(Received for publication, November 2, 1994; and in revised form, December 15, 1994)
From the
The mechanical properties of human erythrocyte membrane are
largely regulated by submembranous protein skeleton whose principal
components are - and
-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
spectrin on
mechanical properties of intact erythrocyte membrane. We have been able
to document that membrane mechanical stability is exquisitely regulated
by phosphorylation of
-spectrin by membrane-bound casein kinase I.
Increased phosphorylation of
-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.
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 -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 -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
-spectrin
was the primary protein phosphorylated in intact membranes and
demonstrated that the degree of
-spectrin phosphorylation
modulates the membrane mechanical stability of intact red cell
membranes.
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.
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, 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.
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 (
), band 3 (
), and protein 4.1
(
) were analyzed for
P content by liquid
scintillation counting. C, dependence of
P
incorporation into
-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
-spectrin (
), band 3 (
), protein 4.1 (
), and
protein 4.2 (
) 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
-spectrin
phosphorylation (C).
To
determine the correlation between membrane mechanical stability and
phosphorylation of -spectrin, T
values shown
in Fig. 1B were plotted against
P
incorporation into
spectrin shown in Fig. 2C (Fig. 2D). T
decreased
linearly with increased net incorporation of
P into
-spectrin, indicating that the phosphorylation of
-spectrin
decreases membrane mechanical stability.
Figure 3:
Identification of the kinase responsible
for spectrin phosphorylation. A, effects of KCl concentration
in the presence () or absence (
) of okadaic acid (124
µM), the protein phosphatase inhibitor on
P
incorporation into
-spectrin. B, inhibition of casein
kinase I inhibitor (CKI-7) on membrane mechanical stability and
P incorporation into
-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 (
) and
P incorporation into
-spectrin
(
) were quantitated.
Figure 4:
Chemiluminescence analysis of
phosphorylation and dephosphorylation of -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.
Figure 5:
Correlation between membrane mechanical
stability and phosphate content of -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.
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
-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
-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
-spectrin, confirming no endogenous phosphatase activity in our
membrane preparations. In contrast, exogenously added purified protein
phosphatase decreased the extent of
-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
-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
-spectrin(45) . We also found
a net incorporation of 1 mol of phosphoryl group per mol of
-spectrin using isotope-labeled MgATP.
The extent of change in
-spectrin phosphorylation was also assessed using
antiphosphoserine antibody which enabled the identification of
inherently present phosphate radicals and the effectiveness of
dephosphorylation of
-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
-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
-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
-spectrin we documented is regulated by casein
kinase I(49) .
While all of the data we have outlined are
consistent with a role for -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
-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
-spectrin. Although we have not identified the site of
-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
-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
-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
-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 -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.