©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Modulation of Erythrocyte Membrane Mechanical Function by -Spectrin Phosphorylation and Dephosphorylation (*)

(Received for publication, November 2, 1994; and in revised form, December 15, 1994)

Sumie Manno Yuichi Takakuwa Kaoru Nagao Narla Mohandas (1)

From the Department of Biochemistry, Tokyo Women's Medical College, 8-1 Kawada-Cho, Shinjuku-Ku, Tokyo, 162, Japan and the Lawrence Berkeley Laboratory, Life Science Division, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The mechanical properties of human erythrocyte membrane are largely regulated by submembranous protein skeleton whose principal components are alpha- and beta-spectrin, actin, protein 4.1, adducin, and dematin. All of these proteins, except for actin, are phosphorylated by various kinases present in the erythrocyte. In vitro studies with purified skeletal proteins and various kinases has shown that while phosphorylation of these proteins can modify some of the binary and ternary protein interactions, it has no effect on certain other interactions between these proteins. Most importantly, at present there is no direct evidence that phosphorylation of skeletal protein(s) alters the function of the intact membrane. To explore this critical issue, we have developed experimental strategies to determine the functional consequences of phosphorylation of betaspectrin on mechanical properties of intact erythrocyte membrane. We have been able to document that membrane mechanical stability is exquisitely regulated by phosphorylation of beta-spectrin by membrane-bound casein kinase I. Increased phosphorylation of beta-spectrin decreases membrane mechanical stability while decreased phosphorylation increases membrane mechanical stability. Our data for the first time demonstrate that phosphorylation of a skeletal protein in situ can modulate physiological function of native erythrocyte membrane.


INTRODUCTION

The mechanical properties of deformability and mechanical stability of human erythrocytes are largely regulated by the submembranous protein skeleton(1) . Spectrin, actin, protein 4.1, adducin, and dematin are the principal components of the membrane skeleton. Lateral interactions among these proteins constitute the composite structure designated as the membrane skeletal network. This network is anchored to the bilayer through vertical interactions, one involving beta-spectrin, ankyrin, and band 3, and the other through an interaction between protein 4.1 and glycophorin C(2, 3) .

All of the components, except actin, of the membrane skeleton are phosphoproteins(4, 5) . The phosphate groups of these proteins undergo turnover in the intact cell as a result of the action of multiple kinases and phosphatases that have been identified in erythrocytes. Based on in vitro studies using purified skeletal proteins and kinases, there is a strong suggestion that phosphorylation may modify binary and ternary interactions between proteins in solution, leading in most cases to a reduced affinity for the interaction(s) (6, 7, 8, 9, 10, 11, 12, 13) . However, the effect of protein phosphorylation on mechanical function of intact membrane has not previously been unequivocally documented. In the present study, we explored the involvement of phosphorylation and dephosphorylation of beta-spectrin in modulating membrane mechanical stability of intact human erythrocyte membranes. In order to obtain unambiguous information on the influence of protein phosphorylation on membrane function, it is necessary to relate the documented changes in membrane function to the measured changes in the phosphate bound to the protein and not just the turnover of phosphate. To achieve this objective we considered the following conditions to be requisite for determining the effects of phosphorylation of a membrane protein on membrane properties: 1) an intact membrane instead of an isolated protein or a kinase must be used in the experiments, 2) the specimen must not possess any protein phosphatase activity, 3) only a phosphate radical donor should be used for phosphorylation, 4) the type of kinase involved in the phosphorylation of protein(s) must be specified, 5) the amount of phosphate incorporated must be quantitated, and 6) all phosphorylation-induced changes in membrane function must be reversed by dephosphorylation with the addition of protein phosphatase. In this context, we developed experimental conditions in which beta-spectrin was the primary protein phosphorylated in intact membranes and demonstrated that the degree of beta-spectrin phosphorylation modulates the membrane mechanical stability of intact red cell membranes.


EXPERIMENTAL PROCEDURES

Materials

Vanadate-free ATP obtained from Sigma was used in all experiments. [-P]ATP was purchased from DuPont NEN. Sodium orthovanadate (vanadium (V) as Na(3)VO(4)) was dissolved in deionized water to a final concentration of 0.1 M. This stock solution of vanadate was diluted to 0.1 mM in 5 mM Tris-HCl (pH 7.4) containing 5 mM KCl (5T5K buffer). N-(2-Aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7), (^1)the casein kinase I inhibitor, was purchased from Seikagaku Co. Okadaic acid, a protein phosphatase inhibitor, was obtained from Sigma. These reagents were dissolved in dimethyl sulfoxide. PVDF membrane (Immobilon P) was purchased from Millipore Corp. The antibodies against phosphoserine, phosphothreonine, and phosphotyrosine were obtained from Sigma. The chemiluminescence reagent was obtained from Dupont NEN.

Methods

Erythrocyte Ghosts Preparation

After obtaining informed consent, human venous blood was freshly drawn from healthy volunteers and was used in all the studies outlined. Erythrocytes were collected and washed three times with Tris-buffered saline. Intact cells were lysed and washed five times in 35 volumes of 5T5K buffer at 4 °C to remove cytosolic components including ATP, cytosolic protein kinases, and phosphatases. The white membranes thus obtained were incubated at 0 °C for 10 min with MgATP at various concentrations and additives such as vanadate, CKI-7, and protein phosphatase. The mixture was gently stirred during this 10-min incubation period. The ghost membranes were subsequently resealed by adding a mixture of KCl, MgCl(2), and dithiothreitol (final concentrations: 150, 1, and 1 mM, respectively) followed by incubation at 37 °C for 40 min as described previously(14) .

Measurement of Membrane Stability

The resealed ghosts were suspended in 45% dextran, and the membrane mechanical stability was quantitated using an ektacytometer, as described previously(14, 15) . Briefly, the suspended ghosts were subjected to a constant shear stress of 750 dynes/cm^2, and changes with time in laser diffraction patterns were measured by recording the rate of change of the deformability index. The rate of decrease of the deformability index is a measure of the rate of membrane fragmentation and thus a quantitative measure of membrane mechanical stability(15) . The time required for the deformability index to reach half-maximum value is designated as T. To examine changes in membrane mechanical stability, T values were compared for variously phosphorylated membrane preparations. A decrease in T value represents mechanical destabilization of the membrane while an increase in T value reflects improved mechanical structural integrity.

Phosphorylation of Erythrocyte Ghosts

To study the phosphorylation of membrane protein, ghosts were prepared as described above except that P-labeled ATP (400-600 cpm/pmol) was used instead of unlabeled ATP. All reactions were terminated by adding an electrophoresis sample buffer. Radiolabeled proteins were resolved by SDS-polyacrylamide gel according to Laemmli (16) on 7.5% acrylamide gel containing 1% SDS. The gels were stained for protein with Coomassie Brilliant Blue and dried. Phosphorylated membrane proteins were identified by autoradiograghy. Radioactivity on membrane proteins was determined by excising protein bands from dried gels and counting in a liquid scintillation counter.

Phosphorylated membrane proteins separated on SDS-polyacrylamide gels were also blotted on PVDF membranes using semi-dry transblots (Nihon Eido Co). Briefly, electrotransfer of the proteins was carried out in 25 mM Tris containing 20% methanol and 40 mM -amino-n-caproic acid for 1 h at 250 mA. The membranes were washed once in 0.05% Tween 20 and blocked with skim milk solution (Block-Ace, Yukijirushi Co) for 1 h, followed by incubation for 1 h at room temperature with antiphosphoserine antibody (1/5000 dilution) in skim milk solution. Antigen-antibody complexes were developed using a peroxidase conjugate substrate kit from Dupont NEN. Briefly, the immunoblots were incubated at room temperature for 1 h with rabbit anti-mouse IgG-conjugated peroxidase and developed using the substrate solution of a chemiluminescence reagent kit. The membranes were exposed to Kodak x-ray film.

Preparation of Protein Phosphatase in Human Erythrocytes

Protein phosphatase IV was purified from human erythrocyte cytosol and treated with 80% ethanol to derive the catalytic subunit (M(r) = 31,000) from the enzyme according to Usui et al.(17, 18) . Phosphoprotein phosphatase activity was monitored with P-labeled H1-histone phosphorylated by cAMP-dependent protein kinase and phosphorylated P as substrates under the conditions of this study. One unit of enzyme was defined as that amount which would catalyze the release of 1 mol of phosphate from 1 mol of spectrin per h.

Measurement of Dephosphorylation of Spectrin Dimer

Fresh ghosts were dephosphorylated with various amounts of protein phosphatase under the resealing conditions. The spectrin dimer was isolated by the method of Harris and Lux (19) and Harris et al.(20) with minor modifications. Dephosphorylated ghosts were washed once in 5 mM sodium phosphate (pH 8.0) and collected by centrifugation at 18,000 times g for 15 min at 4 °C. To extract spectrin and actin, the ghosts were washed once in 0.1 mM sodium phosphate, pH 8.0, by centrifugation at 45,000 times g for 10 min at 4 °C. The ghosts in the pellet were collected and incubated at 37 °C for 10 min in a shaking water bath. Spectrin-depleted ghost membranes were pelleted at 100,000 times g for 1 h at 4 °C, and the supernatant containing spectrin and actin was collected. These two proteins were separated by molecular sieving on a Sepharose 4B column (2.6 times 35 cm) in 150 mM NaCl, 0.1 mM EDTA, 10 mM Tris-HCl (pH 8.0). After measuring the protein concentration of the pooled spectrin dimer fraction by the method of Lowry et al.(21) , an aliquot (20-300 ml) was dot-blotted on a PVDF membrane, and dephosphorylated spectrin was analyzed using antiphosphoserine antibody specified above. The membrane was exposed to Kodak x-ray film, and the reactive spots were quantitated by densitometric scanning (ATTO AE-6900M). Density was found to increase linearly with the amount of spectrin dimer up to 5 µg (data not shown).


RESULTS

Effects of MgATP on Membrane Stability

Representative data for membrane mechanical stability of ghosts prepared in the presence and absence of MgATP are shown in Fig. 1A. In ghosts, 10 µM vanadate was present to prevent a change in shape induced by MgATP. When the control resealed ghosts (without vanadate and MgATP) were subjected to shear stress of 750 dynes/cm^2, the membranes underwent deformation and fragmented, resulting in a T value of 40 s. Ghosts prepared with MgATP in the absence of vanadate underwent extensive endocytosis with concomitant loss of surface area and hence did not deform in the fluid shear field. As such, these membranes did not fragment. Treatment with vanadate inhibited endocytosis and loss of surface area, and such membrane preparations exhibited deformability characteristics identical with resealed ghosts prepared in the absence of vanadate and MgATP. Ghosts prepared with MgATP and vanadate fragment at a faster rate than the control ghosts, so that the T value decreased to 28 s. Vanadate by itself had no effect on membrane mechanical stability. The correlation between membrane mechanical stability (T values) and MgATP concentration used during the resealing step is shown in Fig. 1B. With increasing MgATP concentration, T decreased to 60% of the control value.


Figure 1: Effect of vanadate and various concentrations of MgATP on membrane stability. A, resealed ghosts prepared in the presence or absence of vanadate (10 µM) were measured with an ektacytometer as described under Methods. Under a high constant shear stress of 750 dynes/cm^2, the control membranes without vanadate (NONE) and that with vanadate (VANADATE) showed the same profiles of membrane stability and started to fragment at 10 s with a T of 40 s. Ghosts with MgATP and without vanadate (MgATP) could not fragment due to loss of membrane surface area as a result of endocytosis. This is reflected by the marked decrease in the initial deformability index of this preparation. Ghosts with MgATP and vanadate (MgATP VANADATE) did not undergo endocytosis and had same initial deformability index values as control ghosts. These membranes were more unstable than the control membranes, and T decreased to 28 s. B, in the presence of vanadate, membrane stability (T) progressively decreased with increasing MgATP concentrations. All values were normalized to T values for the vanadate (A)-treated membrane preparations.



Phosphorylation of Erythrocyte Ghosts with [-P]MgATP

The time-dependent incorporation of P into membrane proteins is shown in Fig. 2, A and B. Membranes were incubated under the same conditions as those used for determining membrane mechanical stability, with [-P]MgATP being used instead of unlabeled MgATP. Several membrane proteins incorporated P, and the extent of incorporation increased with time. After a 30-min incubation, beta-spectrin exhibited the highest degree of phosphorylation, attaining a maximal level of 0.6 mol of P/mol of beta-spectrin at an MgATP concentration of 0.5 mM. As beta-spectrin and ankyrin migrate close together on SDS-polyacrylamide gel electrophoresis by the Laemmli method (7.5% acrylamide concentration)(22, 23) , it could be argued that a small degree of P incorporation into spectrin could potentially be attributed to ankyrin. To resolve this issue, we used the SDS-polyacrylamide gel electrophoresis system of the Fairbanks method (3.5-17% acrylamide concentration linear gradient gel) (24) in which beta-spectrin is well separated from ankyrin. In this system we found that the P was indeed associated with beta-spectrin. Furthermore, we showed that ankyrin purified from phosphorylated ghosts contained less than 0.1 mol of P/mol of ankyrin. To determine the concentration dependence of MgATP on P incorporation, ghosts were incubated in the presence of various concentrations of [-P]MgATP for 40 min. With MgATP concentrations up to 1.0 mM, P incorporation into beta-spectrin increased, becoming maximal at 0.9 mol/mol (Fig. 2C). Incorporation ranged from 0.75 to 0.98 mol of P/mol at an MgATP concentration of 1.0 mM, depending on the membrane preparation used. In contrast to beta-spectrin, P incorporation into band 3, protein 4.1, protein 4.2, and dematin was less than 0.1 mol of P per mol of each of the proteins (Fig. 2, B and C). In this region of the gel where band 3 is stained by Coomassie Brilliant Blue, two phosphorylated bands were noted. These are likely to be alpha- and beta- adducin (Fig. 2A).


Figure 2: Phosphorylation of membrane proteins. A, time course of incorporation of P into membrane proteins. Fresh ghosts were phosphorylated with 0.5 mM MgATP (containing [-P]ATP) as described under Methods. The gel on the left was stained with Coomassie Brilliant Blue. The nine gels on the right are autoradiograms. B, following autoradiography (A), spectrin (bullet), band 3 (circle), and protein 4.1 () were analyzed for P content by liquid scintillation counting. C, dependence of P incorporation into beta-spectrin on MgATP concentration. Resealed ghosts were prepared in the presence of vanadate and various concentrations of MgATP (0-1 mM, containing [-P]ATP). Radioactivity incorporated into beta-spectrin (bullet), band 3 (circle), protein 4.1 (), and protein 4.2 (box) was analyzed after SDS-polyacrylamide gel electrophoresis in 7.5% polyacrylamide by the Laemmli method. D, correlation between membrane stability (Fig. 1B) and the extent of beta-spectrin phosphorylation (C).



To determine the correlation between membrane mechanical stability and phosphorylation of beta-spectrin, T values shown in Fig. 1B were plotted against P incorporation into betaspectrin shown in Fig. 2C (Fig. 2D). T decreased linearly with increased net incorporation of P into beta-spectrin, indicating that the phosphorylation of beta-spectrin decreases membrane mechanical stability.

Identification of the Kinase Responsible for beta-Spectrin Phosphorylation

Membrane-associated protein kinases have been shown to be casein kinases(25, 26, 27, 28, 29, 30, 31) , cAMP-dependent kinase(32) , tyrosine kinase(33) , and protein kinase C(4, 34) . Casein kinases and cAMP-dependent kinase phosphorylate spectrin(29, 35, 36) . In this study, we focussed our attention on membrane-bound casein kinases I and II since cAMP, an activator of cAMP-dependent kinase, is not present in our membrane preparations. Phosphorylation of beta-spectrin increased with increasing salt concentrations (Fig. 3A), a characteristic of casein kinases suggesting that the kinase involved in beta-spectrin phosphorylation should belong to this family of kinases. To directly test this thesis, we examined the effects of inhibitors of casein kinases, CKI-7 (37) for casein kinase I and heparin (31, 38) and quercetin (39) for casein kinase II on protein phosphorylation and membrane function. The most effective inhibitor of MgATP-induced beta-spectrin phosphorylation and associated decreases in membrane mechanical stability was CKI-7 (Fig. 3B). Quercetin had no effect on beta-spectrin phosphorylation (data not shown), indicating that the phosphorylation of beta-spectrin is not likely to be due to casein kinase II. Heparin was effective at very high concentrations in inhibiting beta-spectrin phosphorylation (data not shown), but this inhibition was most likely due to competition with ATP and not through inhibition of casein kinase II. P incorporation into spectrin was not affected by okadaic acid (124 mM)(40, 41) , a potent inhibitor of protein phosphatases (Fig. 3A), implying that intact membrane preparations used in our studies do not contain phosphatases. The P incorporation we observed must thus reflect a net increase in P incorporation into beta-spectrin by membrane-bound casein kinase I and not the result of replacement of the intrinsic phosphate associated with beta-spectrin in the isolated membranes prior to our experimental manipulations.


Figure 3: Identification of the kinase responsible for spectrin phosphorylation. A, effects of KCl concentration in the presence (circle) or absence (bullet) of okadaic acid (124 µM), the protein phosphatase inhibitor on P incorporation into beta-spectrin. B, inhibition of casein kinase I inhibitor (CKI-7) on membrane mechanical stability and P incorporation into beta-spectrin. Fresh ghosts were first incubated for 10 min with 1 mM [-P]ATP and various concentrations of CKI-7 at 0 °C as shown on the graph. Subsequent incubation was conducted at 37 °C under resealing conditions, and membrane stability (bullet) and P incorporation into beta-spectrin (circle) were quantitated.



Identification of the Phosphorylated Amino Acid Residue in beta-Spectrin

To identify the amino acid residue in beta-spectrin phosphorylated under the experimental conditions employed, antiphosphoserine antibody was used for Western blot analysis of membrane proteins (Fig. 4A). This antibody reacted with beta-spectrin from ghosts prepared in the absence of MgATP, indicating that beta-spectrin is natively phosphorylated. Densitometric analysis showed increased staining of the serine residue of beta-spectrin with increasing MgATP concentrations. betaSpectrin also reacted with the antiphosphothreonine antibody, but the reactivity was weak, and, most importantly, there was no change in the extent of this reactivity with an increase in MgATP (data not shown). The antiphosphotyrosine antibody did not react with beta-spectrin (data not shown).


Figure 4: Chemiluminescence analysis of phosphorylation and dephosphorylation of beta-spectrin. A, chemiluminescence profiles of the phosphorylation of spectrin at various MgATP concentrations. Resealed ghosts were prepared in the presence of vanadate and various concentrations of MgATP (0-1 mM). Membrane proteins were separated on SDS gels in 7.5% polyacrylamide by the Laemmli method, blotted onto PVDF membranes, and probed with antiphosphoserine antibody to detect phosphoserine groups in spectrin. B, spectrin dimer from phosphorylated or dephosphorylated ghosts were isolated by the method described by Harris and Lux(19) . Equivalent quantities of spectrin dimer from various experiments were applied to PVDF membrane with a dot-blot template and probed with antiphosphoserine antibody. The membranes were fluorographed on Kodak x-ray film, and luminated bands were quantitated by densitometric scanning as described under Methods. Lane 1, no addition; lane 2, 1 mM MgATP; lanes 3, 4, and 5, 0.66, 1.31, and 2.63 units of protein phosphatase activity.



Effects of Exogenous Protein Phosphatase on Dephosphorylation of Spectrin and on Membrane Mechanical Stability

Since the phosphorylation of beta-spectrin decreased membrane stability, it was critical to demonstrate that dephosphorylation of beta-spectrin would increase membrane stability. To dephosphorylate membrane-associated beta-spectrin, partially purified protein phosphatase was incorporated into ghosts followed by incubation at 37 °C for 40 min. The spectrin dimer isolated from these ghosts with antiphosphoserine antibody showed decreased phosphoserine (Fig. 4B). There was a progressive decrease in beta-spectrin phosphorylation with increasing amounts of protein phosphatase incorporated. This decrease in phosphorylation was accompanied by an increase in membrane mechanical stability. To estimate the extent of the decrease of beta-spectrin phosphorylation, we first quantitated the net increase in the amount of phosphoserine in the spectrin dimer isolated from ghosts prepared with 1 mM MgATP (lane 2), compared to that present in ghosts prepared with no MgATP (lane 1). This net increase in spectrin phosphorylation (b in Fig. 4B) was taken to reflect the 0.75 to 0.98 mol of phosphate incorporated/mol of beta-spectrin previously shown using P incorporation (Fig. 2C). Based on these estimates, it can be estimated that the spectrin dimer in the control ghosts should contain roughly 2.12 to 2.77 mol of phosphoserine (a in Fig. 4B). beta-Spectrin contains 4 mol of exchangeable phosphate per beta chain(19, 42, 43) . The present results agree with those reported earlier, about 3 mol for native spectrin and incorporation of a maximum of 1 mol by phosphorylation due to MgATP for a total of 4 mol. In a similar manner, the extent of dephosphorylation of beta-spectrin by phosphatase (c, d, and e in Fig. 4B) was also estimated. Fig. 5shows the correlation between membrane mechanical stability and amount of phosphoserine in the beta-spectrin dimer. It can be seen that the membrane mechanical stability is related to the extent of beta-spectrin phosphorylation. Increased beta-spectrin phosphorylation results in decreased membrane mechanical stability, while decreased beta-spectrin phosphorylation results in increased mechanical stability. These data conclusively demonstrate that the state of beta-spectrin phosphorylation does modulate membrane mechanical cohesion.


Figure 5: Correlation between membrane mechanical stability and phosphate content of beta-spectrin. Phosphorylated and dephosphorylated ghosts were obtained using various concentrations of MgATP and protein phosphatase, respectively. Spectrin dimer was isolated from these ghosts, and the amount of phosphate was quantitated using antiphosphoserine antibody.




DISCUSSION

The effects of phosphorylation on erythrocyte membrane protein-protein interactions in solution have been studied previously using purified proteins in vitro(6, 8, 10, 11, 44) . However, there has not been an unequivocal demonstration of the effect of protein phosphorylation on the function(s) of intact membranes. In the present study we have shown that spectrin phosphorylation and dephosphorylation can modulate the mechanical properties of the intact erythrocyte membrane. Since we were able to demonstrate both the specificity and the quantitative nature of the relationship between beta-spectrin phosphorylation and changes in membrane mechanical stability using a number of different approaches, we are confident that the functional changes in membrane properties we have documented are directly related to beta-spectrin phosphorylation. The data we have outlined is the first demonstration of the effects of erythrocyte membrane skeletal protein phosphorylation on the mechanical function of the intact membrane structure.

While it has long been recognized that many of the skeletal proteins are phosphorylated, the lack of evidence that changes in the state of phosphorylation can induce specific changes in membrane mechanical function have raised concern regarding the physiological contribution of skeletal protein phosphorylation. Since this is an area of much controversy and debate, we wanted to be certain that our studies provide unambiguous data. As such, to establish that the function of the intact membrane is directly related to changes in the phosphorylation state of spectrin, the following approaches were employed in the present study. In contrast to previous studies, we chose to probe phosphorylation-induced changes in intact membranes rather than on protein-protein associations in solution or markedly perturbed membrane preparations. We also established that the membrane preparations used in our study did not contain any phosphatase activity. As more than 97% of spectrin phosphatase activity, which belongs to Type 2A(17, 18) , is present in the cytosol of human erythrocytes, our membrane preparation protocols which involved extensive washing of ghosts ensured its removal from the membrane preparation. Furthermore, okadaic acid, the inhibitor specific to the above-mentioned phosphatase(17, 18) , added to the resealing buffer had no effect on P incorporation into beta-spectrin, confirming no endogenous phosphatase activity in our membrane preparations. In contrast, exogenously added purified protein phosphatase decreased the extent of beta-spectrin phosphorylation. We are thus confident that the changes in phosphorylation measured during the present studies using [-P]MgATP represent the extent of increase in net phosphorylation of beta-spectrin without contribution from dephosphorylation by the phosphatases. Previous studies have shown that incubation with increasing concentrations of ATP results in the net incorporation of 1 mol of phosphate per mol of beta-spectrin(45) . We also found a net incorporation of 1 mol of phosphoryl group per mol of beta-spectrin using isotope-labeled MgATP.

The extent of change in beta-spectrin phosphorylation was also assessed using antiphosphoserine antibody which enabled the identification of inherently present phosphate radicals and the effectiveness of dephosphorylation of beta-spectrin using purified phosphatase. The spectrin dimer possesses an average of four phosphoryl groups which bind to phosphate covalently and exchangeably, near the C terminus(19, 20, 46) . Assuming that isotopically labeled amino acid determined using [-P] MgATP is all phosphoserine, the amount of phosphate was estimated to be about 3 mol per mol of native spectrin dimer in the control unmanipulated membrane preparations, increasing to 4 mol following incubation with MgATP. Taken together these data support the thesis that there is indeed a net increase in the phosphate content of beta-spectrin under our experimental conditions.

Previous studies explored the role of MgATP added to resealed ghosts as a phosphate radical donor on cell shape. The extent of morphologic changes induced in ghosts was noted to depend on the MgATP concentration and the extent of spectrin phosphorylation induced by MgATP(47) . However, as the phosphate radical donor MgATP can also serve as a substrate for ATPase, the observed shape changes could have resulted from MgATP effects on ATPase. Indeed, MgATP-induced shape changes could be prevented by vanadate, an ATPase inhibitor(48) . In contrast, vanadate has no effect on the phosphorylation of spectrin and on the MgATP-induced changes in membrane mechanical behavior we observed. As such, our observations on changes in membrane mechanical function are the result of the effect of MgATP on protein phosphorylation and not due to its effect on ATPase. In fact, MgATP-induced endocytosis and consequent shape changes have hindered our previous attempts to examine the effect of protein phosphorylation on membrane material properties. Loss of cell surface area due to endocytosis prevents the ability of the membrane to undergo deformations necessary to document changes in membrane material properties. The present study, in fact, took advantage of two important features of the effect of vanadate on intact membranes: 1) preventing membrane endocytosis and thus enabling membranes to undergo deformation necessary to document changes in membrane mechanical behavior and 2) not having any effect on protein phosphorylation.

In the present study, we focussed our attention only on the functional effects of beta-spectrin phosphorylation induced by membrane-bound kinases. The documentation of ionic strength-dependent changes in the extent of spectrin phosphorylation (49) and inhibition of spectrin phosphorylation by CKI-7 (37) enabled us to establish that the phosphorylation of beta-spectrin we documented is regulated by casein kinase I(49) .

While all of the data we have outlined are consistent with a role for beta-spectrin phosphorylation in regulating membrane mechanical strength, the most important and crucial evidence is the documenting of the reversible effects of phosphorylation on membrane mechanical stability: increased phosphorylation of beta-spectrin destabilizes the membrane and decreased phosphorylation increases membrane stability. Membrane stability is regulated primarily through membrane protein interactions(1) . The data from the present study indicate that some of these membrane protein interactions can be modulated by the phosphorylation and dephosphorylation of beta-spectrin. Although we have not identified the site of beta-spectrin phosphorylation involved in increased phosphorylation by MgATP, the observed decrease in mechanical stability to about 60% of the normal value implies that phosphorylation of this site on beta-spectrin is important for regulating protein-protein interactions. The observed relationship between changes in membrane mechanical stability and phosphorylation is complex. There does not appear to be a one to one relationship between the extent of phosphate incorporated and membrane stability. The nature of the relationship we observed suggests that some of the sites of phosphorylation in beta-spectrin may be more important in regulating membrane mechanical properties than other sites.

Studies on purified proteins in vitro suggest that the phosphorylation of membrane skeletal proteins can modify interactions among different proteins(5) . Because of the central role of spectrin in erythrocyte skeletal organization, the possibility that its properties may be regulated by phosphorylation has been given considerable attention, and the potential effects of phosphorylation of spectrin have been studied extensively. Phosphorylation of spectrin has been shown not to affect either dimer-dimer associations (44) or spectrin binding to ankyrin in vitro(6) . Spectrin phosphorylation does not appear to have any effect on the capacity of spectrin to bind to F-actin (50) and to red cell inside-out vesicles (51) and on the ability to form higher oligomers(52) . What is clear from our studies, however, is that the state of beta-spectrin phosphorylation can clearly modulate mechanical function of intact membrane structure. The inability of previous studies to document the effects of phosphorylation on spectrin function may in large part be due to the inappropriate choice of membrane functions to be studied or due to the inability to reproduce in solution the complex interactions that can take place in situ on the membrane. Our studies raise the concern that data obtained from in vitro studies using purified proteins may not be directly applicable to defining the physiologic role of protein phosphorylation on membrane function.

The molecular mechanism by which changes in phosphorylation of beta-spectrin in intact membranes modulates membrane mechanical properties is not entirely clear although phosphorylation-induced changes in spectrin oligomerization in situ may account for the observed mechanical behavior. Whether the phosphorylation state of erythrocyte membrane proteins other than spectrin modulate the membrane mechanical properties is an issue which we believe could be explored using approaches similar to the one we have outlined in the present study.


FOOTNOTES

*
This work was supported in part by NIH Grant DK26263. This work was supported by the Director, Office of Energy Research, Office of Basic Energy Science, of the U. S. Department of Energy under Contract No. DE-AC03-76SF00098. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: CKI-7, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; PVDF, polyvinylidene difluoride.


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