THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry ’ and Moleculax ’ Biology, Inc

Vol .266, No. Issue of September 25, pp. 18200-18205,1991 Printed in U. S. A .

In Vitro Proteolysis of Brain Spectrin by Calpain I Inhibits Association of Spectrin with Ankyrin-independent Membrane Binding Site(s)*

(Received for publication, April 10, 1991)

Ren- Ju Hu and Vann Bennett From the Howard Hughes Medical Institute and DeDartment of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

This report demonstrates that specific proteolysis of brain spectrin by a calcium-dependent protease, cal- pain I, abolishes association of brain spectrin with the ankyrin-independent binding site($ in brain mem- branes. Calpain I cleaves the f i subunit of spectrin at the N-terminal end leaving a 218-kDa fragment and cleaves the (Y subunit in the midregion to produce 150- and 130-kDa fragments. Calpain-proteolyzed spectrin almost completely loses the capacity to displace binding of intact spectrin to membranes. Spectrin digested by calpain I under conditions that almost completely de- stroyed membrane-binding remained associated as a tetramer and retained about 60% of the ability to as- sociate with actin filaments. Cleavage of spectrin oc- curred at sites distinct from the membrane-binding site which is located on the fi subunit since the isolated 2 18- kDa fragment of the f i subunit as well as a reconstituted complex of (Y and 218-kDa fi subunit fragment partially regained binding activity. Moreover, cleavage of the (Y

subunit alone reduced the affinity of spectrin for mem- branes by 2-fold. A consequence of distinct sites for calpain I cleavage and membrane-binding is that cal- pain I can digest spectrin while spectrin is complexed with other proteins and therefore has the potential to mediate disassembly of a spectrin-actin network from membranes.

Brain spectrin is a prominent component of plasma mem- branes of neurons and other cells of the central nervous system where it comprises 3% of the total membrane protein. In erythrocytes, a spectrin-based membrane skeleton forms a two-dimensional network that is linked to the plasma mem- brane through a high affinity association with ankyrin which in turn binds to the cytoplasmic domain of the anion exchan- ger. Brain spectrin subunits share sequence and functional homology with subunits of erythrocyte spectrin (Bennett, 1990; Goodman et al., 1988; Coleman et al., 1989), yet are encoded by distinct genes. Both spectrins are elongated mol- ecules with two subunits forming antiparallel dimers and assembled head to head to form tetramers. The spectrins also bind to actin filaments at each end of tetramers and contain binding sites for ankyrin on their p subunits in the midregion of tetramers.

Brain synaptosomal membranes contain ankyrin-inde-

* This research was supported in part by Research Grants AM- 19808 and GM-33996 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

pendent protein sites that bind to brain spectrin with high affinity (& -3-50 nM) under physiological conditions (Stei- ner and Bennett, 1988; Steiner et al., 1989). Ankyrin-inde- pendent binding sites for spectrin are also present in red blood cell membranes, although at less than 10% of the number of ankyrin sites. The high-affinity ankyrin-independent mem- brane binding site of spectrin is located on the p subunit and is distinct from the ankyrin-binding site (Steiner and Bennett, 1988). Association of spectrin with ankyrin-independent membrane site(s) is regulated by calcium/calmodulin, unlike the ankyrin-dependent association (Steiner et al., 1989). Cal- modulin competitively inhibits binding of spectrin to mem- brane proteins with half-maximal inhibition a t 0.2 PM cal- cium. Elevation of intracellular free calcium and subsequent activation of calmodulin thus is a potential mechanism for disassembly of this class of spectrin-membrane associations.

Calcium-dependent proteolysis of spectrin has been impli- cated in a possible regulatory role in chromaffin cell secretion (Perrin et al., 1987), neutrophil degranulation (Jesaitis et al., 1988), and platelet activation (Fox et al., 1987). A correlation has been noted between calcium-dependent spectrin degra- dation and neuronal degeneration induced by excitatory amino acids (Siman et al., 1989). Moreover, lesions of entor- hinal cortex in the brain activate proteolysis of spectrin (Ivy et al., 1988). Calpain I is a protease regulated directly by calcium (Pontremoli and Melloni, 1986) as well as by calmod- ulin (Wang et al., 1989) that is a candidate to mediate calcium- dependent proteolysis of spectrin in cells. The physiological studies suggesting involvement of calcium-dependent prote- olysis of spectrin raise the question of the functional conse- quences of spectrin cleavage by calpain I. Calpain I proteolysis of brain spectrin in the presence of calmodulin and 200 UM calcium resulted in inhibition of ability of proteolyzed spectrin to interact with actin filaments and abolished formation of spectrin tetramers (Harris and Morrow, 1990).

This study characterizes the effect of limited proteolysis of spectrin by calpain I on another protein interaction: binding to ankyrin-depleted brain membranes. The results suggest that in vitro cleavage of spectrin by calpain I inhibits associ- ation of spectrin with membranes through an allosteric mech- anism.

EXPERIMENTAL PROCEDURES

M~teriak-~~~I-Labeled Bolton-Hunter reagent was from ICN. Di- thiothreitol, Triton X-100, Tween 20, diisopropylfluorophosphate, leupeptin, and pepstatin A were from Sigma. Nitrocellulose paper and electrophoresis reagents were from Bio-Rad. Sucrose, urea, and ammonium sulfate were from Schwarz/Mann. Phenyl-Sepharose and DEAE 53 were from Pharmacia LKB Biotechnology Inc.

Methods-Brain spectrin and CY subunit of spectrin were isolated from bovine forebrain as described (Bennett et al., 1986). Spectrin

18200

Calpain 1 Cleavage Inhibits Spectrin Membrane Binding was assayed for ability to compete for binding of lZsI-labeled spectrin to ankyrin-depleted bovine brain membranes as described (Steiner and Bennett, 1988). Intact brain spectrin was labeled with "'1 using Bolton-Hunter reagent (Bennett, 1983). Actin was purified as de- scribed (Pardee and Spudich, 1982). Calpain I was isolated from the red blood cell cytosol based on calcium-dependent binding to phenyl- Sepharose essentially as described (Gopalakrishna and Barsky, 1985). SDSI-polyacrylamide electrophoresis was performed on 3.5-17% ex- ponential gradient slab gels with the buffers of Fairbanks et al. (1971). Native polyacrylamide electrophoresis was performed on 3-10% ex- ponential gradient slab gels with the Fairbanks buffer in the absence of SDS. Protein concentrations were determined by the fluorescarnine method (Bohlen et al., 1973) using bovine serum albumin as a stand- ard. Immunoblot analysis was performed using '2sI-labeled protein A to detect polypeptides electrophoretically transferred from SDS-gels to nitrocellulose paper (Davis and Bennett, 1983).

Proteolysis of Spectrin by Calpain I and the Purification of the 218- kDa 0 Spectrin Fragment-100 pg of spectrin was proteolyzed by 6 pg of calpain I for 60 min at 24 "C in a buffer containing 50 mM Hepes, pH 7.0, 50 mM NaCl, 1 mM NaN3, 1 mM CaCI2, 1 mM DTT. The reaction was stopped by adding 0.5 M NaEGTA to give a final concentration of 15 mM. After proteolysis, the 218-kDa fragment of 0 spectrin (0') was isolated by the following procedure. 0.5 g of ammonium sulfate was added to 2 ml of proteolysis reaction mixture followed by incubation at 4 "C for 15 min. After centrifugation, the pellet was resuspended in dissociation buffer containing 7 M urea, 10 mM sodium phosphate buffer, pH 7.5,lO mM glycine, 1 mM NaEGTA, 0.5 mM DTT, and the samples were dialyzed against the same buffer a t room temperature for 30 min. The digest was fractionated on a Superose 6 gel-filtration column equilibrated with buffer containing 6 M urea, 10 mM sodium phosphate buffer, pH 7.5, 1 mM NaEGTA, 1 mM NaN3, 0.05% Tween 20, and 0.5 mM DTT. 0' eluted from the column first and was separated from other proteolyzed fragments. /3' was stored in 6 M urea, 10 mM sodium phosphate buffer, pH 7.5, 1 mM NaEGTA, 1 mM NaN3, 0.5 mM DTT, 10% sucrose at -70 "C.

Reassociation of 0' and 01 Spectrin-20 pg of 0' subunit and 20 pg of LY subunit were mixed together in the buffer of 6 M urea, 10 mM sodium phosphate buffer, pH 7.5, 1 mM NaEGTA, 1 mM NaN3, 0.5 mM DTT, and 10% sucrose and dialyzed against 10 mM sodium phosphate, pH 7.5,l mM NaEGTA, 1 mM NaN3, 0.5 mM DTT, 0.05% Tween 20, 1 M NaBr, 10% sucrose a t 4 "C for 16 h followed by dialysis against 10 mM sodium phosphate buffer, pH 7.5,50 mM NaCI, 1 mM NaEGTA, 1 mM NaN3, 0.5 mM DTT, 0.05% Tween 20, 10% sucrose a t 4 "C for 4 h.

RESULTS

Cleavage of Brain Spectrin by Calpain I Inhibits Binding of Spectrin to Ankyrin-depleted Brain Membranes-The binding of brain spectrin to ankyrin-depleted brain membranes has been studied previously (Steiner and Bennett, 1988; Steiner et al., 1989). It was found that the high-affinity binding site(s) for membrane protein(s) were located on the p subunit of brain spectrin. To understand more about the regulation of this interaction, brain spectrin was digested by a calcium- dependent protease, calpain I, and the effect of digestion on the spectrin-membrane interaction was examined. In the pres- ence of 1 mM Ca2+, three major proteolyzed fragments of spectrin were generated by calpain I digestion (Fig. LA, lane 2). 12sI-Labeled CY spectrin has been shown previously to associate with p spectrin transferred on nitrocellulose paper, and provides a selective probe for the p subunit (Davis and Bennett, 1983). 12sI-Labeled CY spectrin associated only with a 218-kDa fragment following digestion of spectrin with calpain I in the presence of 1 mM Ca2+ (Fig. lB, lane 2). These results indicate that calpain I cleaves p spectrin near one end of the subunit resulting in a 218-kDa fragment. Comparison of the Commassie Blue staining of intact spectrin and the proteo- lyzed fragments (Fig. lA, lanes 1 and 2) , indicates that most

' The abbreviations used are: SDS, sodium dodecyl sulfate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; DTT, dithio- threitol; NaEGTA, sodium [ethylenebis(oxyethylenenitrilo)]tetraa- cetic acid.

A.

18201

B.

g 1 2 3 1 2 3

I_ ~_, -150 -1 30

90- IJll

FIG. 1. Analysis of calpain I fragments of bovine brain spec- trin. A , Coomassie Blue-stained SDS-polyacrylamide gel of brain spectrin before and after digestion with calpain I in the presence of 1 mM Ca2+. 100 pg of spectrin was proteolyzed by 6 pg of calpain I for 60 min a t 24 "C in a buffer containing 50 mM Hepes, pH 7.0, 50 mM NaC1, 1 mM NaN?, 1 mM CaClz, 1 mM DTT. The reaction was stopped by adding 0.5 M NaEGTA to give a final concentration of 15 mM. Lane I, intact bovine brain spectrin; lane 2, spectrin digested with calpain I followed by addition of NaEGTA to stop the reaction; lane 3, spectrin incubated with calpain I, with 15 mM NaEGTA added before addition of Ca2+;g, protein from red blood cell ghost as markers. B, polypeptides were electrophoretically transferred to a nitrocellu- lose paper and probed with "'I-labeled CY spectrin as described (Davis and Bennett, 1983). Lanes 1-3 correspond to the same lanes in A .

of the p spectrin was cleaved to 218 kDa. The other proteolytic fragment(s) of the p subunit are 40 kDa or less. The 150- or 130-kDa fragments are derived from the CY subunit of spectrin based on 1) their inability to associate with 12sI-labeled CY

spectrin when transferred to nitrocellulose paper, 2) the fact that their sizes are considerably larger than the expected 40- kDa fragment of the /? subunit, and 3) since their sizes are similar to the calpain I-proteolyzed fragments of CY spectrin noted previously by Harris and Morrow (1990).

The effect of proteolysis of spectrin with calpain I and 1 mM Ca2+ on the interaction between brain spectrin and an- kyrin-independent membrane binding sites was evaluated by comparing abilities of intact and calpain I-proteolyzed spec- trin to compete with '2sI-labeled brain spectrin for binding to membranes. Intact brain spectrin displaced binding of "'1- labeled brain spectrin to ankyrin-depleted bovine brain mem- branes, with 50% inhibition at 30 nM. However, brain spectrin digested by calpain I exhibited a 10-fold reduction in activity in this assay (Fig. 2).

The inability of calpain I-digested spectrin to compete for binding is not due to the presence of calpain I in the solution or the association of calpain with spectrin. Addition of 1 mM NaEGTA to chelate Ca2+ before the addition of calpain I prevented the cleavage activity of spectrin by calpain I (Fig. LA, lane 3) and the intact spectrin containing calpain I still exhibited the same competition activity as the intact spectrin alone (Fig. 2). Limited cleavage of spectrin by calpain I at sites on the CY subunit as well as cleavage of the p subunit thus inhibits a binding interaction between the p subunit and membranes.

Calpain 1 Cleavage Inhibits Spectrin Membrane Binding

50 100 150

Spectrin concentration (nM) FIG. 2. Effect of calpain I digestion of brain spectrin on

binding of 'Z"I-labeled brain spectrin to ankyrin-depleted brain membranes. Various concentrations of intact, brain spectrin (O), calpain I-proteolyzed brain spectrin (O), and intact. spectrin with calpain I mixture from lane 3 of Fig. 1 (0) were incubated individually with 7.5 nM ""I-labeled intact brain spectrin and 5-10 pg of ankyrin- depleted bovine brain membrane in 10 mM Hepes, pH 6.8, 50 mM NaCI, 1 mM EGTA, 1 mM NaN:,, 3 mg/ml RSA a t 4 "C. After 1-h incubation, the reaction mixture was loaded onto 10% sucrose cushion and centrifuged a t 5000 rpm for 30 min. The pellet which contained membrane-bound spectrin and supernatant which contained free spectrin were separated and counted. The binding percentage of '"'I- labeled spectrin was plotted as a function of concentrations of intact hrain spectrin, calpain I-proteolyzed brain spectrin, and intact spec- trin with calpain I mixture.

The effect of cleavage of N spectrin on membrane binding was studied further using spectrin digested with calpain I at pCa = 4 which results in cleavage only of the N subunit (Fig. 3). Spectrin with cleaved N subunit exhibited a 2-fold reduc- tion in activity, with the concentration required for half- maximum of displacement increased from 45 to 85 nM. There- fore, cleavage of N spectrin reduces binding activity with membranes in a minor role, while cleavage of both N and /3 subunits results in loss of displacement activity of spectrin.

The Isolated 218-kDa Fragment of p Spectrin and Reconsti- tuted ~/218-kDa Fragment Regain Binding Actiuity-Calpain I cleavage could inhibit membrane binding of spectrin by two mechanisms. One possibility is that calpain I cleavage directly modifies the membrane binding site of @ spectrin. Alterna- tively, calpain I cleavage could occur at a site distinct from the membrane binding site and modify binding activity indi- rectly through an allosteric effect. One prediction if the cleav- age site were the same as the membrane binding site is that the isolated 218-kDa fragment of the p subunit would remain inactive. We purified the 218-kDa fragment of the @ subunit by gel filtration in 6 M urea. We refer to this 218-kDa fragment as the @' subunit. Surprisingly, the isolated /3' partially re- gained the ability to compete with "'I-labeled brain spectrin for the binding to ankyrin-independent membranes (Fig. 4). Half-maximal displacement occurred at 100 nM (Fig. 4) which is only 2-fold less than intact spectrin in this assay. This result strongly suggests that calpain I cleavage of p spectrin does not destroy the membrane-protein contact site, although cleavage may be close to the contact site.

P C P p 100 - C

9 3 4 0

50 100 150

Spectrin Concentration (nM)

FIG. 3. Comparison of the membrane binding activity be- tween a- and &cleaved spectrin and a-cleaved spectrin. Poly- acrylamide gel on left shows spectrin proteolyzed hy calpain I in the presence of either 1 mM Ca" (pca" = 3 ) or 100 pM Ca2+ (pea'+ = 4). g, human ghost as markers. Right, comparison of competition activity of increasing concentrations of intact spectrin (0): hrain spectrin proteolyzed hy calpain 1/1 mM Ca" (0); hrain spectrin proteolyzed by calpain I a t 100 pM Ca" (0).

-I

50 100 150

Spectrin concentration (nM) FIG. 4. Isolated 218-kDa calpain I fragment of @ spectrin

and a reconstituted a/218-kDa @ fragment complex partially regain ability to compete for binding of 12"I-labeled brain spectrin to ankyrin-depleted brain membranes. Isolated 218- kDa calpain I fragment of'B spectrin (B), reconstitr~ted tr/ZlX-kDa /j fragment complex (0) and the intact hrain spectrin (0) were meas- ured for their competition activity for the membrane-binding site(s) with '""I-labeled hrain spectrin. The displacement activity was as- sayed the same as described in Fig. 2. The binding percentage of ","I- labeled spectrin was plotted as a function of concentration of int.act spectrin, and isolated 218-kDa B' fragment. and reconstituted trpL18- kDa B' fragment complex were plotted individually.

The full effect of calpain I digestion of the @ subunit presumably requires participation of the (Y subunit, either constitutively or following cleavage of the N subunit. The effect of the (Y subunit on activity of the digested @ subunit

Calpain 1 Cleavage Inhibits Spectrin Membrane Binding 18203

was evaluated by reconstituting a complex of native N subunit and digested subunit (Fig. 4). The reconstituted N/@' com- plex formed head-to-head tetramers with subunits aligned side-to-side based on electron microscopy and nondenaturing gel electrophoresis (see below). The a/@' complex exhibited nearly identical activity as the p' subunit alone (Fig. 4). Therefore, inhibition of @' activity requires cleavage of the N subunit.

A membrane protection assay also indicated that the cal- pain I-cleavage site on p spectrin is distinct from the mem- brane-protein binding site. ""I-Labeled spectrin was incu- bated with brain membranes for 1 h and after centrifugation on a 10% sucrose cushion, the membrane-bound spectrin was resuspended from the pellet. Both membrane-bound spectrin and free spectrin were examined for calpain I proteolysis (Fig. 5 ) . In both cases, p spectrin was proteolyzed to a 218-kDa fragment and cy spectrin t.o 150- and 130-kDa fragments (Fig. 5 , lanes 2 and 4 ) . Therefore, spectrin continues to be a substrate for calpain I while associated with membranes, yielding equivalent calpain I-proteolytic fragments when com- pared to proteolysis of free spectrin. This indicated that the membrane-binding site on spectrin is distinct from the calpain I-cleavage site, since the binding of membrane protein(s) to spectrin cannot prevent spectrin from calpain I proteolysis. The possibility that spectrin may dissociate from the mem- brane during the period of calpain I digestion has been ex- cluded by the following experiment. After 1-h incubation of '""I-labeled spectrin with membranes, intact cold spectrin was

calpain - + - + 1 2 3 4

Mr x 1 o - ~

260 - 218 -

150 - 130 -

mu? FIG. .5. Spectrin associated with bovine brain membranes is

a substrate for proteolysis by calpain I. Intact '?"I-labeled spec- t rin was incubated with ankyrin-depleted bovine membranes in bind- ing buffer at 4 "C for 1 h. The reaction mixture was loaded onto a 105 sucrose cushion. After centrifugation, the pellet was separated from the supernatant. The pellets was resuspended in 50 mM Hepes, pH 7.0, 50 mM NaCI, 1 mM NaN:,, 1 mM CaCl?, 1 mM DTT buffer and divided into two parts: either proteolyzed by 18.7 pg/ml of calpain I at 24 "C for 1 h or untreated. Pellets were analyzed on a SDS- polyacrylamide gel, and spectrin pol-ypeptides were visualized by autoradiography. Lanes I and 2, free '".'I-labeled brain spectrin with- out incubating with bovine membrane; lanes 3 and 4, memhrane- bound brain spectrin which is in the pellet after centrifugation. Lanes I and 3, intact spectrin without calpain I proteolysis; lanes 2 and 4 , calpain I-proteolyzed spectrin.

added to compete for binding under conditions similar to the calpain I digestion but without enzyme. More than 75% of '"'I-labeled spectrin remained associated with membranes. The result shown here is consistent with the results that both the isolated 218-kDa fragment of p spectrin and the reconsti- tuted cy and 218-kDa fragment retain partial binding activity, suggesting that the calpain I-cleavage site on spectrin is not the membrane-binding site.

Calpain I-proteolyzed Spectrin and Reconstituted alp' Spec- trin Are Tetramers-The oligomeric state of calpain I-proteo- lyzed spectrin and reconstituted cy/@' complex have been studied by native gel electrophoresis (Fig. 6) and electron microscopy (Fig. 7). Brain spectrin, erythrocyte spectrin, and a spectrin were used as standards. Calpain I-proteolyzed spectrin and reconstituted alp' spectrin have similar mobility on native gels compared to brain spectrin tetramer (Fig. 6, lanes 1, 2, and 5 ) . These proteins thus are all tetramers. Furthermore, tetramers can be seen in rotary shadowed im- ages visualized by electron microscopy (Fig. 7). No consistent difference between intact spectrin and calpain I-proteolyzed spectrin could be detected by low-angle rotary shadowing (Fig. 7). The contour length of both cleaved and native spectrin is about 200 nm. The reconstituted a/@' complex also looks like a tetramer, but exhibits a fork on the ends. These data suggest that the cleaved p' subunit still contains an intact C-terminal end which is responsible for interaction with the CY subunit to form tetramers (Morrow et al., 1980), while the N-terminal end of the /3 subunit was cleaved by calpain I to generate a truncated N-terminal end. This truncated N-terminal end apparently has reduced ability to associate with N spectrin, resulting in the fork on the end of tetramer (Fig. 7C, 8' + a) .

Calpain I-proteolyzed Spectrin Exhibits Reduced Ability to Rind to Actin-The calpain I-proteolyzed spectrin was incu- bated with actin, and the reaction mixture was centrifuged under conditions where only the spectrin complexed with actin was sedimented. The pellet and supernatant were ex-

1 2 3 4 5 ' '1

I

tetramer I

dimerhnonomer - "

FIG. 6. Determination of the oligomeric state of brain spec- trin, calpain I-proteolyzed brain spectrin, and reconstituted a/21S-kDa 0 fragment complex by native polyacrylamide gel electrophoresis. Spectrins were analyzed on an exponential 3-10% gradient polyacrylamide gel which was stained with Coomassie Blue. Lane 1, brain spectrin (tetramer); lane 2, calpain I-proteolyzed brain spectrin; lane 3, red blood cell spectrin (dimer); lane 4 , (Y spectrin (monomer); lane 5 , reconstituted (u/218-kDa If fragment complex.

18204 Calpain 1 Cleavage Inhibits Spectrin Membrane Binding

A. Intact spectrin C. . . . . . . . . . . . . .

Intact spectrin

Spectrin cleaved by calpain

s'+ Q

100 nm -

200 nrn -

FIG. 7. Visualization by electron microscopy of native spec- trin, spectrin digested by calpain I/1 mM Ca'+, and reconsti- tuted complex of a/218-kDa fl fragment by low angle rotary shadowing with metal and carbon. 10 pg/ml spectrin was dialyzed against 100 m u ammonium formate, pH 6.9, 30% glycerol (Shotton c't nl., 1979) and sprayed directly onto a carbon-coated 400-mesh grid. The grid was then rotary-shadowed with alloy of platinum and palladium (8020) at an angle of '7. A , shows the intact spectrin at 56,000 X magnification. R is the calpain I-proteolyzed spectrin a t the same magnification as in A . Bar, 200 nm. C, 92400 X magnification. The first roll' shows intact spectrin. The second row is calpain I- proteolyzed spectrin. The third rou: is the reconstituted complex of o/218-kDa /,' fragment. Bar, 100 nm.

amined by SDS-polyacrylamide gel electrophoresis, and the concentration of spectrin both in pellet and supernatant was estimated separately by gel densitometry (Fig. 8). About 40% of intact spectrin was cosedimented with actin, while only 25% of calpain I-proteolyzed spectrin and 13% of reconsti- tuted alp' complex were cosedimented with actin. Actin is known to bind to spectrin via the end of the tetramer, and the N-terminal end of the p subunit is responsible for this interaction (Karinch et al., 1990). The reduced actin-binding activity of calpain I-proteolyzed spectrin and the alp' com- plex is consistent with the localization of the site of spectrin proteolysis near the N-terminal end of the p subunit. The smaller proteolyzed p fragment from the N-terminal end may still be attached to the a subunit and retain some ability to associate with actin. However, this cleavage is near the actin binding site and could reduce ability of the proteolyzed spec- trin to bind to actin. The reconstituted alp' tetramer no longer contains the small fragment of p subunit on its N- terminal end which could explain the further reduction in ability to bind actin. The remaining 13% binding capacity of the alp' spectrin may be due to the possibility that a portion of the actin-binding domain is still retained in the p' fragment following calpain I cleavage. Alternatively, the residual activ- ity may be due to an actin binding site on the a subunit. Reduction of actin-binding activity suggests two conclusions. 1) Calpain I cleaves spectrin on the N-terminal end of the p subunit. An N-terminal cleavage site also can be deduced from the results of electron microscopy and native gel electro- phoresis which show that the C terminus is still intact to give tetramer structure. 2) Calpain I cleavage of spectrin reduces association of spectrin with actin in addition to the loss of binding to membranes. In confirmation of the data from Harris and Morrow (1990), these results suggest that the

2 3 4 5 spectrin spectrinl tr/l%' 1%' n

calpain complex FIG. 8. The effect of spectrin proteolysis by calpain I/1 mM

Ca'+ on the binding of spectrin to actin. Actin was polymerized by adding KC1 to 0.1 M, MgCI? to 5 mM, and ATP to 1 mM before use. 0.5 p~ actin was incubated with 50 nM spectrin at 4 "c for 1 h in buffer containing 30 mM Hepes, pH 7.2, 50 mM KC1, 20 mM NaC1, 1 mM MgCI,, 1 mM NaEGTA, 0.05% Tween 20, 0.2 mM DTT, 0.5 mM ATP, 30 mg/ml BSA, 10% sucrose. The reaction mixture was loaded onto a 20% sucrose cushion and centrifuged a t 40,000 rpm for 30 min. The pellets which contained actin and actin-bound spectrin were resuspended in 5% SDS, 25% sucrose, 50 mM Tris, pH 8.0, 0.5 mM NaEDTA, and 10 mg/ml bromphenol blue, and analyzed by SDS- polyacrylamide gel electrophoresis to compare the ratio of spectrin in the pellet over the total amount of spectrin. The amount of protein in each lane was estimated by laser scanning densitometry. The amount of actin-bound spectrin which sedimented in the pellet di- vided by the amount of total spectrin is the percentage of spectrin cosedimented with actin which is shown on the y axis. Column I represents the binding percentage of intact spectrin with actin. Col- umns 2-5 represent the percentage of cosedimentation with actin filaments of brain spectrin proteolyzed by calpain I, the recombinant complex of (Y subunit/218-kDa 0 subunit fragment, isolated 218-kDa f l spectrin, or isolated n subunit of spectrin, respectively.

binding of brain spectrin with actin can be regulated by calpain I cleavage of brain spectrin.

DISCUSSION

This study demonstrates that limited digestion with calpain I abolishes the ability of spectrin to associate with ankyrin- independent membrane binding sites in brain membranes. Calpain I in the presence of 1 mM Ca2+ cleaves the p subunit at the N-terminal end to a 218-kDa fragment and cleaves the a subunit to 150- and 130-kDa fragments. This proteolyzed spectrin almost completely loses the capacity to displace binding of intact spectrin to membranes. Spectrin digested by calpain I under conditions that almost completely destroyed membrane binding remained associated as a tetramer and retained about 60% of the ability to associate with actin filaments. An unanticipated finding was that cleavage of spectrin occurred a t sites distinct from the membrane-binding site which is located on the p subunit. This conclusion is based on the observation that the isolated p' subunit fragment as well as reconstituted complex of a and p' subunit partially regained binding activity. Loss of binding induced by calpain I cleavage thus requires participation of the cleaved a subunit.

Calpain 1 Cleavage Inhibits Spectrin Membrane Binding 18205

An important consequence of distinct sites for calpain I cleavage and membrane binding is that spectrin can function as a substrate for calpain I while spectrin is associated with membranes. Calpain I thus has the potential to digest spectrin while spectrin is complexed with other proteins and to mediate disassembly of spectrin from membranes of cells.

Calmodulin, in the presence of submicromolar Ca2+, is a competitive inhibitor of the binding of brain spectrin to ankyrin-depleted brain membranes (Steiner et al., 1989). Cal- modulin, in the presence of higher concentrations of calcium, also promotes cleavage of spectrin by calpain I leading to dissociation of spectrin into dimers and loss of ability of spectrin to interact with actin filaments (Harris and Morrow, 1990; Harris et al., 1989). These data combined with the findings of this study suggest that calcium acting through calpain I and calmodulin could abolish several critical protein interactions of spectrin that potentially would lead to disso- lution of a membrane-associated spectrin-actin network. The system has the potential for several levels of response to calcium beginning with reversible inhibition of the spectrin- membrane interaction mediated by calmodulin alone at sub- micromolar levels of calcium. Higher levels of calcium and the presence of calpain I lead to cleavage of the a subunit with a 2-fold reduction in affinity of spectrin for the mem- brane site(s) as well as a reduction in association with actin filaments. The next level of response occurs at still higher levels of calcium which result in cleavage of both (Y and 6 subunits by calpain I with loss of membrane binding, and further reduction in ability to bind to actin. Finally, the combination of calpain I with calmodulin and high levels of calcium results in loss of actin association and dissociation of spectrin into dimers (Harris and Morrow, 1990).

The physiological significance of in vitro observations of calpain-cleaved spectrin remains to be established in future studies. Experimental challenges will be to demonstrate that cleavage of spectrin is required for processes where calcium- dependent cleavage of spectrin has been noted, such as secre- tion (Perrin et al., 1987), platelet activation (Fox et al., 1987), and neuronal degeneration (Siman et al., 1989). Useful re- agents for these studies would include antibodies that react selectively with cleaved spectrin to verify the localization at expected sites in cells and inhibitors of calpain I that selec- tively block cleavage of spectrin but not other protein sub- strates for calpain I. Ultimately it may be possible to evaluate consequences of transfecting cells or animals with genes en- coding calpain-resistant forms of spectrin.

The membrane binding protein(s) for spectrin that are responsible for associations in these assays have not yet been well defined. One candidate is a 180-kDa component of N- CAM (Pollerberg et al., 1987). The studies of the biochemistry interaction and calpain I regulation between N-CAM 180 and brain spectrin will promise a further understanding of the spectrin-based membrane skeleton interaction.

Mammalian brain contains three isoforms of spectrin: a brain isoform (240/235) which represents the major spectrin, an erythroid isoform (240/235E), and an astrocyte-specific isoform (240/235A) (Goodman et al., 1989). In the cerebellum, the major brain isoform is present predominantly in neuronal axons, while the erythroid isoform is present in neuronal cell bodies and dendrites (Riederer et al., 1986). In this paper, the

focus has been on the major isoform of brain spectrin. It will be interesting in future experiments to evaluate the effect of calpain I cleavage on membrane association of the erythroid isoform of spectrin.

The a subunit of spectrin apparently can regulate activity of the f i subunit following cleavage of the a subunit by calpain I. This conclusion is based on the fact that the calpain I- digested D subunit regains activity following isolation, and that cleavage of the (Y subunit alone was sufficient to lower binding activity of the membrane-binding site which is located on the D subunit. One possible explanation for this phenom- enon is that, following cleavage the (Y subunit, fragments reorient with respect to the adjacent (3 subunit such that the binding site of the f i subunit is blocked. Spectrin thus exhibits an unanticipated level of organization as an integrated unit that goes well beyond what might be expected from a simple a-helical fibrous protein. The results of this study and that of Harris and Morrow (1990) demonstrate that calpain I cleavage of spectrin also inhibits association with actin by an allosteric mechanism. Calpain I, as the result of long range effects on spectrin, is capable of coordinated inhibition of two independ- ent protein associations. These considerations suggest the hypothesis of a specific and elegant regulatory mechanism for disassembly of a spectrin-based membrane skeleton.

Acknowledgments-Helpful discussions with Joe Steiner are grate- fully acknowledged. We also thank Rashmi Joshi for helping actin and calpain I preparation, Jonathan Davis for rotary shadowing discussion, Gerda Vergara for helping in rotary shadowing electron microscopy studies and preparation of figures, and Brenda Sampson for helping in preparing manuscript.

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