Calmodulin inhibits the phosphorylation of spectrin in vitro

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 246, No. 1, April, pp. 469-4’7’7, 1986

Calmodulin Inhibits the Phosphotylation DIETER MARETZK12 Department

of Biochemistry,

AND

of Spectrin in Vitro’

HANS U. LUTZ3

Swiss Federal Institute of Technology, ETH-Zen&urn, CH 8092 Zurich, Switzerland Received November

lo,1985

In vitro phosphorylation of purified spectrin dimer was studied in the presence of Ca2+-calmodulin (CaM). CaM inhibited autophosphorylation of the fi subunit of spectrin. The inhibitory effect (65% at a 32-fold molar excess) appeared to be due to a weak interaction of CaM with spectrin. CaM was similarly effective in a phosphatase-stimulated autothiophosphorylation of the p subunit with [T-~~S]ATP. Hence, its inhibitory effect was not due to stimulation of a spectrin-associated phosphatase activity. Phosphorylation of spectrin by the catalytic subunit of a CAMP-dependent protein kinase occurred in both subunits (1984, FEBS Lett. 169,323). CaM selectively inhibited a CAMPdependent phosphorylation of the (Ysubunit of spectrin to 30% at two CaM per spectrin. It was ineffective on the CAMP-dependent phosphorylation of the p subunit up to a 32fold molar excess. These results yield functional evidence for a CaM-spectrin interaction. They further suggest that CaM can regulate the extent of a CAMP-dependent phosphorylation of the (Ysubunit of spectrin. o 1986 Academic press, I,,~. The maintenance of red cell shape requires cellular ATP (1). Its effect cannot be explained entirely on the basis of noncovalent interactions (2, 3), implying that phosphorylation of membrane components plays an important role. Nevertheless, none of the in vitro interactions of skeletal proteins was functionally dependent on protein phosphorylation: spectrin binding to actin (4) as well as to inside-out vesicles was independent of spectrin being phosphorylated in its /3 subunit (5). Likewise, the shape change from discocytes to echinocytes occurred long before the fi subunit of spectrin was dephosphorylated (5). Thus, spectrin phosphorylation appeared unlikely to mediate skeletal anchorage and binding to actin. However, the functional aspects had only been studied on two phos1 Dedicated to Professor Carl Martius on the occasion of his 80th birthday. ‘Permanent address: Institute of Physiology and Biological Chemistry, Humboldt University, Hessisches Strasse 3, DDR 1040 Berlin, Germany. 3 To whom correspondence should be sent. 46 9

phoforms of spectrin, a CAMP-independent and an autophosphorylated form of spectrin (4-7). Both types of phosphorylation result in exclusive labeling of a C-terminal segment of the p subunit of spectrin, whether studied on purified spectrin or in ghosts (8,9). A CAMP-dependent phosphoform of spectrin was not studied. It was considered unlikely to occur, because low ionic strength extracted spectrin was labeled to comparable extent whether obtained from membranes incubated with [y32P]ATP in the presence or absence of CAMP (10). In contrast to this, Yawata et al. (11) and Greenquist and Shohet (12) found a CAMP-dependent phosphorylation of both spectrin subunits in isolated membranes. Moreover, purified spectrin dimer was in fact a substrate for a CAMP-dependent protein kinase and was phosphorylated in both subunits primarily in peptides that did not show autophosphorylation (13). Both lines of evidence for a CAMPdependent phosphorylation of spectrin remained a curiosity of little relevance to the in vivo situation, because others could not 0003-9861/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

470

MARETZKI

detect [3zP]phosphate labeling of the (Ysubunit of spectrin (8-10, 14). The contradictory results might be due to experimental differences, because EGTA was present only in the incubation mixtures used by Yawata et al. This suggested to us that isolated membranes contained an inhibitor of a CAMP-dependent phosphorylation of spectrin which was inactivated by EGTA. This treatment was likely to abolish the effect of Ca’+-calmodulin (CaM)4 that has recently been shown to interact with spectrin (15-18). Therefore, we investigated the effect of CaM on in vitro phosphorylation of spectrin. In vitro phosphorylation of purified spectrin dimer avoided the difficulties encountered in phosphorylation of membranes and allowed assessment of the effect of CaM on the generation of two different phosphoforms of spectrin, an autophosphorylated and a CAMP-dependent phosphoform (13). Here we report that CaM selectively inhibited the CAMP-dependent phosphorylation of the cxsubunit of spectrin already at low CaM/spectrin ratios, while it inhibited autophosphorylation of the p subunit only at unphysiological CaM concentrations. The findings clarified the inhibitory potential of CaM and may explain inconsistent results with respect to CAMP-dependent phosphorylation of the a subunit of spectrin in isolated membranes. MATERIALS

AND

METHODS

Human erythrocytes from fresh blood collected in citrate-phosphate-dextrose fortified with adenine were freed from white cells using &cellulose and microcrystalline cellulose (19). Erythrocytes were washed three times with buffer containing 140 mM NaCl, 5 mM KCl, 10 mrd sodium phosphate, 0.5 mM EDTA, 3 mM glucose (pH 7.4). Membranes were prepared by Steck’s modification (20) of Dodge’s procedure (21) with 5 mM sodium phosphate buffer containing 1 mM EDTA (pH ‘7.4), except that the first

’ Abbreviations used: y-S-ATP, adenosine 5’-0-(3thiotriphosphate; [Y-~S]ATP, adenosine 5’-0-(3-[?S]thiotriphosphate); CaM, calmodulin in the presence of CaClz as specified; DTT, dithiothreitol; EDTA, ethylendiaminetetraacetic acid, EGTA, ethylene glyco1 bis(@-aminoethyl ether) N,N,l\r,hr-tetraacetic acid; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

AND

LUTZ

wash buffer contained 0.4 mM diisopropylfluorophosphate (22). Spectrin dimer was purified as described (13) according to the method of Bennett and Branton (23). In two preparations the protease inhibitors diisopropylfluorophosphate and phenylmethylsulfonyl fluoride were omitted (where indicated). Purified spectrin dimer from Sepharose CL-4B column fractions was dialyzed against 50 mM Tris-HCl, 10 mM KCl, 40 mM NaCl, 0.2 mM DTT (pH 7.4) and concentrated by ultrafiltration to l-2 mg protein/ml. Calmodulin was prepared from ox brain by phenylSepharose affinity chromatography (24). Bovine erythrocyte calmodulin was purchased from Fluka AG, Buchs. A reference sample of highly purified calmodulin was a gift from Dr. J. Krebs, ETH, Zurich. Calmodulin samples were homogenous as judged by SDS-PAGE. Phmphmylatim of sped& diwm. Phosphorylation of spectrin was assayed in 50 mM Tris-HCl, 10 mM KCl, 40 mM NaCl, 5 mM MgClz, 1 mM DTT, (pH 7.4) at 30°C in a total volume of 50 pl(l3) containing 1330 pg of spectrin dimer. [y-82p]ATP (Amersham, U. K.) was added at 0.1 mM with specific activities of 250 to 450 cpm/pmol. For thiophosphorylation 0.03 to 0.1 mM yS-ATP (Boehringer-Mannheim) was mixed with adenosine 5’-[y-%S]thiotriphosphate (Amersham) to give a specific activity of 2 nCi/pmol at 100 pM ySATP in the 507.d assay. The catalytic subunit of the CAMP-dependent protein kinase from bovine heart (Sigma, St. Louis, MO.) was dissolved in 50 mM DTT and added with a capacity to transfer about 50 pmol of phosphate per minute at 30°C. Calmodulin was added at the given concentrations together with CaClz in concentrations equivalent to the four binding sites of this protein. In autophosphorylation assays 5 pg ovalbumin (Sigma) was included. Ovalbumin was without influence on the pattern and extent of spectrin phosphorylation; however, it eliminated the diffuse background on autoradiographs from SDS-PAGE lanes loaded with autophosphorylated samples. Reactions were initiated by the addition of labeled ATP and stopped with electrophoresis sample buffer containing 50 mM Tris-HCl, 10 mM EDTA, 50 mM DTT, 5% glycerol, 2% SDS, and 0.1 mg bromophenol blue (pH 6.8). Samples were denatured for 3 min in a boiling water bath. The reduced material was alkylated with a slight excess of N-ethylmaleimide. SDS-polyaerylamide gel electrophoresis and incorporation of label Alkylated samples of phosphorylated spectrin dimer were electrophoresed on 4.5% polyacrylamide gels utilizing the discontinous buffer system of Neville (25) as outlined elsewhere (26). The gels were stained with Coomassie blue and dried. Incorporation of 82p label into polypeptides was revealed qualitatively by autoradiographs using Kodak X-Omat SO 282 films, exposed in intensifier cassettes at -70°C. [azPlphosphate incorporation into spectrin was quantitated by Cerenkov emission from gel pieces. The a

CALMODULIN

INHIBITS

SPECTRIN

and p subunits of spectrin were excised separately from dried gels. Gel pieces were vortexed at 50°C in distilled water for 2 b prior to counting. The incorporated label above background and the specific activity of labeled ATP served to calculate the extent of phosphorylation in moles of [32P]phosphate. The number of moles of each subunit of spectrin was determined from the amount of spectrin dimer loaded (M, = 460,000) onto the gels. The data are given in moles of phosphate incorporated per mole of spectrin subunit, if not otherwise indicated. Thiophosphorylation of spectrin with [y-?S]ATP was analyzed as follows: gels run with such samples were immersed in Amplify (Amersham). Dried gels were exposed to X-ray films at -70°C for qualitative analyses. Thiophosphorylation of the @ subunit was quantitated by determining the label in gel pieces containing the stained fl subunit of spectrin. The gel pieces were rehydrated and treated with NCS solubilizer (Amersham) as outlined elsewhere (27) prior to scintillation counting. Control pieces of the same size were cut from the gel to determine the background which was measurable particularly when the samples were alkylated. Therefore, samples labeled with [y%S]ATP were reduced, but not alkylated, in some experiments. RESULTS

Autophosphorglatim

of spectrin

Purified

spectrin dimer catalyzed self-phosphorylation of its /3subunit (Fig. 1) in a strongly ATP-dependent way (13). Autophosphorylation was pronounced at 1 mM [y-32P]ATP (0.18 mol of phosphate/m01 of spectrin), but consumed large amounts of labeled ATP. Therefore, we examined autophosphorylation of spectrin at 0.1 mM labeled ATP. At this concentration 21-52 mmol of phosphate were incorporated per mole, if spectrin was prepared in the presence of protease inhibitors as outlined. In their absence or upon storage of spectrin, the extent of labeling declined (Table I). Autophosphorylation required a phosphoryl transfer, because [Y-~~P]ATP could be replaced by [Y-~~S]ATP (Table II), but not by [w~~P]ATP (not shown). Furthermore, autothiophosphorylation of spectrin with [y?S]ATP was greatly enhanced by a simultaneous dephosphorylation of spectrin (Table II). Autophosphorylation was inhibited by CaM (Figs. lb-d). The inhibition was dependent on the concentration of CaM and reached 65% at a 32-fold molar excess of

471

PHOSPHORYLATION

l2-

a

b

c

d

e

f

a’

b’

d’

e’

FIG. 1. In vitro phosphorylation of spectrin dimer by autophosphorylation and by the catalytic subunit of a CAMP-dependent protein kinase in the presence and absence of CaM. Fifteen micrograms of spectrin dimer was incubated for 90 min at 30°C with 0.1 mM [y-32P]ATP as outlined for autophosphorylation (a-f) and in the presence of the catalytic subunit of a CAMPdependent protein kinase in al-e’. Calmodulin and CaClz were present in the incubation mixtures at the indicated concentrations: (b, b’), 20 j~~/80 pM, (c) 5 @/20 pM, (d, d’) 1 &4 PM. In e and e’ CaClz was added at 80 pM, and in f, CaClz (80 PM) and 1 mM EGTA were present. Reactions were stopped by adding electrophoresis sample buffer. The samples were electrophoresed on SDS-PAGE containing a total concentration of 4.5% acrylamide in the running gel. At the end of the run the lower part of the gel was cut off to remove the bulk of unreacted [y-?]ATP and free “‘Pi. The rest of the gel was stained, dried, and exposed to X-ray films. An autoradiograph (‘*P) and the stained gel are shown. The numbers 1 and 2 mark the positions of the 01and 0 subunits of spectrin.

CaM (20 PM) over that of spectrin (Fig. 2A). The inhibition by CaM was not eliminated by extending the incubation times (Fig. 3A). The inhibitory effect was similar with three different calmodulin preparations (see Materials and Methods and Table I). Ca2+ ions alone were ineffective (Table I and Fig. le). Autothiophosphorylation of spectrin was inhibited to the same extent as autophosphorylation with [y-32P]ATP, whether assayed in a phosphatase-stimulated or unstimulated assay system (Table III). The effect of CaM was therefore not due to stimulation of a cryptic, spectrin-associated phosphatase. Inhibition was most

472

MARETZKI

AND

TABLE

LUTZ

I

PHOSPHATE INCORPORATION INTO PURIFIED SPECTRIN DIMER INCUBATED WITH 0.1 mM [y-32PjATP WITHOUT PROTEIN KINASE (AUTOPHOSPHORYLATION) AND IN THE PRESENCE OF THE CATALYTIC SUBUNIT OF A CAMP-DEPENDENT PROTEIN KINASE’ CAMP-dependent phosphorylation Autophosphorylation In @subunit

5Pe of spectrinb

Addition None

In p subunit

n

27-56 24 13 28-36 18-24 15

81-140 95 80 85-110 83-96 89

24 2 3 4 2

21-46 26 5 18-45 6-16 9

PI c a

Ca*+ (80 /.tM) Calmodulin (20 pM) + Ca*+ (80 PM)

In (Y subunit

’ Spectrin dimer preparations were incubated as described for 90 min at 30°C. In each experiment the [saP]phosphate incorporation was determined for the (Y and fi subunits separately in different spectrin preparations. The data are given in millimoles of phosphate incorporated per mole of spectrin subunit. The data represent the range of values obtained with the highest and lowest value given, if more than two (n) experiments were carried out. Averages are given for n = 2. ‘Type of spectrin preparation: (a) in the presence of protease inhibitors as outlined under Materials and Methods; (b) without protease inhibitors; (c) prepared as in (a), but stored for 2 weeks at 4°C.

likely due to CaM binding to spectrin, although with a low affinity. Cyclic AMP-dependent

incorporation into the /3subunit of spectrin exceeded autophosphorylation by fivefold in the presence of the catalytic subunit of a CAMP-dependent protein kinase at 0.1 mM [T-~~P]ATP. In contrast to autophosphorylation, a CAMP-dependent thiophosphorylation of the /3subunit of spectrin was not stimulated by dephosphorylation of

phosphmylation

of spectrin. Purified spectrin dimer was labeled in both subunits when phosphorylated in the presence of a CAMP-dependent protein kinase or its catalytic subunit [Ref. (13) and Fig. la]. The extent of phosphate TABLE

II

THIOPHOSPHOR~LATION OF THE p SUBUNIT OF SPECTRIN DIMER WITH [y-%]ATP INTHEPRESENCEOFANALKALINE PHOSPHATASE"

Addition

Incubation time (min)

None Phosphatase 1 unit 2 units

60 120 60 120

Autophosphorylation

CAMP-dependent phosphorylation

100 (2400 cpm)

100 (87’70 cpm)

139 210 288 (n = 2) 432 (n = 2)

106 + 14 (n = 4)

a Spectrin dimer was incubated with or without the catalytic subunit of a CAMP-dependent protein kinase as outlined, 0.03 to 0.05 mM [y-%]ATP and an alkaline phosphatase (No. 108138, Boehringer-Mannheim) as indicated. Incorporation of label into the /3 subunit of spectrin was quantified and is shown as a percentage of the corresponding control. An average value of incorporated label in cpm is given for the controls to show the extent of stimulation by the catalytic subunit. The number of independent experiments (n) including corresponding controls is indicated.

CALMODULIN

INHIBITS

10

SPECTRIN

30

473

PHOSPHORYLATION

10

50

Calmodulin/Spcctrin

(moles/mol)

FIG. 2. Inhibitory effect of CaM on spectrin autophosphorylation and on spectrin phosphorylation by the catalytic subunit of a CAMP-dependent protein kinase. Type a spectrin dimer (see Table I) was phosphorylated in the presence of increasing concentrations of Ca*+-calmodulin. Phosphate incorporation into spectrin from [T-~P]ATP was determined as a percentage of the appropriate control following incubation for 90 min at 30°C. The results are given as percentages inhibition and represent the averaged values from two independent experiments. (A) Autophosphorylation, phosphorylation of the /J’subunit@); (B) CAMP-dependent phosphorylation of the (Y subunit (A) and of the /3 subunit (0) of spectrin.

spectrin (Table II). This together with previously shown phosphopeptide maps (13) demonstrates that the introduced label was located primarily in sites other than those undergoing autophosphorylation. The formation of a CAMP-dependent phosphoform of the (Ysubunit of spectrin was inhibited by CaM (Figs. lb’, d’, 3). Inhibition of (Y subunit phosphorylation

reached 30% with two calmodulins per spectrin and 50% at a ratio of 30 or more (Fig. ZB). The concomitant labeling of the p subunit of spectrin was slightly reduced in the presence of CaM (Figs. 1, b’, d’, 2). The slight reduction was due to inhibition of ongoing autophosphorylation as evident from phosphopeptide analyses (Fig. 4) that revealed a decrease in labeling of those

6 i?!iz 1

z 8 t 8 .c-

0.6

s0 ‘p

0.4

ir

01.2 L 0

30

60

90

120

Incubation

0 time

30

60

90

im

(min)

FIG. 3. Time course of phosphate incorporation into spectrin in the presence and absence of CaM. Reaction mixtures contained 14.5 pg spectrin dimer, 0.1 mM [y-81p]ATP (A), and the catalytic subunit of a CAMP-dependent protein kinase (B), and were incubated with (filled symbols) or without (open symbols) Caa+-calmodulin (20 PM, 32-fold molar excess) for the given times. (A) Autophosphorylation of the /I subunit of spectrin (0, n ); (B) CAMP-dependent phosphorylation of the (Y subunit (A, A) and of the @subunit (0,O) of spectrin in the presence of the catalytic subunit of a CAMP-dependent protein kinase.

474

MARETZKI TABLE

III

AND

LUTZ

strength buffer rather than with, e.g., urea, we cannot exclude that minute amounts of EFFECTOF CaM ONAUTOTHIOPHOSPHORYLATION a CAMP-independent protein kinase were OF THE fi SUBUNIT OF SPEC~RIN IN THE PRESENCE copurified. We think, however, that autoANDABSENCEOFANALKALINE PHOSPHATASE' phosphorylation is more than an operaNO tional term, because the ATP concentration Addition phosphatase With phosphatase required for substantial labeling of spectrin (13) was considerably higher than that None 100 (2400 cpm) 100 (7880 cpm) required for a CAMP-independent protein Calmodulin (20 PM) Ca8’ (80 phi) kinase, as was reported earlier for an iso32 (n = 2) 232,14(7&=4) tonically prepared spectrin (7). Autophosa Spectrin dimer was incubated with or without 2 units of phorylation and a CAMP-independent an alkaline phosphatase (Table II), 0.03 to 0.05 mx [-r-86sfiTP phosphorylation of spectrin introduced laas outlined, and CaM where indicated. After incubation for bel into the same C-terminal region of the 2 h the samples were electrophoresed and the radioactivity in the fl subunit was determined. The data are given as perp subunit which normally contains phoscentages of control. Average cpm for controls are listed to phate (9). This is further supported by the illustrate the extent of stimulation by the added phosphatase; finding that autothiophosphorylation of n = number of experiments. the p subunit of spectrin was enhanced by a simultaneous dephosphorylation of native spectrin dimer. Autophosphorylation peptides that were also labeled in autoof the /3 subunit was due to a phosphoryl phosphorylated spectrin (note the phostransfer on the basis of experiments with phopeptide marked by stars in Fig. 4). [y-%]ATP and [LY-~~P]ATP. Thus, CaM did not inhibit the formation of Autophosphorylation was inhibited suba CAMP-dependent phosphoform of the /3 stantially by CaM at a molar ratio of 32 subunit of spectrin. It selectively inhibited CaM to spectrin. In view of the stimulatory the CAMP-dependent phosphorylation of role of CaM on certain phosphatases (2% the (Y subunit. This finding suggests that 30), it was important to study whether inthe CAMP-dependent phosphorylation of hibition was due to stimulation of a conthe two subunits proceeds by different mechanisms. This is not unlikely, because taminating, spectrin-associated phosphathe (Ysubunit of spectrin was labeled nei- tase. This was not the case, because CaM ther at 1 mM nor at 0.03 mM [T-~~S]ATP in inhibited a phosphatase-stimulated autothiophosphorylation of spectrin to the the presence of the catalytic subunit same extent as the unstimulated one. The of CAMP-dependent protein kinase (not highest concentration of CaM (20 pM) shown). tested yielded a 65% inhibition of the reaction. This concentration of CaM exceeded DISCUSSION its in vivo concentration by a factor of 8 In vitro phosphorylation of purified (15). Though high, the concentration was spectrin dimer was helpful in verifying the required in view of the low affinity of CaM possible modes of phosphorylation and to spectrin [14 and 22 PM (31), 15 pM (32)]. their regulation. This approach reduced the The physiological relevance of this type of number of variables as compared to phos- inhibition is unknown, because there is unphorylation of isolated membranes. Nev- certainity about the accessibility and loertheless, the simplified system was yet cation of the CaM binding domains on more complex than was hitherto recog- spectrin. While some authors reported CaM binding to native spectrin dimer (16, 31, nized. Phosphorylation of the /3 subunit of 32), others could only verify CaM binding spectrin in the absence of added kinase is to both subunits in their denatured form called autophosphorylation here. Since (15). Sears et al. (18) localized one CaM spectrin dimer was purified from a low binding domain to the C-terminal region ionic strength extracted spectrin by gel fil- of the p subunit of spectrin. This same retration in the presence of a moderate ionic gion of native spectrin also contains the

CALMODULIN

INHIBITS

.

SPECTRIN

Coomassie m

a

b

c

b’

c’

FIG. 4. One-dimensional peptide and 32P-phosphopeptide maps from electrophoretically separated spectrin subunits. Samples (22 rg) from two different preparations of spectrin dimer (type a) were phosphorylated in the absence (a) or the presence of the catalytic subunit of a CAMP-dependent protein kinase (b, b’, c, c’) either with 20 pM ealmodulin and 80 PM CaClz (c, c’) or without (b, b’) as outlined. Samples containing 13 pg protein were electrophoresed on 4.5% SDS-PAGE. The regions of the gel containing the two spectrin subunits were cut out and were processed as outlined elsewhere (13) for subsequent, one-dimensional peptide mapping on 14% SDS-PAGE loaded with 8 pg V,, protease from Staphyloccceus aweus The Coomassie blue-stained gel reveals rows of peptides which were generated from the electrophoretically separated spectrin subunits. Each row of peptides is marked with 1 or 2 to indicate the parent subunit from which the peptides were generated. The autoradiograph (9) shows the [“PIphosphate-labeled peptides and was exposed for 4 days for b, c, b’, c’, and twice as long for a. The heavy arrows on the righthand side of the panels mark corresponding peptides and =P-phosphopeptides. The small arrows in b’ point to corresponding peptides and phosphopeptides of the (Ysubunit of a CAMP-dependent phosphoform of spectrin. The stars mark the position of the major auto-

PHOSPHORYLATION

475

four (8) or more (33) phosphate residues, a tenth of which is exchangeable in in vitro phosphorylation assays (9). Thus, it seems probable that CaM binding to this domain could inhibit autophosphorylation of spectrin. The CAMP-dependent phosphorylation of both spectrin subunits has some interesting features: phosphorylations of the (Y and /3subunit differ in their mode of phosphoryl transfer and their inhibition by CaM. While both forms were obtained with [T-~‘P]ATP, [r-%S]ATP exclusively thioph,osphorylated the B subunit. CaM selectively inhibited CAMP-dependent phosphorylation of the (Ysubunit. Therefore, it is unlikely that inhibition was due to CaM binding to added kinase. Thus, CaM most likely exerted its effect by binding to spectrin. CaM binding to spectrin was observed with affinities ranging from >150 nM to 22 PM (17, 31), but little is known of the localization of the binding sites and their individual affinities (17,lS). None of the CaM binding studies offers a high-affinity binding site on the CYsubunit of spectrin. Since spectrin is a heterodimer such a site is not necessarily required on the (Y subunit to explain a selective inhibition of the (Ysubunit phosphorylation. CaM binding to the p subunit could impair the accessibility of the kinase to the LYsubunit. Likewise, small molecular weight proteins in spectrin extracts which have a high affinity for CaM (17) and which may bind to spectrin could enhance the apparent affinity of spectrin for CaM in vivo. We do not think that this possibility could explain the selective inhibition of the CAMP-dependent in vitro phosphorylation of the (Ysubunit, because we used purified spectrin dimer, and because the minor polypeptides smaller than spectrin (Fig. 1) were spectrin breakdown products, which were neither cofactors for a CAMP-dependent phosphorylation nor mediators for a CaM-induced inhibition of the CAMP-dependent phosphorylation. CaM acted similarly on spectrin containing phosphorylated peptide of the B subunit of spectrin. (a) Autophosphorylated spectrin; (b, b’) CAMP-dependent phosphoform of spectrin; (c, c’) CAMP-dependent phosphoform of spectrin in the presence of CaM.

476

MARETZKI

no visible breakdown products (not shown). The ability of CaM to interfere with the CAMP-dependent phosphorylation of a protein is not unique for spectrin; it was also reported for the CAMP-dependent phosphorylation of phosphorylase kinase with a 66% inhibition at 5.5 PM (34). The inhibitory effect of CaM on the CAMP-dependent phosphorylation of the (Y subunit of spectrin was clearly not due to stimulation of a spectrin-associated phosphatase. This was expected, because native spectrin dimer purified from low ionic strength extracts did not contain any phosphate in the (Ysubunit and was exclusively labeled in the fl subunit (8,9). While the latter data seem to exclude an in viva significance for a CAMP-dependent phosphoform of spectrin, they are not conclusive evidence against its existence, because a CAMP-dependent phosphoform could differ in its extractability properties from a CAMP-independent phosphoform of spectrin. A CAMP-dependent phosphoform of spectrin was indeed generated in isolated membranes (11,12). It resisted a low ionic strength extraction and remained associated with inside-out vesicles (Lutz et al., unpublished result). A CAMP-dependent phosphorylation of both spectrin subunits (11) or an enhanced labeling in both subunits together (35) was found in [y-3?P]ATP-labeled membranes by those investigators, who used EGTA or EDTA in their assay or during membrane preparation. This treatment may have eliminated the inhibitory effect of CaM on the CAMP-dependent phosphorylation of the a subunit of spectrin, which occurred at a low CaM-to-spectrin ratio, as shown here. This could explain why others, using different protocols, did not find a CAMPdependent labeling of both spectrin subunits (8-10, 14). The results shown here suggest, however, that a CAMP-dependent phosphorylation of the j3 subunit should have been detected in isolated membranes, irrespective of whether EGTA was added or not, because the CAMP-dependent phosphorylation of the p subunit was not inhibited by CaM. While this was not studied, it was found that phosphorylation by a CAMP-independent and phosphorylation

AND

LUTZ

by a CAMP-dependent protein kinase were additive (14,36). Thus, a CAMP-dependent phosphorylation of spectrin may yet be relevant to our understanding of spectrin phosphorylation. Although the functional role of a CAMPdependent phosphoform of spectrin remains to be established, there is little doubt that CAMP and CaM are involved in membrane-skeleton interactions (3’7-39), suggesting a relationship between calmodulin binding, presumably to spectrin, and its state of phosphorylation. ACKNOWLEDGMENTS We thank Miss Pia Stammler for her excellent technical assistance and Dr. Joachim Krebs, ETH Zurich, for a sample of highly purified calmodulin from bovine brain. This work was supported by a FEBS fellowship to D. Maretzki and by a grant to H. U. Lutz from the Swiss National Science Foundation, Berne. REFERENCES 1. NAKAO, M., NAKAO, T., AND YAMAZOE, S. (1969) Nature (London) 187,945-946. 2. JINBU, Y., SATO, S., AND NAKAO, M. (1984) Nature (London) 307,376-3’78. 3. FAIRBANKS, G., PATEL, V. P., AND DINO, J. E. (1981) Stand J. Clin Invest. 41, Suppl. 156,139-144. 4. BRENNER, S. L., AND KORN, E. D. (1979) J. Biol Chem. 254,8620-8627. 5. ANDERSON, J. M., AND TYLER, J. M. (1980) J. Biol. Chem. 255,1259-1265. 6. TAO, M., CONWAY, R., CHIANG, H.-C., CHETA, S., AND YAN, T. F. (1981) in Protein Phosphorylation (Krebs, R., ed.) Cold Spring Harbor Conf. Vol. 8, pp. 1301-1312, Cold Spring Harbor Lab., Cold Spring Harbor, N. Y. 7. IMHOF, B. A., ACHA-ORBEA, H. J., LIEBERMANN, T. A., REBER, B. F. X., LANZ, H. W., WINTERHALTER, K. H., AND BIRCHMEIER, W. (1980) P~oc. Nat1 Acad Sci. USA 77.3264-3268. 8. HARRIS, H. W., AND Lux, S. E. (1980) J. Bi01 Chem 255,11512-11520. 9. HARRIS, H. W. JR., LEVIN, N., AND Lux, S. E. (1980) J. Biol. Chem. 255,11521-11525. 10. FAIRBANKS, G., AND AVRUCH, J. (1974) Biochemistry 13.5514-5521. 11. YAWATA, Y., KORESAWA, S., AND MIYASHIMA, K. (1980) Hemoglobin 4,717-734. 12. GREENQUIST, A. C., AND SHOHET, S. B. (1974) FEBS Lett. 48, 133-135. 13. LUTZ, H. U. (1984) FEBS I&t. 169.323-329. 14. HOSEY, M. M., AND TAO, M. (1977) J. Bid Chem 252.102-109. 15. SOBUE, K., MURAMOTO, Y., FUJITA, M., AND KAK-

CALMODULIN

16. 17. 18. 19. 20.

21. 22. 23. 24. 25. 26. 27.

INHIBITS

SPECTRIN

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