In vitro digestion of spectrin, protein 4.1 and ankyrin by erythrocyte calcium dependent neutral protease (Calpain I)

hf. J. Eiochem. Vol. 22, No. 12, pp. 1479-1489, 1990 Printed in Great Britain. All rights reserved

0020-711x/90$3.00+ 0.00 Copyright 0 1990Pergamon Press plc

IN VITRO DIGESTION OF SPECTRIN, PROTEIN 4.1 AND ANKYRIN BY ERYTHROCYTE CALCIUM DEPENDENT NEUTRAL PROTEASE (CALPAIN I) P. BOIVIN, C. GALAND and D. DHERMY INSERM U 160 and Association Cl, Bernard Hopital Beaujon, 92118 Clichy Cedex, France [Tel. (l)-140-8755-01; Fax 47-90-94-403 (Receiued 18 April 1990) Abstract-l. In whole ghosts, ankyrin, protein 4.1, protein band 3 and spectrin are lysed by purified calpain I in the presence of calcium. 2. Limited calpain lysis of purified ankyrin results in several peptides, including a 85 kD peptide bearing the ankyrin interaction site for the protein band 3 internal fragment (43 kD), and a 55 kD peptide carrying the ankyrin-spectrin interaction site. 3. These pe-ptides are differently phosphorylated: the 85 kD by cytosol casein kinase, and the 55 kD by membrane casein kinase. 4. Protein 4.1 lysis mainly produces a 30 kD peptide resistant to proteolysis. 5. The spectrin B-chain is more sensitive to calpain cleavage than the a chain; both chains seem to be cleaved in a similar sequential manner. 6. Limited proteolysis of spectrin dimer does not impede tetramerization in vitro.

INTRODUCTION The red cell membrane

is, like others, composed

of a

lipid bilayer plus transmembrane proteins which traverse the bilayer. The main transmembrane protein is the anion channel or electrophoretic protein band 3. The membrane skeleton forms a hexagonal meshwork composed of spectrin, actin, protein 4.1 and 4.9 on the internal surface of the bilayer leaflet. The skeleton is anchored to transmembrane proteins at several sites: the main one links the spectrin /I chain to the internal segment of protein 3. This anchoring system is made up of a major component, ankyrin or protein 2.1 (215,000 M,), and several minor components called proteins 2.2, 2.3 and 2.6 (syndeins) (Bennett and Stenbuck, 1979; Luna et al., 1979; Yu and Goodman, 1979; Bennett and Stenbuck, 1980a,b). The membrane skeleton controls the shape and probably the deformability of the red cell. The structural and functional integrity of skeleton proteins, which is necessary for this control can be influenced by proteolytic enzymes. There are several proteolytic enzymes in the red cell cytosol and membrane. The main one is a cytosolic neutral calcium-dependent protease, calpain I. Calpains are present in most cells; calpain I requires micromolar concentrations of Ca2+, while calpain II needs millimolar calcium concentration. Only calpain I is present in the red cell (Murakami et af., 1981; Melloni et al., 1982a,b; Hatanaka et al., 1983; Pontremoli et al., 1984). It has been purified from the cytosol, but some enzyme remains bound to the Abbreviations: EGTA, ethylene glycol bis (-aminoethyl ether)-N, N, N’, N’-tetraacetic acid; EDTA, ethylene diamine tetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; BME, p mercaptoethanol.

membrane (Hatanaka et aI., 1984; Pontremoli et al., 1985a,b; Croall et al., 1986) where it is activated by partial autolysis. The physiological activity of calpain in the red cell is controlled by a thermostable inhibitor, which has itself been purified (Melloni et al., 1982a; Murakami et al., 1981). The present work investigates the pattern of the in oitro digestion of the membrane proteins spectrin, protein 4.1 and ankyrin by calpain in order to determine the influence of proteolysis by calpain on some protein interactions. The effect of limited proteolysis of the spectrin dimer in the tetramerization process in oitro was examined and the peptides of ankyrin digestion bearing the sites of ankyrin interaction with spectrin and protein band 3 were identified. Finally, it was demonstrated that cytosol and membrane casein kinases phosphorylated distinct sites on the ankyrin molecule. MATERIALS AND

METHODS

(A) Materials DE 52, CL4B Phenyl-Sepharose and CNBr-activated CL4B Sepharose were purchased from Pharmacia, and AcA34 Ultrogel was from IBF (Industrie Biologique FranGaise, France). H3B Procion Red was a gift from IBF. Casein was from Merck and CAMP-dependent protein kinase from Sigma. Acrylamide and bisacrylamide were from BDH, [y3*P]ATP was from Amersham and other reagents were from Merck. (B) Purification of cytosol calpain A 50 ml sample of packed human red blood cells was washed three times with 0.15 M NaCl to remove white blood cells and platelets, lysed at 4°C in 500 ml of buffer A (5 mM Tris, 1 mM EGTA, 5 mM /IME, pH 8) and centrifuged at 14,000 rpm for 45 min. The hemolysate was made 50 mM in NaCI. A 35 g sample of DE 52 (Pharmacia) was equilibrated

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P.

~IVIN

with buffer A + 50 mM NaCl and added to the hemolysate. The mixture was stirred for 90min at 4°C and the DE 52 collected on a fritted glass Biichner funnel. The DE 52 was packed into a 2.5 x 17.5 column and washed with buffer A + 50 mM NaCl until the OD 200 nm of the eluat was below 0.05. The column was then eluted with buffer A + 150 mM NaCI. The fractions under the protein peaks were collected and the proteins precipitated by adding ammonium sulfate (final 45% saturation) stirring for 1 hr and centrifuged at 20,000 rpm. The precipitate was dissblved in 4 ml buffer B (20 mM Tris. 1 mM EGTA. 5mM OME. 50mM NaCI. DH 7.4). The 4 ml protein’ solution was’placed on a 115-x 90&n column of AcA 34 (Ultrogel IBF France) equilibrated with buffer B and eluted 10 ml/hr. Fractions (5 ml) were collected and the calpain activity in each fraction measured. The calpain-containing fractions were brought to 200 mM NaCI. lavered onto a 1.5 x IOcm column of CL4B phenyl-sepharose (Pharmacia) equilibrated with 20 mM Tris,.1 m%l EGTA, 5 mM /?ME, 200 mM NaCl, pH 7.4 and eluted with a linear NaCl gradient (200-O mM) in buffer B at 30 ml/hr. The fractions containing calpain activity were pooled and placed on a 1.5 x 7.5 cm column of H3B Procion Red-sephrose equilibrated with buffer B without NaCI. The column was eluted with 1mM urea in buffer B at 30 ml/hr. The fractions under the protein peak were pooled and concentrated with G 200, dialyzed overnight against 500 ml buffer B without NaCl and for 5 hr against 300 ml of the same buffer and stored at 4°C. The calpain activity thus obtained was stable for several months. (C) Measurement of calpain acliuity Calpain activity was measured with alkali-denatured Hammarsten casein (Merck reference 2242) as a substrate. The reaction mixture containing 0.4ml of the substrate solution (casein 0.4%, 50mM Tris, 1OmM BME, 1 mM CaCl,, 0.2 mM EGTA, pH 7.5) and 0.1 ml of enzyme solution was incubated on 30 min at 30°C and the reaction stopped by adding 0.5 ml 7.5% trichloracetic acid. The precipitated protein was removed by centrifugation and the optical density (OD) of the supernatant was measured at 280 nm. One arbitrary unit was defined as that giving an OD of I under the above experimental conditions. (0) Preparation of ankyrin and protein 4.1 The technique used was that of Tyler et al. (1980) with slight modifications. DE 52 column was eluted stepwise with KC1 concentrations of 50 mM, 120 mM (elution of protein 4.1). 200 mM (elimination of contaminants), 300 mM (elution of ankyrin). Purified ankyrin was stored at -80°C in 5 mM phosphate, 20 mM KCI, 1 mM EDTA, 0.5 mM BME, pH 7.6. Purified protein 4.1 was stored at -80°C in the same buffer containing 120 mM KC1 instead of 20 mM KCI. Ankyrin and protein 4. I were equilibrated by dialysis with 50 mM Tris, 10 mM BME, pH 7.5 prior to digestion with calpain.

et d.

ditions are indicated in the appropriate figure legends. The resulting peptides were separated by SDS-polyacrylamide gel electrophoresis according to Fairbanks et al. (1971) or Laemmli (1970) at the acrylamide concentrations and gradients indicated. The gels were stained with Coomassie Blue or with silver (Morrissey, 1981).

RESULTS I. Calpain digestion of whole ghost membrane Ankyrin to calpain

proteins

was the membrane protein most sensitive (Fig. 1). The Fairbanks gels indicated that

ankyrin was degraded at calpain/membrane protein ratio of l/l000 and the ankyrin completely disappeared at an enzyme/substrate ratio of l/100. This loss of ankyrin was accompanied by an initial increase in band 2.3 up to an enzyme/substrate ratio of l/250. Both band 2.3 and band 2.6 decreased and were lost at higher calpain concentrations. Protein band 3 (the anion channel) was lysed at calpain/protein ratios above l/500. The Laemmli gels showed that band 3 decreased and finally disappeared at high calpain concentration, as did protein 4.1; the two components of this band (4. la and 4.lb) decreased simultaneously. Laemmli gels also clearly displayed the mode of spectrin proteolysis. The p chain was more sensitive to proteolysis than the c( chain; it was cleaved into two components at calpain/protein ratios above l/500. The concentration of abnormal components increased and that of normal p chain decreased with increasing calpain concentration. The c( chain was cleaved only at high calpain concentration, at which ankyrin (and its derivatives) band 3 and protein 4.1 were completely degraded. Actin and glyceraldehyde 3-phosphate dehydrogenase (bands 5 and 6) were not sensitive to proteolysis. The gels contained many extra minor bands at high calpain/membrane protein ratios; there were degradation products of the high molecular weight components (spectrin, ankyrin) and band 3. Similar sequences of ankyrin, protein band 3 and protein 4.1 proteolysis occured when inside-out vesicles (IOV) were incubated with calpain under similar conditions (not shown). II. Calpain digestion

of puriJied spectrin

(Figs 2 and 3)

Crude spectrin was more resistant to proteolysis by calpain than by trypsin. High calpain concentrations produced high molecular weight peptides and a peptide of 74 kD. (E) Preparalion of spectrin Although actin is not sensitive to calpain in ghost Spectrin was obtained by extracting red cell ghosts in membranes it was degraded in crude spectrin prep0.3 mM phosphate, 0.1 mM PMSF, 0.1 mM BME, 0.1 mM arations (i.e. in water-soluble proteins) by high calEDTA, PH j.8 at 37-C for 30min (Liu el ai., 1982) and stored at -80°C in 50 mM Tris. 10 mM BME. Suectrin G( pain concentrations. Treatment of purified c( chain with a high calpain and /I chains were separated by HPLC in 3 M urea on a mono Q column, desalted on a PD 10 column and diluted concentration gave several peptides between 100 and to 0.05 mg/ml for calpain digestion (Yoshino and Marchesi, 200 kD, the 74 kD seen in whole spectrin digests and 1984; Lecomte ef al., in press). two other major peptides of ca 40 and 34 kD. The /I The concentrations of spectrin, ankyrin and protein 4.1 chain was more extensively cleaved by high calpain were measured at OD 280 using t = 10 for spectrin, c = 66,5 concentrations than was the a chain, and gave rise to for ankyrin and t = 8 for protein 4.1. several major peptides of ca 150, 74 and 30 kD. These (F) Calpain digestion qf ghosts and purified proteins results were very different from those obtained by limited tryptic digestion (Speicher et al., 1983) and no The details of calpain, ghosts or protein concentrations, reaction time, temperatures and other experimental concomparison could be made between the two.

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Fig. I. SDSPAGE pattern of peptides from calpain digestion of red cell ghosts. Red cells from volunteer donors were washed with 0.15 M NaCl and lysed in 5 mM Tris, 1mM EDTA, 0.3 mM PMSF pH 8. Ghosts were washed once with 5 mM TRIS, 1mM EDTA pH 8 and twice with 10 mM TRIS, 10 mM BME pH 7.5 and incubated with purified calpain at calpaimmembrane protein (w/w) ratios of l/5 to /IO00 for I hr at 0°C in IOmM Tris, IOmM BME pH 7.5 with (final concentration) I mM CaCl,, 0.2 zn M EGTA. Digestion was stopped by adding SDS-BME-sucrose solution and membrane proteins were separated by SDS-PAGE according to Laemmli (right) with a 5%-15% acrylamide gradient and to Fairbanks (left) with a 3.5512% acrylamide gradient. Lane I, control undigested membranes. Calpain/membrane protein ratio: lane 2, l/1000; lane 3, l/500; lane 4, l/250: lane 5, l/100; lane 6, l/25; lane 7, l/S.

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10

II

12

13

14

15

kD

Fig. 2. Digestion of spectrin and a and fi chains with calpain. Spectrin and isolated a and p chains were digested in 50 mM Tris, IO mM flME, I mM CaClr, 0.1 mM EGTA, pH 7.5, for 1 hr at 0°C with calpain/protein ratios of l/2, l/IO, l/SO, l/250 and the peptides were separated by SDS-PAGE according to Laemmli in 9% acrylamide gels. Lane I, untreated a chain; lanes 2-5, a chain peptides; lane 6, molecular weight standards (phosphorylase b 94,000; bovine serum albumin 67,000; ovalbumin 43,000; carbonic anhydrase 30,000; soybean trypsin inhibitor 20,100; a lactalbumin 14,400). Lanes 7-10, /I chain peptides; lanes I I-14, whole spectrin peptides; lane 15, untreated /I chain. Calpain/protein ratio: lanes 2, 7, 1l--1/2; lanes 3, 8, 12-l/10; lanes 4, 9, 13-l/50; lanes 5, 10, 14-l/250.

1481

?

2

3

4

5

6

7

a

9

10

11

kD 94 67

43

30

Fig. 3. Caipaln (and cf ohymotrypticf digestion of protein 4.1. Digestion was performed in 50mM Tris, IO mM ME, 0.8 mM CaCl,, 0. I mM EGTA, pH 7.5 far 1 hr at 0°C. Lanes I,6 and 1I-molecular weight standards (idem Fig. 2); lanes 2 and 3--undigested protein 4.1; lanes 4 and 5---a chymotrypsin peptides at enzyme/substrate ratio of i/200 (iane 4) and l/SO0 (lane 5); lanes 7-it&calpain peptides at caIpainj4. I ratios of Ii2 (lane 7) I/i0 @me 8): l/SO (Lane 9) and L/250 (lane IO).

1

2

3

4

5

6

-

20.1 -

14.4 Fig. 4. Peptides produced by c&pain digestion of ankyrin. Five different purified ankyrin samples were digested with catpain in 50 mM Tris, IO mM FME, I mM CaCI,, 0.2 mM EGTA, pH 7.5 for I hr at 0°C with at a catpain/ankyrin ratio of l/l0 (w/w). Peptides were separated by SDS-PAGE according to Laemmli on 5-U% acrylamide gradient and silver stained.

1482

Digestion of spectrin, protein 4.1 and ankyrin by calpain I III. Eflect of spectrin dimer limited digestion upcin the tetramerization process (Table 1)

Proteolysis caused a loss of both spectrin dimer and spectrin tetramer (not shown). The dimer/ tetramer ratio was slightly changed in a manner which suggested a moderate effect of dimer digestion on tetramer formation. The differences were small and noticeable only at highest calpain concentrations. IV. Digestion of protein 4.l(Fig.

4)

The peptide patterns produced by CLchymotrypsin and calpain digestion were different. The main digestion product at all calpain concentrations was a 30 kD peptide, which is also found after GI chymotrypsin lysis (Leto and Marchesi, 1984). Several minor bands, corresponding to peptides of h4, 50,00&72,000 were also produced by calpain digestion; but they differed from the peptides produced by c( chymotrypsin. V. Digestion of ankyrin (A) Purified ankyrin was digested by calpain at enzyme/substrate ratios from l/2 to l/500. Digestion

with a calpain/ankyrin ratio of l/10 produced a reproducible electrophoretic pattern (Fig. 4) which remained unchanged when digestion was continued for l-4 hr at 0°C. This pattern contained about 20 different peptides, most of them having molecular weights of lo&50 kD. The two most important peptides had mol. wt of 85,000 and 55,000 and were the most resistant to prolonged proteolysis. The other peptides, always present in lesser amounts, had mol. wt of 150,000, 95,000, 72,000, 67,000 and 51,000. Two lower mol. wt peptides (32,000 and 16,000 M,) were always produced. The higher molecular weight peptides were lysed during prolonged digestion with an increase in the 16,000 M, component. However, the 85,000 and 55,000 peptides remained without noticeable change up to 20 hr of digestion. (B) Identijcation of peptides bearing the spectrin and protein 3 interaction sites. The peptides bearing

the interaction sites with spectrin and protein 3 were identified by affinity chromatography on spectrinTable

I, Effect

of calpain on the spectrin tetramerization

Control without calpain digestion Calpain/dimer l/IO Calpain/dimer l/50 Calpain/dimer I /250 Calpain/dimer l/l000

% Dimer

% Tetramer

55.4 59 51.9 57.4 52. I

44.6 41 42. I 42.6 47.9

test in vitro

Ratio dimer/tetramer I .24 I .43 I .3-l I .34 I .08

Spectrin dimers were digested with calpain in 50 mM Tris, IO mM BME, I mM CaCI,, 0.2 mM EGTA pH 7.5 for I hr at 0°C. Digestion was stopped by adding EGTA (IO mM final concentration) and the digested dimers were dialyzed overnight against phosphate buffered saline (PBS) containing 0.1 mM PMSF, 0.1 mM BME and IO mM EGTA to eliminate Cd’+. The relative percentages of spectrin dimers and tetramers were determined by scanning of Coomassie Blue stained non-denaturing gels (3.5% acrylamide) after the in vitro tetramerization test (Dhermy et al., 1982) performed with dimers digested with different concentrations of calpain.

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sepharose and 43 kD-sepharose. Crude tetramerit spectrin was prepared by buffer extracting membranes by dialysis against low ionic strength at 4°C (Marchesi et al., 1970). The 43 kD fragment was purified by CI chymotrypsin proteolysis and DE 52 chromatography (Bennett and Stenbuck, 1980b). A 3Opg sample of ankyrin was digested by calpain with a l/5 enzyme/ substrate ratio for 30 min at 0°C. A 150 ~1 sample of ankyrin digest was incubated with 100~1 spectrinor 43 kD sepharose in 10 mM Tris, 100 mM KCl, 20 mM NaCl, 0.5 mM BME, 1 mM PMSF, pH 7.4 for 2 hr at 0°C. The sepharose was removed by centrifugation and the peptides in the supernatant were electrophoresed. The control was prepared by incubating the same amount of digested ankyrin with CNBr-activatedSepharose 4B, the sites of which had been blocked by triethanolamine. The gels were silver stained and the relative amounts of the main peptides were determined by densitometry. The peak areas corresponding to the main peptides remaining after contact with the affinity materials were compared with the peak areas obtained from the control. The results (Fig. 5) show that the 85 kD peptide was entirely bound to the 43 kD-sepharose and that 55% of the 55 kD peptide was bound to the spectrin-sepharose. Thus, the 85 kD peptide carries the ankyrin 43 kD-binding site and the 55 kD peptide carries an ankyrin spectrin-binding site. (C) Identzjication of peptides bearing the phosphorylation sites for cytosol and membrane protein kinases. Purified ankyrin was phosphorylated by the

cyclic AMP-dependent protein kinase extracted from erythrocyte membrane (Boivin and Galand, 1978), by the catalytic subunit of CAMP-dependent protein kinase (Sigma Chemical Co) or by casein-kinases I and II purified in our laboratory from erythrocyte membrane and cytosol, respectively (Boivin and Galand, 1979; Boivin et al., 1980). The main peptides phosphorylated by the CAMPdependent protein kinase had mol. wt of 85,000, 72,000, 67,000 and 55,000; there were other minor bands corresponding to mol. wt of ca 46,000 and 43,000. It was difficult to distinguish different peptides in the lower molecular weight region as there was a single diffuse spot between 16,000 and 30,000. The main peptide phosphorylated by the red cell membrane casein kinase had a M, 55,000. There were other bands of M, 72,000, 67,000 and 46,000 and several minor low molecular weight bands. The 85,000 M, peptide was not phosphorylated. Noticeably different results were obtained when ankyrin was phosphorylated by cytosol caseinkinase. Two major phosphorylated peptides had molecular weights of 85,000 and 67,000; the former correspond to the peptide bearing the 43 kD interaction site. There was considerable phosphorylation at 28,000 M,, due to auto-phosphorylation of the protein kinase subunit. Thus, the peptide carrying the interaction site with 43 kD fragment of protein 3 is phosphorylated by the CAMP-dependent protein kinase and the cytosolic casein kinase. The 55,000 peptide carrying the spectrin interaction site is phosphorylated by the CAMP-

P. BOIVIN et al.

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Fig. 5. Density scans of ankyrin peptides not bound to affinity chromatography columns after polyacrylamide gel electrophoresis and silver staining. From top to bottom: control: sepharose CNBr column saturated with triethanolamine; peptides not bound to the 43 kD-sepharose column; peptides not bound to the spectrin-sepharose column; peak 1: 85 kD peptide-peak 2: 72 kD peptide-peak 3: 55 kD peptide. The 85 kD peptide was completely extracted by the

43 kD-sepharose column; most of the 55 kD peptide was bound to the spectrin-sepharose column. dependent protein-kinase, as previously reported (Weaver ef al., 1984a,b) and by the membrane casein kinase (Fig. 6). DISCUSSION

There is very rapid proteolysis of ankyrin, protein 4.1, anion channel (protein band 3) and to a lesser degree spectrin when red cell ghosts are prepared without EDTA or with Ca*+ added in the hemolysis and washing media. The same lysis occurs when resealed ghosts or IOVs are incubated with purified calpain or when leaky ghosts are incubated with calpain in low Ca*+ medium. Our results are thus similar to those previously reported (Pant et al., 1983; Croall et al., 1986). However, we did not observe the 195,000 M, proteolytic fragment of ankyrin reported by Hall er al. (1987). Spectrin was relatively resistant to calpain proteolysis: its digestion in the membrane gave rise to successive shorter fragments derived from the a and /? chains, suggesting sequential similar lysis of both chains. But the /l chain was more sensitive to

proteolysis than the a chain and its degradation products appeared before those of the a chain. Other membrane proteins, such as actin and glyceraldehyde 3-phosphate dehydrogenase are calpain-resistant in whole membranes. But actin is degraded when it is freed from the membrane by extraction at low ionic strength. Many of the substrates of calpain are calmodulinbinding proteins [see review by Wang et al. (1989)]. The a chain of fodrin (brain spectrin) binds calmodulin at the junction of the eleventh and twelfth 106-aminoacid repeats and calpain cleaves a fodrin (Siman et al., 1984) by acting on a site located 10 residues from the calmodulin-binding domain (Harris et al., 1988, 1989). This domain contains an aminoacid sequence rich in proline, glutamic and aspartic acid, serine and threonine (the PEST sequence) which is recognized by calpain (Rogers et al., 1986). This sequence does not exist at the same site in the erythrocyte a chain. Many short-lived proteins contain PEST-sequences (Rogers et al., 1986). Thus a PEST hypothesis could fit well with the very fast degradation of excess c[ and /l chains synthesized in erythroid precursors during the biosynthesis of the red cell membrane skeleton. Unassembled /I chains are degraded in < 15 min and a chains in ca 2 hr (Woods and Lazarides, 1985; Lazarides and Woods, 1989), but spectrin oligomers are stable in the membrane and after extraction from circulating red blood cells. The presence or absence of PEST sequences remains to be determined as the complete aminoacid sequences of the a and b chains is not yet available. The c( and fi chains of erythrocyte spectrin bind calmodulin (Sobue et al., 1981; Berglund et al., 1986; Boivin and Galand, 1987), but it is doubtful that this binding plays a role in calpain digestion since purified spectrin and isolated a and /l chains are lysed by calpain in the absence of calmodulin. Limited digestion of spectrin dimers by calpain does not severely impair tetramerization in vitro. This result could indicate that the sites of dimer-dimer interactions must be relatively protected from calpain at least up to a certain amount of proteolysis. The peptides lost during the initial steps of a and /l chain shortening are probably not derived from the heads of the two chains, which bear the tetramerization sites. In this context it is worth noting that, as reported by Harris et al. (1989) cleavage of the fodrin /3 subunit alone does not destroy the tetrameric form of the molecule, whereas proteolytic cleavage of both subunits impairs tetramerization. However, while calmodulin and Ca*+ are required for degradation of the fodrin p chain by calpain, the erythrocyte spectrin p chain in very sensitive to calpain in the presence of Ca*+ even without calmodulin. Ankyrin is very sensitive to proteolytic agents. Indeed this sensitivity led to the discovery of ankyrin and related proteins 2.2, 2.3 and 2.6 via the 72,000 M, fragment obtained by proteolysis (Bennett, 1978). The ankyrin on open red cell membranes and IOVs is lysed by calpain in the presence of Ca*+. Proteolysis of purified ankyrin by trypsin, a chymotrypsin and NTCB was used to identify relatively resistant peptides, among which were the functional domains interacting with spectrin and the 43 kD fragment

94 67

43

30 20.1

Fig. 5. Identification of ankyrin peptides phoshorylated by cAMP dePendent protein kinase and membrane and cytosol casein kinases. Samples (12.5 fig) of ankyrin were phospho~lated in 20 mM Tris, 5 mM BME, 0.5 mM EGTA, pH 7.4 plus 3.25 PM Y[~~P]ATP and added to the above buffer: 0.15 M KCI, &Of3 M Mg acetate for the membrane and cytosof casein kinase; 0.013 M Mg acetate and S ,uM CAMP for the CAMP dependent protein kinase. Reaction mixtures were incubated for 30 min at 3o”C, dialyzed in a microdialyzer (Pierce) for 1 hr against 20 mM Tris, I mM EGTA, 5 mM BME, pH 7.5 buffer and digested with calpain in 50 mM Tris, 10 mM /?ME, 0.8 mM CaCl,, 0.2 mM EGTA, pH 7.5 for 30 min at WC with a I/S ~~yme/substmte ratio. Reactions was stopped by adding SDS and /?ME (final concentrations 2% SDS and 5% @ME) and the reaction mixture, was boiled for 3 min. Peptides were separated by Laemmli SDS-PAGE on a 5-t 5% acrylamide gradient gel and identified by autoradiography on X-Omat Kodak film using a amplifying screen (DuPont cbronex). Left, Coomassie Blue stained gets after phospho~Iation and digestion: lane t, molecular weight standard; lane 2, ph~pho~lation by membrane casein kinase; lane 3, phosphorylation by cytosol kinase; lane 4, phosphorylation by CAMP-dependent protein kinase. Right, lanes 2, 3, 4 after autoradiography.

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Digestion of spectrin, protein 4.1 and ankyrin by calpain I

(Wallin et al., 1984). The electrophoretic patterns of peptides resulting from calpain digestion are not similar to those obtained by tryptic or CL chymotryptic digestion. But the two main peptides from calpain digestion carry interaction sites for spectrin and 43 kD fragment, as do those of a chymotryptic digestion, although their molecular weights are somewhat different. It is also noteworthy that ankyrin does not bind calmodulin, despite its high sensitivity to calpain proteolysis. One of the most interesting aspects of the digestion of ankyrin by calpain concerns the peptides carrying the phosphorylation sites. The ankyrin on whole red cells and ghosts is phosphorylated by the CAMP-dependent protein kinase on a 55,000 M, tryptic peptide containing the spectrin interaction site. Purified ankyrin can by phosphorylated in vitro by both red cell membrane and cytosol casein kinases I and II (Lu et at., 1985). Both kinases should have the same functional effect on ankyrin: phosphorylation decreases the affinity of ankyrin for the 43 kD fragment, for the spectrin tetramer, but not for the dimer (Lu et al., 1985; Cianci et al., 1988). However, the phosphorylation sites for the CAMP-independent kinases had not been determined nor compared with the known phosphorylation sites for the CAMP-dependent kinase. Analysis of the peptides from calpain digestion of ankyrin phosphorylated with the membrane and cytosol casein kinases shows that the phosphorylation site varies with the kinase used. Our results indicate that the spectrin binding site is phosphorylated by CAMP-de~ndent protein kinase [as previously demonstrated by Weaver and Marchesi (1984) and Weaver er al. (1984)] and by the membrane casein kinase. The anion channel binding site is phosphorylated mainly by the cytosol casein kinase, which is a casein kinase II phosphorylating serine and threonine, but also by the CAMP-dependent protein kinase. As ankyrin phospho~lation reguiates its molecular interaction with spectrin and band 3, the membrane casein kinase would be involved in regulating the ankyrin-spectrin relationship, while cytosol casein kinase would be implicated in the ankyrin-band 3 interaction. However, calpain might regulate anky~n-Satan interaction in other ways. Very limited digestion of ankyrin yields a 20,000 M, fragment, while the affinity of the remaining 195,000 fragment for IOVs is reduced to l/S of that of the whole protein 2.1. Protein 2.2 is an activated form since its affinity for spectrin is three times that of protein 2.1 and it has twice as many high a~nity sites for the 43,000 M, fragment (Hall and Bennett, 1987). But protein 2.2 is not a product of protein 2.1 proteolysis (Croall et al., 1986). Calpain might have several functions in the red cell besides its possible role in stabilizing the cytoskeleton and in membrane physical properties (Shields et al., 1987). It seems to regulate the activity of cytosol pyruvate-kinase (Dahlquist-Ed~rg and Ekman, 1981); it activates Ca’+ ATPase by partial lysis so intervening in cell calcium regulation (Au, 1987; Wang et al., 1988a,b; Au et aI., 1989; James et al., 1989); it could modify, by limited proteolysis (Kishimoto et al., 1984), the activation of protein

1487

C, as it does in neutrophils (Melloni et al., 1986) and that of a calmodulin-dependent phosphatase (Tallant ef al., 1988). However, the normal Ca2+ level in the red cell is very low and before calpain could play a physiolo~~l role it would have to be autolyzed after membrane binding and find a sufficient Ca2+ concentration (Pontremoli et al., 1985a,b; Cong et al., 1989; Inomata et al., 1989). Such conditions may well occur in pathological states such as drepanocytosis, hereditary spherocytosis and other situations in which there are primary or secondary membrane abnormality and an increase in intracellular Ca2+ concentrations. This particular aspect of red cell calpain proteolysis is now being examined in our laboratory. kinase

SUMMARY

Purified erythrocyte calpain I has been used to study the lysis sequences of several red cell membrane proteins and the resulting peptides. The most calpainsensitive protein in whole membrane was ankyrin, followed by protein 4.1, protein band 3 and spectrin. Spectrin was relatively resistant to proteolysis, but, the spectrin fl chain was more sensitive than the u chain, although the two chains seemed to have similar sequential lysis patterns. Limited calpain proteolysis of normal spectrin dimer did not impede tetramerization in vitro. Purified protein 4.1 was rapidly lysed by calpain. The resulting peptides differed from those obtained by CLchymotr~sin lysis; the main one was a 30 kD peptide relatively resistant to lysis. Lysis of ankyrin in well-deflned experimental conditions furnished a reproducible peptide pattern with two main peptides, 85 and 55 kD. Affinity chromatography on spectrin and 43 kD-sepharose identified the 85 kD peptide as that bearing the 43 kD-ankyrin binding site and the 55 kD as the peptide carrying the spectrin-ankyrin interaction site. The 85 kD peptide was selectively phosphorylated by cytosol casein kinase and the 55 kD by the membrane casein kinase. These results indicate that interactions between ankyrin and spectrin may depend on the phosphorylation of ankyrin by membrane casein kinase, and the interaction between ankyrin and protein band 3 internal fragment (43 kD) on phospho~lation by cytosol casein kinase. REFERENCES Au K. S. (1987)

Activation of erythrocyte membrane Ca*+-

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