. Thus, A1.1 cells were induced to die by culturing in wells coated with anti-CD3 mAb, and cell lysates were made and then separated by SDS-polyacrylamide gel electrophoresis, followed by probing for alpha-fodrin expression. Fig. 1illustrates that A1.1 cells underwent apoptosis following stimulation on anti-CD3 mAb-coated plates or during culture with dexamethasone. Furthermore, cells were prevented from undergoing anti-CD3 mAb-induced, but not dexamethasone-induced, apoptosis by preincubation with antisense oligonucleotides targeted to c-myc (see ``Materials and Methods''), as described previously (28) .


Figure 1: Apoptosis of A1.1 cells induced by CD3 ligation and dexamethasone treatment. Cells of the A1.1 murine T cell hybridoma line were induced to undergo apoptosis by exposure to plate-immobilized anti-CD3 mAb or 1 µM dexamethasone (Dex). A, morphology of A1.1 cells either untreated or exposed to anti-CD3 mAb for 7 h, as indicated. Magnification, times400. B, light scattering properties of A1.1 cells, as assessed by flow cytometry, after exposure to either immobilized anti-CD3 mAb or dexamethasone (1 µM) for 7 h, in the presence or absence of antisense (AS) oligonucleotides directed against c-myc (3.5 µM), or a scrambled nonsense (NS) control oligonucleotide (3.5 µM). Apoptotic cells appear as a well defined cell peak with decreased forward scatter (FSC) properties, roughly equating with decreased cell size, as indicated. The percentages of apoptotic cells in each culture are indicated in parentheses. 5000 cells were analyzed in each condition. C, cell death, as assessed by the uptake of PI dye in A1.1 cell cultures exposed to the same treatments as indicated in B. 5000 cells were analyzed in each condition.



Lysates made from these cultures were probed for alpha-fodrin expression by immunoblotting. As shown in Fig. 2, cleaved fodrin was detected in lysates made from apoptotic cells, yielding a major detectable fragment of 150 kDa. In contrast, in cell populations with few apoptotic cells, a band of 240 kDa, corresponding to intact alpha-fodrin, was the major band in evidence (Fig. 2A). Cleavage of fodrin during anti-CD3 mAb-induced apoptosis was largely inhibited by preincubation of cells with antisense oligonucleotides targeted to c-myc (Fig. 2B), whereas dexamethasone-induced fodrin cleavage was unaffected by similar concentrations of the same oligonucleotide (Fig. 2C). Control oligonucleotides had no effect on either apoptosis or fodrin cleavage ( Fig. 1and Fig. 2). These results are in agreement with the differential inhibitory effects of myc antisense oligonucleotides on these different forms of apoptosis(28) .


Figure 2: Cleavage of fodrin during anti-CD3 and dexamethasone-induced apoptosis of A1.1 cells and inhibition of this cleavage under conditions in which apoptosis is inhibited. Lysates were prepared from control A1.1 cells and from A1.1 cells induced to undergo apoptosis as indicated in Fig. 1legend. Proteins were solubilized as described under ``Materials and Methods'' and separated by SDS-polyacrylamide gel electrophoresis followed by Western blotting. Blots were then probed with an antibody to the fodrin alpha subunit. A, A1.1 cells were incubated for 7 h in either medium alone (untreated), or in the presence of immobilized anti-CD3 mAb or 1 µM dexamethasone, as indicated. B, A1.1 cells were incubated for 7 h in either medium alone (untreated), c-myc antisense oligonucleotide (3.5 µM), or a nonsense control oligonucleotide (3.5 µM), in the presence or absence of plate-immobilized anti-CD3 mAb, as indicated. C, A1.1 cells were incubated for 7 h in either medium alone (untreated), c-myc antisense oligonucleotide (3.5 µM), or a nonsense control oligonucleotide (3.5 µM), in the presence or absence of 1 µM dexamethasone, as indicated.



Since it has been demonstrated recently that anti-CD3 mAb-induced apoptosis of T cell hybridoma cells is due to a Fas and Fas ligand interaction(30) , then it follows that direct ligation of Fas on a cell line constitutively expressing this molecule should also result in proteolysis of fodrin. Thus, CEM cells, which are a T cell leukemia line that expresses Fas, were induced to undergo apoptosis in the presence of anti-Fas IgM (Fig. 3, A and B) and fodrin expression in these cells was examined as before (Fig. 3C). In addition, to test whether diverse apoptosis-inducing stimuli stimulated fodrin cleavage, CEM cells were also treated with the cell-permeable synthetic ceramide N-acetylsphingosine (C(2)-ceramide) or with staurosporine, both of which are potent inducers of apoptosis in many cell types(32, 33) . These experiments revealed that, once again, the fodrin alpha subunit was cleaved in association with apoptosis, whether induced by Fas ligation, treatment with C(2)-ceramide, or staurosporine. Interestingly, in CEM cultures that had undergone extensive apoptosis, fodrin was cleaved to a single detectable fragment of 120 kDa, whereas in cultures containing fewer apoptotic cells, a larger fragment of 150 kDa was also observed. It is plausible that the 120-kDa fragment is a further breakdown product of the 150-kDa fodrin fragment.


Figure 3: Cleavage of fodrin during Fas, ceramide and staurosporine-induced apoptosis of CEM cells. Human T cell leukemia CEM cells were induced to undergo apoptosis by exposure to either anti-Fas mAb (250 ng/ml), C(2)-ceramide (50 µM), or staurosporine (0.5 µM) for 16 h, as indicated. Western-blotted protein extracts made from these cells were then probed with an antibody to the fodrin alpha subunit. A, morphology of CEM cells undergoing apoptosis in response to the indicated stimuli. Note the abundance of cells with with many blebs in treated cultures. Magnification, times200. B, light scattering properties of CEM cells, as assessed by flow cytometry, after 16 h of exposure to the indicated stimuli. The percentages of apoptotic cells in each culture are indicated in parentheses. 5000 cells were analyzed in each condition. Results are from a representative experiment. C, detection of alpha-fodrin in cell lysates made from CEM cells treated as in B.



To further extend these findings, we also induced apoptosis in murine P815 cells that had been trans-infected with human Fas (Fig. 4, A and B) and, once again, found that fodrin became cleaved in association with apoptosis induced either by Fas ligation or staurosporine treatment (Fig. 5A), confirming the widespread nature of this observation. Furthermore, probing of cell extracts made from apoptotic and control cell populations of P815 and CEM cells with an antibody that specifically recognizes the peptide (GMMPR) that forms the N-terminal sequence of the of calpain I-generated fragment of fodrin(24) , revealed that this antibody also recognized the fodrin fragments generated in apoptotic cells (Fig. 5, A and B). These data suggest that proteolysis of fodrin during apoptosis may be a consequence of calpain activation.


Figure 4: Induction of apoptosis in P815 cells by Fas ligation and staurosporine-treatment. Murine P815 cells were induced to undergo apoptosis by exposure to 250 ng/ml anti-Fas mAb or 0.5 µM staurosporine. A, morphology of a group of four P815 cells prior to and after several hours of treatment with anti-Fas mAb. Magnification, times400. (B) Light scattering properties of P815 cells, as assessed by flow cytometry, 7 h after exposure to the indicated stimuli. The percentages of apoptotic cells in each culture are indicated in parentheses. 5000 cells were analyzed in each condition.




Figure 5: Fodrin proteolysis during P815 and CEM cell apoptosis is catalyzed by calpain. Murine P815 cells were induced to undergo apoptosis as described in Fig. 4legend and CEM cells as described in Fig. 3legend. Western-blotted protein extracts made from these cells were probed with an antibody to the fodrin alpha subunit, followed by stripping of the blots and reprobing with an antibody specific for the peptide GMMPR, which matches the N-terminal sequence of the calpain-catalyzed proteolytic fragment of the fodrin alpha subunit. A, detection of alpha-fodrin in cell lysates made from P815 cells treated as in Fig. 4B. B, detection of alpha-fodrin in cell lysates made from CEM cells treated as in Fig. 3B.



Fodrin, a multifunctional protein with spectrin-like properties, is a major component of the cortical cytoskeleton of most eukaryotic cells (34) . Fodrin, like spectrin, is a rod-shaped protein consisting of alpha and beta subunits, which form heterodimers aligned in a side-to-side manner. Fodrin heterodimers then further associate end-on-end to form tetramers, which cross-link actin filaments at their ends(35) . Fodrin possesses binding sites for many proteins, including actin(35) , calmodulin(36) , CD45(37) , and possibly also for members of the phospholipid-binding annexin family of proteins(38) , consistent with its perceived role in the organization of receptor domains and vesicle traffic at the plasma membrane(35) . Proteolysis of fodrin by calcium-dependent proteases has been observed during several processes, notably, in the context of the present study, during ischemia-induced cell death in the rodent forebrain(24) .

The direct consequences of fodrin proteolysis are still unknown. However, given its role as a major component of the plasma membrane-associated cytoskeleton, and since dramatic plasma membrane blebbing and reorganization are a consistent feature of apoptosis, it is conceivable that fodrin proteolysis during apoptosis may contribute to these rearrangements. Fodrin is also cleaved during platelet activation(39) , a process that results in exposure of phosphatidylserine on the outer leaflet of the plasma membrane of these cells. Such an event also occurs during apoptosis and seems to trigger recognition of apoptotic cells by phagocytes(6) . Thus it is plausible that fodrin proteolysis may be linked to the externalization of phosphatidylserine in both processes. These possibilities are currently under investigation.


FOOTNOTES

*
This research was supported by a Wellcome Trust International Travelling Fellowship (to S. J. M.) and National Institutes of Health Grant GM52735 and American Cancer Society Grant CB-82 (both to D. R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, 11149 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-558-3500; Fax: 619-558-3525.

(^1)
E. Gulbins, R. Bissonnette, A. Mahboubi, S. Martin, W. Nishioka, T. Brunner, G. Baier, G. Bitterlich-Baier, C. Byrd, F. Lang, R. Kolesnick, A. Altman, and D. R. Green, submitted for publication.

(^2)
The abbreviations used are: mAb, monoclonal antibody; PI, propidium iodide.


ACKNOWLEDGEMENTS

We thank Dr. Pierre Henkart for helpful advice and comments.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.