Department of Molecular Sciences, University of Tennessee Health Science
Center, 858 Madison Avenue, Memphis, Tennessee 38163, USA
* Present address: MCBL, Salk Institute for Biological Studies, 10010 N. Torrey
Pines Rd, La Jolla, CA 92037-1099, USA
Author for correspondence (e-mail:
jcox{at}utmem.edu)
Accepted 9 August 2002
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Summary |
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Key words: Cytoskeleton, Ankyrin, Regulation, Erythroid, Epithelial
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Introduction |
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Our recent studies have shown that the spectrin-binding properties of
chicken erythroid ankyrin are regulated by phosphorylation
(Ghosh and Cox, 2001).
Treatment of chicken embryonic erythroid cells with serine and threonine
phosphatase inhibitors stimulated the hyperphosphorylation of the 225 kDa and
205 kDa erythroid ankyrin isoforms. Ankyrin hyperphosphorylation correlated
with a reduced association of ankyrin-AE1 complexes with cytoskeletal
spectrin. In vitro binding studies have shown that the dissociation of
ankyrin-AE1 complexes from the spectrin cytoskeleton is at least partially due
to the reduced ability of hyperphosphorylated ankyrin to bind to spectrin.
Thus, ankyrin phosphorylation represents a critical mechanism for regulating
the cytoskeletal association of ankyrin-bound membrane proteins.
The studies described here have shown that a kinase activity that mediates the in vivo phosphorylation of ankyrin is constitutively associated with ankyrin-containing complexes isolated from chicken embryonic erythroid cells. Immunological and biochemical assays have shown that this associated kinase has properties identical to protein kinase CK2. Studies using CK2-specific inhibitors have suggested that all of the phosphorylation events associated with both basally phosphorylated and hyperphosphorylated ankyrin in vivo are dependent upon CK2. Furthermore, binding studies have indicated that the CK2-dependent phosphorylation of erythroid ankyrin regulates its ability to associate with spectrin in vitro. Additional analyses revealed that CK2 is constitutively associated with ankyrin 3 (ank3)-containing complexes isolated from Madin Darby canine kidney (MDCK) epithelial cells, and it phosphorylates this epithelial ankyrin isoform in vivo. The association of CK2 with ankyrin-containing complexes in erythroid and epithelial cells provides a mechanism for rapidly altering the organization of the membrane cytoskeleton in these cell types through the CK2-dependent phosphorylation of ankyrin.
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Materials and Methods |
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32P-orthophosphate labeling of chicken erythroid
cells
Erythroid cells from 10-day-old chicken embryos or MDCK cells were
incubated in DMEM containing 1 mCi/ml 32P-orthophosphate at
37°C for various times in the presence or absence of 100 nm calyculin A.
Some of the cells were treated with 4,5,6,7-tetrabromobenzotriazole (TBB), a
CK2-specific inhibitor (Sarno et al.,
2001; Battistuta et al.,
2001
), during the labeling period. After labeling, the cells were
detergent-fractionated, and ankyrin immunoprecipitates were prepared and
analyzed on a 6% SDS polyacrylamide gel. The gels were stained with GelCode
Blue (Pierce) and dried. 32P-labeled species were detected by
autoradiography.
In vitro kinase assays
Immunoprecipitates prepared as described above were washed three times with
kinase buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 2 mM
EGTA, 6 mM ß-mercaptoethanol). The precipitates were then resuspended in
100 µl of kinase buffer containing 10 µCi of
[-32P]-ATP or [
-32P]-GTP and incubated at
37°C for 30 minutes. The reactions were terminated by the addition of SDS
sample buffer. The samples were analyzed on a 6% SDS polyacrylamide gel, and
32P-labeled species were detected by autoradiography. In some
instances, 5 mM EDTA, 50 µg of heparin, 10 µM emodin
(Battistuta et al., 2000
) or 10
µM TBB was added during the in vitro kinase reaction. Alternatively,
immunoprecipitates were boiled for 10 minutes, which eliminated the activity
of the coprecipitating kinase. These heat-treated precipitates were incubated
with 15 mU of purified CK2 from rat liver (Sigma) for 30 minutes at 37°C
and analyzed as described above.
In gel kinase assays
A whole cell lysate was prepared from erythroid cells isolated from
10-day-old chicken embryos. This lysate and an ankyrin immunoprecipitate
prepared from the lysate were electrophoresed on a 12.5% SDS polyacrylamide
gel containing either 50 µg/ml myelin basic protein or 50 µg/ml of
chicken erythroid membranes (Cox et al.,
1985). After electrophoresis, the gels were washed with 20%
isopropanol in 50 mM Tris-HCl pH 8.0 followed by two washes in 50 mM Tris-HCl
pH 7.5 and 5 mM ß-mercaptoethanol. Proteins in the gel were denatured by
incubating the gel in 6 M guanidinium-HCl for 1 hour. The denaturation buffer
was discarded and the proteins were allowed to renature in 50 mM Tris-HCl, 5
mM ß-mercaptoethanol and 0.05% Tween-20 overnight at 4°C. After two
washes in kinase buffer, the gels were incubated in kinase buffer containing
25 µM ATP and 100 µCi [
-32P] ATP for 45 minutes at
37°C. The gels were then washed extensively with 5% TCA containing 1%
sodium pyrophosphate, dried and exposed to Biomax MS X-ray film.
Immunoblotting analysis
Immunoprecipitates were prepared from a whole cell lysate from 10-day-old
embryonic erythroid cells using ankyrin preimmune and immune antisera. The
precipitates were electrophoresed on a 12.5% SDS polyacrylamide gel and
transferred to nitrocellulose. The filter was incubated with a 1:1,000
dilution of a rabbit polyclonal antibody that recognizes the and
' catalytic subunits of CK2
(Kikkawa et al., 1992
). The
filter was then washed and incubated with protein A conjugated to horseradish
peroxidase. Following washing, immunoreactive species were detected by
enhanced chemiluminescence. Immunoblotting analyses were used to determine the
proteins in MDCK cells that are recognized by the ank3-specific antiserum
(Doctor et al., 1998
). A whole
cell lysate from MDCK cells was electrophoresed on a 6% SDS polyacrylamide gel
and processed for immunoblotting using a 1:2,000 dilution of the ank3-specific
serum.
One-dimensional phosphopeptide mapping
Ankyrin immunoprecipitates were prepared from the detergent-soluble and
-insoluble fractions of control or calyculin-A-treated erythroid cells. Some
of the precipitates were prepared from cells labeled with
32P-orthophosphate as described above. The remaining precipitates
were prepared from unlabeled cells. Unlabeled precipitates were incubated in
100 µl of kinase buffer containing 10 µCi [-32P]-ATP
or heat-treated prior to incubation with purified CK2 from rat liver in kinase
buffer containing [
-32P]-ATP. In each instance, the
precipitates were resolved on a 6% SDS polyacrylamide gel. Following GelCode
Blue staining, individual ankyrin isoforms were excised from the gel and
electroeluted in a buffer composed of 125 mM Tris-HCl pH 6.8, 0.1% SDS, 1.0 mM
EDTA and 30 mM DTT. The eluted proteins were digested with 10 ng of
Staphylococcus aureus strain V8 endoproteinase at 37°C for 30
minutes. The resulting phosphopeptides were analyzed on 18% SDS polyacrylamide
gels. The gels were dried, and 32P-labeled peptides were detected
by autoradiography.
Ank3 immunoprecipitates prepared from unlabeled MDCK cells were incubated
in kinase buffer containing [-32P]-ATP or heat-treated prior
to incubation with purified CK2 from rat liver (Promega) in kinase buffer
containing [
-32P]-ATP. The precipitates were electrophoresed
on a 6% SDS polyacrylamide gel, and individual ank3 isoforms were isolated and
digested with V8 protease as described above.
In vitro binding assay
An immunoprecipitate was prepared with an -spectrin monoclonal
antibody (ICN) from the detergent-insoluble fraction of erythroid cells that
were lysed in isotonic buffer containing 1% Triton X-100. This precipitate was
washed into a low salt buffer (10 mM NaCl, 10 mM Tris pH 7.5, 5 mM
MgCl2, 2 mM EGTA, 6 mM ß-mercaptoethanol, and 1% Triton
X-100). The precipitate was then incubated overnight at 4°C with ankyrin
that was immunopurified from control erythroid cells or from erythroid cells
that were incubated with calyculin A, calyculin A plus TBB or TBB alone as
described below. Following extensive washing in low salt buffer, the
-spectrin immunoprecipitates were processed for immunoblotting analysis
using a 1:2,000 dilution of the ankyrin-specific antiserum or a 1:1,000
dilution of
-spectrin-specific monoclonal antibody.
Control erythroid cells, or cells that were incubated in the presence of
100 nM calyculin A, 100 nM calyculin A plus 60 µM TBB or 60 µM TBB for 2
hours were lysed in isotonic buffer containing 1% Triton X-100. These cells
were separated into soluble and insoluble fractions by centrifugation, and
ankyrin antibodies directly conjugated to cyanogen-bromide-activated Sepharose
4B beads were used to immunoprecipitate ankyrin from the detergent insoluble
fraction. Following washing in isotonic buffer containing 1% Triton X-100,
immunoprecipitated ankyrin was eluted from the beads in 0.2 M glycine, pH 2.3.
The eluted ankyrin was dialyzed against low salt buffer and quantified by
immunoblotting analysis. An equivalent amount of ankyrin from each sample was
incubated with an -spectrin immunoprecipitate in a total volume of 250
microliters.
Quantitative densitometry
Coomassie-stained gels were scanned using DeskScan II 2.2 software and
quantitative densitometry was performed using NIH Image. The incorporation of
32P into the erythroid and epithelial ankyrin isoforms was
quantified by phosphoimager analysis using Image-Quant software (Molecular
Dynamics).
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Results |
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Characterization of a kinase activity that coprecipitates with
ankyrin using an in gel kinase assay
To characterize the kinase activity that coprecipitates with ankyrin, in
gel kinase assays were performed. The substrate used for these assays was a
crude membrane preparation isolated from chicken erythroid cells. Analysis of
a lysate from 10-day-old embryonic erythroid cells using this in gel assay
revealed polypeptides of 140 kDa,
100 kDa and
45 kDa with
kinase activity (Fig. 2, lane
2). Although the
140 kDa and
100 kDa polypeptides could also
phosphorylate the promiscuous substrate myelin basic protein, the
45 kDa
kinase only utilized erythroid membranes as a substrate
(Fig. 2, lanes 1 and 2). In
addition, none of these proteins were detected when substrate was omitted from
the gel matrix (data not shown). A similar analysis with an ankyrin
immunoprecipitate prepared from a whole cell lysate from 10-day-old embryonic
red cells detected a single polypeptide of
45 kDa that could utilize the
erythroid membrane preparation as a substrate for phosphorylation
(Fig. 2, lane 3).
|
The immunological and biochemical properties of the kinase that
coprecipitates with ankyrin are similar to CK2
Purified CK2 from human red blood cells phosphorylates human erythroid
ankyrin in vitro (Wei and Tao,
1993). Interestingly, the
45 kDa polypeptide in ankyrin
immunoprecipitates that was detected in the in gel kinase assay is similar in
size to the predicted molecular weight of the
catalytic subunit
(Mr 45, 190) of chicken CK2
(Maridor et al., 1991
). To
investigate the relationship between the coprecipitating kinase and CK2, an
ankyrin immunoprecipitate prepared from a whole cell lysate from embryonic
erythroid cells was processed for immunoblotting using antibodies that
recognize the
and
' catalytic subunits of CK2
(Kikkawa et al., 1992
). This
analysis revealed that the CK2 antibody detected a polypeptide of
45 kDa
in the ankyrin immunoprecipitate (Fig.
3, lane 3) that was not detected in the precipitate prepared with
preimmune sera (Fig. 3, lane
2). This suggests that the
or
' CK2 subunit, or a related
CK2 isoform, is associated with ankyrin-containing complexes in these
cells.
|
Having demonstrated that a CK2-like protein coprecipitates with erythroid ankyrin, we investigated whether the kinase that phosphorylates ankyrin in vitro possessed biochemical properties similar to CK2. The catalytic activity of CK2 is Mg2+-dependent, and the addition of EDTA to the in vitro kinase reaction completely inhibited ankyrin phosphorylation (Fig. 4A, lane 2). The activity of CK2 is also heparin-sensitive, and the inclusion of heparin in our in vitro kinase assays almost entirely blocked the ability of the coprecipitating kinase to phosphorylate ankyrin (Fig. 4A, lane 3). Another hallmark of CK2 is its ability to use GTP as well as ATP as a phosphate donor during the phosphorylation reaction. The kinase that coprecipitates with ankyrin efficiently utilized GTP as a phosphate donor during in vitro assays (Fig. 4A, lane 4).
|
We also examined the effect of the CK2-specific inhibitor TBB on the
activity of the coprecipitating kinase. Until recently, highly specific
inhibitors for CK2 have not been available. However, other investigators have
shown that 10 µM TBB inhibits 87% of the in vitro activity of rat liver CK2
while having little or no effect on 32 other kinases that were assayed
(Sarno et al., 2001). As shown
in Fig. 4B (lane 2), 10 µM
TBB significantly blocked the activity of the coprecipitating kinase.
Quantification of multiple experiments has revealed that TBB inhibits
90.2±1.2% (n=2) of the coprecipitating kinase activity. A
similar result was observed with emodin
(Fig. 4B, lane 3), another CK2
inhibitor (Battistuta et al.,
2000
). Taken together, these data indicate that CK2 is
constitutively associated with erythroid ankyrin-containing complexes and
mediates the phosphorylation of ankyrin in our in vitro assays.
To directly determine whether chicken erythroid ankyrin can serve as a substrate for CK2-dependent phosphorylation, ankyrin immunoprecipitates prepared from whole cell lysates from control or calyculin A treated cells were boiled prior to the vitro kinase reaction. This treatment completely eliminated the activity of the coprecipitating kinase (Fig. 4C, lanes 3 and 4). These heat-inactivated precipitates were used as a substrate for purified CK2 isolated from rat liver. This analysis revealed that rat liver CK2 could phosphorylate each of the ankyrin isoforms from control cells in vitro (Fig. 4C, lane 5). In addition, hyperphosphorylated ankyrin from calyculin-A-treated cells was efficiently utilized as a substrate by purified CK2 (Fig. 4C, lane 6).
CK2 is the major ankyrin kinase in chicken erythroid cells
One-dimensional peptide mapping was used to compare the phosphorylation
sites in ankyrin immunoprecipitated from control and calyculin-A-treated
erythroid cells. This analysis yielded identical phosphopeptide maps for the
detergent-soluble and -insoluble 225 kDa ankyrin isoforms precipitated from
cells labeled with 32P-orthophosphate in the presence of calyculin
A (Fig. 5, lanes 3 and 4). Some
of the phosphorylated peptides from hyperphosphorylated ankyrin were held in
common with the phosphopeptides generated from the detergent-insoluble 225 kDa
isoform precipitated from 32P-orthophosphate-labeled control cells
(Fig. 5, lane 5). However,
several phosphopeptides were uniquely associated with ankyrin precipitated
from control or calyculin-A-treated cells. These distinct patterns of
phosphorylation probably contribute to the differing abilities of basally
phosphorylated and hyperphosphorylated ankyrin to associate with spectrin
(Ghosh and Cox, 2001).
|
The profile of phosphopeptides observed for the in vitro phosphorylated 225 kDa isoform (Fig. 5, lane 6) was similar to the phosphopeptide map of the in vivo phosphorylated 225 kDa isoform precipitated from control cells (Fig. 5, lane 5; comigrating peptides are marked with asterisks). This result suggests that the kinase activity that coprecipitates with ankyrin can mediate most of the ankyrin phosphorylation events that occur in control cells in vivo. In addition, the in vitro phosphorylated 225 kDa (Fig. 5, lane 6) and 220 kDa (Fig. 5, lane 7) isoforms precipitated from control cells shared similar phosphopeptide maps. This indicates that the lack of phosphorylation of the 220 kDa isoform in vivo is not simply due to the fact that this isoform lacks the sequences that are phosphorylated in the 225 kDa isoform in vivo. The phosphopeptide map of the in vitro phosphorylated 225 kDa ankyrin isoform from control cells (Fig. 5, lane 6) lacked all of the phosphopeptides uniquely associated with 32P-labeled ankyrin precipitated from calyculin-A-treated cells (Fig. 5, lanes 3 and 4). However, many of these unique phosphopeptides (marked with dashes in Fig. 5) were generated when ankyrin precipitated from calyculin-A-treated cells was used as a substrate for in vitro phosphorylation (Fig. 5, lane 2).
To investigate whether purified CK2 phosphorylates a similar array of sites on ankyrin as the coprecipitating kinase, we compared the one-dimensional phosphopeptide maps of the 225 kDa ankyrin isoform that had been phosphorylated by these kinases. This analysis revealed very similar phosphopeptide maps for the 225 kDa ankyrin isoform from control cells that was phosphorylated by the coprecipitating kinase (Fig. 5, lane 6) or by purified CK2 (Fig. 5, lane 8). Interestingly, purified CK2 also phosphorylated many of the phosphopeptides uniquely associated with the hyperphosphorylated 225 kDa ankyrin isoform (Fig. 5, lanes 3 and 4) when ankyrin from calyculin-A-treated cells was used as a substrate (Fig. 5, lane 1).
The data described above strongly suggest that the majority of the phosphorylation events associated with both basally phosphorylated and hyperphosphorylated erythroid ankyrin in vivo are mediated by CK2. Furthermore, the in vivo phosphorylation sites in the 225 kDa ankyrin isoform that are not observed in vitro may be caused by bound antibody blocking the phosphorylation of these sequences in vitro. To further investigate the role of CK2 in directing ankyrin phosphorylation, we examined the effect of the CK2-specific inhibitor, TBB, on ankyrin phosphorylation in vivo. This analysis revealed that TBB completely blocked the in vivo phosphorylation of ankyrin in control and calyculin-A-treated erythroid cells (Fig. 6), suggesting that the phosphorylation of ankyrin in chicken erythroid cells is entirely CK2 dependent. This reagent also prevented the shift of ankyrin from the detergent-insoluble to the soluble pool that results when erythroid cells are treated with calyculin A (Fig. 6, compare the Coomassie-stained profiles in lanes 5 and 6 with those in lanes 7 and 8).
|
The ability of TBB to block the calyculin-A-induced changes in the
solubility of ankyrin suggested a critical role for CK2 in regulating the
cytoskeleton-binding properties of erythroid ankyrin. To address this
possibility, binding studies have examined whether the CK2-dependent
phosphorylation of ankyrin directly affects its capacity to associate with
spectrin in vitro. For these analyses, erythroid cells were lysed in isotonic
buffer containing 1% Triton X-100, and -spectrin immunoprecipitates
were prepared from the detergent-insoluble fraction in the same buffer. The
precipitates were washed into low salt buffer and incubated with ankyrin that
was immunopurified from control erythroid cells or from cells that were
treated with calyculin A, calyculin A plus TBB or TBB alone as described in
the Materials and Methods. Following washing of the
-spectrin
immunoprecipitates in low salt buffer, they were subjected to immunoblotting
analysis with ankyrin-specific or
-spectrin-specific antibodies
(Fig. 7). As shown previously,
chicken erythroid ankyrin dissociates from spectrin when cells are lysed in
isotonic buffer containing 1% Triton X-100
(Fig. 7, lane 1). When ankyrin
from control cells was incubated with the
-spectrin in low salt buffer,
the 225 kDa ankyrin isoform coprecipitated with spectrin
(Fig. 7, lane 4). Longer
exposure of the ankyrin immunoblot revealed that the 205 kDa ankyrin isoform
from control cells also bound to spectrin (data not shown). In contrast, there
was no detectable binding of hyperphosphorylated ankyrin that had been
immunopurified from calyculin-A-treated cells to spectrin
(Fig. 7, lane 2). However,
ankyrin immunopurified from cells treated with calyculin A plus TBB
(Fig. 7, lane 3) or TBB alone
(Fig. 7, lane 5) bound to
spectrin to a similar extent to ankyrin from control cells. These results,
which were obtained in two independent experiments, indicate that the
CK2-dependent hyperphosphorylation of ankyrin dramatically inhibits its
ability to associate with spectrin in vitro. These data further suggest that
CK2 is the major regulator of the cytoskeleton-binding properties of ankyrin
in chicken embryonic erythroid cells.
|
CK2 phosphorylates ankyrin 3 isoforms in MDCK kidney epithelial
cells
We also studied whether CK2 serves as a regulator of ankyrin function in
cell types other than erythroid cells. For these experiments, we used
antibodies that recognize the major epithelial ankyrin isoform, ank3.
Immunoblotting analysis of a whole cell lysate from MDCK cells with these
antibodies revealed two major ank3 isoforms of 215 kDa and 200 kDa
(Fig. 8A, lane 1), as well as
two minor species of 170 kDa and 120 kDa (data not shown). To assess whether
these ank3 isoforms were phosphorylated in a CK2-dependent manner, ank3
immunoprecipitates were prepared from 32Porthophosphate-labeled
MDCK cells incubated in the absence or presence of the CK2-specific inhibitor,
TBB. Coomassie staining of these immunoprecipitates revealed a profile similar
to the immunoblot, with two major species of 215 kDa and 200 kDa
(Fig. 8A, lanes 2 and 3).
Although both isoforms were phosphorylated in vivo, the extent of
phosphorylation was significantly inhibited in cells treated with TBB
(Fig. 8A, lanes 4 and 5).
Quantification of multiple experiments identical to that shown in
Fig. 8A indicated that TBB
treatment of cells resulted in a 53.9±8.7% (n=2) decrease in
the phosphorylation of the ank3 isoforms. The residual ankyrin phosphorylation
observed in TBB-treated cells suggests that other kinases are involved in the
phosphorylation of ank3 in this epithelial cell type.
|
In vitro kinase assays with ank3 immunoprecipitates examined whether the kinase(s) involved in ank3 phosphorylation in vivo physically associate with ankyrin-containing complexes in MDCK cells. This analysis revealed that a kinase activity coprecipitated with ank3 and phosphorylated both the 215 kDa and 200 kDa ank3 isoforms in vitro (Fig. 8B, lane 2). This coprecipitating kinase also phosphorylated a species of 210 kDa, which was not visible in the Coomassie staining pattern of the ank3 immunoprecipitate (Fig. 8B). Like the erythroid ankyrin kinase, this MDCK cell kinase was heparin sensitive (Fig. 8B, lane 3), and incubation of a heat-inactivated precipitate with purified CK2 resulted in a profile of phosphorylated species identical to that observed with the coprecipitating kinase (Fig. 8B, lane 5). Finally, one-dimensional phosphopeptide mapping revealed that purified CK2 and the coprecipitating kinase phosphorylated similar sites in the 215 kDa (Fig. 9) and 200 kDa (data not shown) ank3 isoforms from MDCK cells. These results strongly suggest that CK2 is constitutively associated with ank3-containing complexes in MDCK cells and is likely to be involved in regulating ankyrin function in this epithelial cell type.
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Discussion |
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CK2 is a ubiquitous, highly conserved kinase involved in multiple cellular
processes including cell division and proliferation
(Li et al., 1999) and membrane
protein trafficking (Cotlin et al.,
1999
; Mauxion et al.,
1996
; Shi et al.,
2001
). This multisubunit kinase is composed of
and
' catalytic subunits and a regulatory ß subunit
(Sarno et al., 2000
), which
associate to form
2ß2,
'2ß2, and
'ß2 complexes that may have distinct
functions in vivo (Dobrowolska et al.,
1999
). Although the subunit composition of the CK2 holoenzyme in
chicken erythroid cells has not been defined, in gel kinase and immunoblotting
analyses have shown that the size of the catalytic subunit of the
ankyrin-associated kinase is very similar to the predicted size of the
catalytic subunit of chicken CK2 (Maridor
et al., 1991
).
The in vitro phosphorylation of ankyrin from control cells by coprecipitating CK2 did not yield the slower-migrating hyperphosphorylated form of ankyrin that is observed when erythroid cells are treated with phosphatase inhibitors in vivo. Furthermore, the phosphopeptides uniquely associated with hyperphosphorylated ankyrin from calyculin-A-treated cells could not be recapitulated when ankyrin from control cells was phosphorylated in vitro by coprecipitating CK2. These results must be reconciled with the inhibitor studies, which suggested that all of the phosphorylation events associated with both basally phosphorylated and hyperphosphorylated ankyrin in vivo are CK2 dependent. One explanation that could account for these results is that another CK2-dependent kinase is required to generate some of the unique phosphorylation events associated with hyperphosphorylated ankyrin in vivo. Alternatively, CK2 undergoes phosphorylation in calyculin-A-treated erythroid cells (data not shown). This modification may slightly alter the specificity of this kinase, leading to the phosphorylation events detected in hyperphosphorylated ankyrin.
The cytoskeletal 225 kDa ankyrin polypeptide from TBB-treated erythroid
cells (Fig. 6, lane 4) migrated
as a discrete species that comigrated with the lower half of the 225 kDa
ankyrin species from untreated cells (Fig.
6, lane 2). This result suggests that cytoskeletal ankyrin in
untreated cells exists in both a phosphorylated and a dephosphorylated state.
Although the physiological consequences of the CK2-dependent phosphorylation
events associated with basally phosphorylated ankyrin are not known, recent
studies have shown that cytoskeletal ankyrin in chicken erythroid cells turns
over with a relatively short half-life
(Ghosh and Cox, 2001).
Interestingly, CK2-dependent phosphorylation is involved in regulating the
degradation of other ANK-repeat containing proteins, such as
Drosophila Cactus (Liu et al.,
1997
) and its mammalian counterpart I
B
(Lin et al., 1996
;
McElhinny et al., 1996
).
Whether the basal phosphorylation events associated with a subset of
cytoskeletal ankyrin polypeptides are involved in regulating their stability
remains to be determined.
Unlike the other chicken erythroid ankyrin isoforms, the 220 kDa isoform is exclusively detergent insoluble, and treatment of cells with phosphatase inhibitors does not alter its solubility. This isoform is also unique in that it is not phosphorylated in vivo, although it can serve as a substrate for CK2 in vitro. The lack of phosphorylation of this isoform in vivo could be due to the fact that this protein is sequestered in a cellular compartment devoid of CK2, or alternatively the protein may assume an in vivo conformation that can not undergo phosphorylation. Further studies will be required to determine whether these or other mechanisms account for the observed in vivo properties of the 220 kDa isoform.
Crystal structure data have suggested that CK2 is constitutively active
(Niefind et al., 2001). The
fact that this kinase is also constitutively associated with ankyrin raises
the question of how the level of ankyrin phosphorylation is regulated. The
concentration of calyculin A used during our in vivo studies typically
inhibits the multimeric serine and threonine phosphatases, PP1 and PP2A.
Although it is not known whether the phosphorylation status of ankyrin in
cells is directly regulated by PP1, PP2A or another cellular phosphatase,
additional analyses have shown that in vitro phosphorylated ankyrin can be
directly dephosphorylated by a phosphatase present in chicken erythroid cell
lysates (data not shown).
Our previous studies demonstrated that chicken erythroid ankyrin-containing complexes can undergo dynamic rearrangements in response to changes in ankyrin phosphorylation. The results described here suggest that most, if not all of the phosphorylation events associated with erythroid ankyrin are mediated by CK2. Furthermore, we show that CK2 is constitutively associated with ankyrin in both erythroid and kidney epithelial cells. This is the first report of a kinase constitutively associating with elements of the membrane cytoskeleton in these cell types. Through this physical association, cells can rapidly regulate the interaction between ankyrin and spectrin and as a consequence alter the organization of their membrane cytoskeleton in response to extracellular signals.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Battistuta, R., de Moliner, E., Sarno, S., Zanotti, G. and
Pinna, L. A. (2001). Structural features underlying selective
inhibition of protein kinase CK2 by ATP site-directed
tetrabromo-2-benzotriazole. Protein Sci.
10,2200
-2206.
Battistuta, R., Sarno, S., de Moliner, E., Papinutto, E.,
Zanotti, G. and Pinna, L. A. (2000). The replacement of ATP
by the competitive inhibitor emodin induces conformational modifications in
the catalytic site of protein kinase CK2. J. Biol.
Chem. 275,29618
-29622.
Bennett, V. and Chen, L. (2001). Ankyrins and cellular targeting of diverse membrane proteins to physiological sites. Curr. Opin. Cell Biol. 13, 61-67.[CrossRef][Medline]
Bourguignon, L. Y., Zhu, H., Shao, L., Zhu, D. and Chen, Y. (1999). Rho-kinase promotes CD44V3,8-10-ankyrin interaction and tumor cell migration in metastatic breast cancer cells. Cell Motil. Cytoskeleton. 43,269 -287.[CrossRef][Medline]
Cotlin, L. F., Siddiqui, M. A., Simpson, F. and Collawn, J.
F. (1999). Casein kinase II activity is required for
transferrin receptor endocytosis. J. Biol. Chem.
274,30550
-30556.
Cox, J. V., Moon, R. T. and Lazarides, E. (1985). Anion transporter: Highly cell-type specific expression of distinct polypeptides and transcripts in erythroid and nonerythroid cells. J. Cell Biol. 100,1548 -1557.[Abstract]
De Matteis, M. A. and Morrow, J. S. (2000).
Spectrin tethers and mesh in the biosynthetic pathway. J. Cell
Sci. 113,2331
-2343.
Dobrowolska, G., Lozeman, F. J., Li, D. and Krebs, E. G. (1999). CK2, a protein kinase of the next millennium. Mol. Cell. Biochem. 191,3 -12.[CrossRef][Medline]
Doctor, R. B., Chen, J., Peters, L. L., Lux, S. E. and Mandel,
L. J. (1998). Distribution of epithelial ankyrin (Ank3)
spliceoforms in renal proximal and distal tubules. Am. J.
Physiol. 274,F129
-F138.
Fukata, Y., Oshiro, N., Kinoshita, N., Kawano, Y., Matsuoka, Y.,
Bennett, V., Matsuura, Y. and Kaibuchi, K. (1999).
Phosphorylation of adducin by Rho-kinase plays a crucial role in cell
motility. J. Cell Biol.
145,347
-361.
Ghosh, S. and Cox, J. V. (2001). Dynamics of
ankyrin-containing complexes in chicken embryonic erythroid cells: Role of
phosphorylation. Mol. Biol. Cell
12,3864
-3874.
Ghosh, S., Cox, K. H. and Cox, J. V. (1999).
Chicken erythroid AE1 anion exchangers associate with the cytoskeleton during
recycling to the Golgi. Mol. Biol. Cell
10,455
-469.
Husain-Chishti, A., Faquin, W., Wu, C. C. and Branton, D.
(1989). Purification of erythrocyte dematin (protein 4.9) reveals
an endogenous protein kinase that modulates actin-bundling activity.
J. Biol. Chem. 264,8985
-8991.
Khanna, R., Chang, S. H., Andrabi, S., Azam, M., Kim, A.,
Rivera, A., Brugnara, C., Low, P. S., Liu, S. C. and Chishti, A. H.
(2002). Headpiece domain of dematin is required for the stability
of the erythrocyte membrane. Proc. Natl. Acad. Sci.
USA 99,6637
-6642.
Kikkawa, U., Mann, S. K., Firtel, R. and Hunter, T. (1992). Molecular cloning of casein kinase II alpha subunit from Dictyostelium discoideum and its expression in the life cycle. Mol. Cell. Biol. 12,5711 -5723.[Abstract]
Li, D., Dobrowolska, G., Aicher, L. D., Chen, M., Wright, J. H.,
Drueckes, P., Dunphy, E. L., Munar, E. S. and Krebs, E. G.
(1999). Expression of the casein kinase 2 subunits in Chinese
hamster ovary and 3T3 L1 cells provides information on the role of the enzyme
in cell proliferation and the cell cycle. J. Biol.
Chem. 274,32988
-32996.
Lin, R., Beauparlant, P., Makris, C., Meloche, S. and Hiscott, J. (1996). Phosphorylation of I kappa B alpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. Mol. Cell. Biol. 16,1401 -1409.[Abstract]
Liu, Z. P., Galindo, R. L. and Wasserman, S. A.
(1997). A role for CKII phosphorylation of the cactus PEST domain
in dorsoventral patterning of the Drosophila embryo. Genes
Dev. 11,3413
-3422.
Malhotra, J. D., Koopmann, M. C., Kazen-Gillespie, K. A.,
Fettman, N., Hortsch, M. and Isom, L. L. (2002). Structural
requirements for interaction of sodium channel beta 1 subunits with ankyrin.
J. Biol. Chem. 277,26681
-26688.
Maridor, G., Park, W., Krek, W. and Nigg, E. A.
(1991). Casein kinase II. cDNA sequences, developmental
expression, and tissue distribution of mRNAs for alpha, alpha', and beta
subunits of the chicken enzyme. J. Biol. Chem.
266,2362
-2368.
Mauxion, F., le Borgne, R., Munier-Lehmann, H. and Hoflack,
B. (1996). A casein kinase II phosphorylation site in the
cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor
determines the high affinity interaction of the AP-1 Golgi assembly proteins
with membranes. J. Biol. Chem.
271,2171
-2178.
McElhinny, J. A., Trushin, S. A., Bren, G. D., Chester, N. and Paya, C. V. (1996). Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation. Mol. Cell. Biol. 16,899 -906.[Abstract]
Nelson, W. J. and Hammerton, R. W. (1989). A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity. J. Cell Biol. 108,893 -902.[Abstract]
Niefind, K., Guerra, B., Ermakowa, I. and Issinger, O. G.
(2001). Crystal structure of human protein kinase CK2: insights
into basic properties of the CK2 holoenzyme. EMBO J.
20,5320
-5331.
Sarno, S., Marin, O., Boschetti, M., Pagano, M. A., Meggio, F. and Pinna, L. A. (2000). Cooperative modulation of protein kinase CK2 by separate domains of its regulatory beta-subunit. Biochemistry 39,12324 -12329.[CrossRef][Medline]
Sarno, S., Reddy, H., Meggio, F., Ruzzene, M., Davies, S. P., Donella-Deana, A., Shugar, D. and Pinna, L. A. (2001). Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 (casein kinase-2). FEBS Lett. 496, 44-48.[CrossRef][Medline]
Shi, X., Potvin, B., Huang, T., Hilgard, P., Spray, D. C.,
Suadicani, S. O., Wolkoff, A. W., Stanley, P. and Stockert, R. J.
(2001). A novel casein kinase 2 alpha-subunit regulates membrane
protein traffic in the human hepatoma cell line HuH-7. J. Biol.
Chem. 276,2075
-2082.
Subrahmanyam, G., Bertics, P. J. and Anderson, R. A. (1991). Phosphorylation of protein 4.1 on tyrosine 418 modulates its function in vitro. Proc. Natl. Acad. Sci. USA 88,5222 -5226.[Abstract]
Tse, W. T. and Lux, S. E. (1999). Red blood cell membrane disorders. Br. J. Haematol. 104, 2-13.[Medline]
Tuvia, S., Garver, T. D. and Bennett, V.
(1997). The phosphorylation state of the FIGQY tyrosine of
neurofascin determines ankyrin-binding activity and patterns of cell
segregation. Proc. Natl. Acad. Sci. USA
94,12957
-12962.
Wei, T. and Tao, M. (1993). Human erythrocyte casein kinase II: characterization and phosphorylation of membrane cytoskeletal proteins. Arch. Biochem. Biophys. 307,206 -216.[CrossRef][Medline]