From the Department of Internal Medicine and the Cancer Research
and Treatment Center, University of New Mexico School of Medicine,
Albuquerque, New Mexico 87131 and the Division of Cell
Biology, La Jolla Institute for Allergy and Immunology,
La Jolla, California 92121
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ABSTRACT |
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Moesin, a member of the
ezrin-radixin-moesin (ERM) family of membrane/cytoskeletal linkage
proteins, is known to be threonine-phosphorylated at
Thr558 in activated platelets within its conserved
putative actin-binding domain. The pathway leading to this
phosphorylation step and its control have not been previously
elucidated. We have detected and characterized reactions leading to
moesin phosphorylation in human leukocyte extracts. In
vitro phosphorylation of endogenous moesin, which was identified
by peptide microsequencing, was dependent on phosphatidylglycerol (PG)
or to a lesser extent, phosphatidylinositol (PI), but not
phosphatidylserine (PS) and diacylglycerol (DAG). Analysis of charge
shifts, phosphoamino acid analysis, and stoichiometry was consistent
with a single phosphorylation site. By using mass spectroscopy and
direct microsequencing of CNBr fragments of phospho-moesin, the
phosphorylation site was identified as KYKT*LRQIR (where * indicates
the phosphorylation site) (Thr558), which is conserved in
the ERM family. Recombinant moesin demonstrated similar in
vitro phospholipid-dependent phosphorylation compared with the endogenous protein. The phosphorylation site sequence of
moesin displays a high degree of conservation with the pseudosubstrate sequences of the protein kinase C (PKC) family. We identified the
kinase activity as PKC- on the basis of immunodepletion of the
moesin kinase activity and copurification of PKC-
with the enzymic
activity. We further demonstrate that PKC-
displays a preference for
PG vesicles over PI or PS/DAG, with minimal activation by DAG, as well
as specificity for moesin compared with myelin basic protein, histone
H1, or other cellular proteins. Expression of a human
His6-tagged PKC-
in Jurkat cells and purification by
Ni2+ chelate chromatography yield an active enzyme that
phosphorylates moesin. PG vesicle binding experiments with expressed
PKC-
and moesin demonstrate that both bind to vesicles independently
of one another. Thus, PKC-
is identified as a major kinase within cells with specificity for moesin and with activation under
non-classical PKC conditions. It appears likely that this activity
corresponds to a specific intracellular pathway controlling the
function of moesin as well as other ERM proteins.
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INTRODUCTION |
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Moesin is a member of the ERM1 family of actin cytoskeletal-membrane linkage proteins. Although closely related, there is evidence that these proteins have distinct functions, based upon their patterns of intracellular and tissue distribution. Moesin is named for its association with extracellular extensions (membrane-organizing-extension spike protein). ERM proteins have N-terminal membrane-binding domains and C-terminal actin-binding domains. Relatively little is known about their intracellular control, although phosphorylation of moesin Thr558 by an unidentified kinase in activated platelets has recently been described (1). This residue is within the previously identified actin-binding domain of ezrin, which is highly conserved in moesin (2).
This laboratory has been studying a distinctive protein phosphorylation
reaction characterized by stimulation of more than 10-fold with the
addition of appropriate phospholipid vesicles in the presence of
Mn2+, in preference to Mg2+, but not requiring
Ca2+ (3-5). Under these conditions phosphorylation was
strikingly specific for two major endogenous substrates from human
leukemic cell extracts, which were 47 and 73 kDa by SDS-PAGE. This
activity was found to be most highly expressed in both normal and
malignant hemopoietic cells and tissues of both human and murine
origin. We noted the activation requirements of these reactions to
contrast with those of classical PKC isozymes and therefore embarked
upon the purification and identification of the components of this system from human leukocytes. Previously, we identified the 47-kDa substrate as the small calpain fragment of talin, a protein which has
homology to ERM proteins and is involved in focal adhesions of cells to
substratum (6). We here report the purification and identification of
the 73-kDa substrate as moesin. We furthermore have identified the
moesin kinase present in our cell extracts as protein kinase C-, a
novel PKC isozyme, which is known to be highly expressed in hemopoietic
lineage cells but is of unknown function. These results thus provide
evidence for a novel pathway of control of ERM proteins by PKC-
. The
results also provide insights into the specificity of PKC-
for
moesin and the activation requirements for PKC-
, which has not
previously been shown to be activated by Mn2+ and
PG.
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MATERIALS AND METHODS |
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All electrophoresis materials were supplied by Bio-Rad, and all
other chemicals, as well as the catalytic subunit of PKA, were supplied
by Sigma unless otherwise noted. Protein kinase C (PKC), purified from
rat brain, was obtained from Lipidex (Westfield, NJ) and is a mixture
of isozymes. Isozyme-specific anti-PKC sera used for immunodepletion
experiments were obtained from R & D Ab Diagnostics (Berkeley, CA).
These antibodies, with the exception of anti-PKC-, are capable of
immunoprecipitation. Anti-PKC-
specific for the V3 region of PKC-
was used as described previously (7); anti-PKC-
(F-13) specific for
the C-terminal residues 691-703, anti-PKC-
(C-20) specific for
C-terminal residues 651-672, and anti-His6 probe (H-15)
antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Antibodies and reagents for ECL detection were obtained from
Amersham.
In Vitro Phosphorylation-- Phosphorylation reactions were carried out as described previously (6), except that the phospholipid was sonicated in water for 30 min to produce a suspension of vesicles. After the reaction, proteins were precipitated by adding 1/10 volume of 0.15% deoxycholic acid, mixing, and then quickly adding 1/10 volume of 100% (w/v) trichloroacetic acid, mixing, and centrifuging in a microcentrifuge for 5 min. This method improves protein yield and results in a stable precipitate (8). The precipitate was rinsed once with 20 µl of water, dissolved in 10 µl of 1× Laemmli sample buffer at 56 °C for 10 min, and subjected to 10% SDS-PAGE as described (9). The gels were stained with Coomassie Blue and dried between cellophane sheets, and autoradiograms were made. Protein determinations were done as described (10).
Purification of the pp73-- Purification of the 73-kDa substrate was carried out as described previously, including ammonium sulfate fractionation and chromatography of proteins on CM-cellulose (6), which was based on earlier work (11). Fractions from a gradient elution of the CM-cellulose column were assayed for PG-stimulable phosphorylation of a 73-kDa substrate, and the peak fractions (about 0.1-0.15 M NaCl) were combined and concentrated using a PM-30 membrane (Amicon, Beverly, MA); 40 ml of pelleted acute myelogenous leukemia (AML) cells from a patient gave 4 ml of CM-cellulose fraction with 14 mg of protein. 400 µl of this was adsorbed to PG vesicles as in the assay (800 µl total), incubated at 30 °C for 5-15 min, put on ice for 5-15 min, and then spun in a microcentrifuge for 10 min. The supernatant was removed, and the PG vesicles including adsorbed proteins were suspended in 200 µl of 0.5% Nonidet P-40, 0.6% Ampholines, pH 6-7 (Serva; Crescent Chemicals, Hauppauge, NY), 0.4% Ampholines, pH 3.5-10, 8% sucrose. This was divided between two isoelectric focusing tubes 0.15 cm inner diameter by 18 cm, containing a 0-40% sucrose gradient plus the Ampholines listed above, as described (12). 100 V was applied for 18 h and then 0.25-ml fractions were collected through a 26-gauge needle and analyzed by SDS-PAGE. The three fractions containing pp73 (determined by a 32P-radiolabeled tracer and SDS-PAGE) were co-precipitated with deoxycholate plus trichloroacetic acid as described above and rinsed on ice with 50 µl of cold acetone. The precipitates were submitted to the W. M. Keck Biotechnology Resource Laboratory, Yale University (New Haven, CT) for trypsin digestion, HPLC separation of the resulting peptides, and Edman sequencing of two of the peptides. One peptide gave a major and a minor sequence. To identify the phosphorylation site, 32P-labeled moesin was digested with CNBr, and the peptides were purified by reverse phase HPLC on a Vydac C18 column using a gradient of acetonitrile in 0.1% trifluoroacetic acid. The radioactive peak was analyzed by mass spectroscopy using matrix-assisted laser desorption (performed at the Yale University facility). This system is capable of 1-Da accuracy at 3500 Da. Manual amino acid sequencing of the 32P-labeled moesin peptides was performed as described (13).
A further partial purification of moesin, starting at the CM-cellulose pool, resulted in separation of moesin, moesin kinase, and phosphatase activity. The CM-cellulose pool was equilibrated in 0.02 M Tris, pH 7.5, applied to a column of DEAE 8HR (Millipore, Bedford, MA), and eluted with a linear gradient to 0.5 M NaCl. moesin eluted at about 0.25 M NaCl, just before the endogenous kinase activity.Bacterial Expression of Human Moesin-- cDNA to human moesin was generously given by H. Furthmayr (Stanford University) in plasmid form. Double-stranded cDNA was generated by the polymerase chain reaction, making the two BglI fragments of moesin, using the following two primer pairs: 5'-CATGCCCAAAACGATCAGTGT plus 5'-CAAGGCCTCCTTGGCCTCT and 5'-TTACATAGACTCAAATTCGTCAAT plus 5'-AGAGGCCAAGGAGGCCTTG. The products were cut with BglI (14) and ligated, and after agarose gel electrophoresis, full-length moesin cDNA was obtained using Geneclean (Bio 101, La Jolla). Primers were synthesized to allow insertion into the NheI to KpnI site of the bacterial expression vector pRSETA (Invitrogen, San Diego): 5'-CCGGCTAGCATGCCCAAAACGATCAGTGT plus 5'-GCGGGTACCTTACATAGACTCAAATTCGTCAAT. The resulting DNA was ligated into the vector and used for transformation of Escherichia coli BL21(DE3) containing pLys S for expression of protein (15). This sequence codes for moesin preceded by the protein "tag" MRGSHHHHHHGMAS; the six-histidine (His6) tag binds tightly to a nickel (Ni2+) chelate column (see below).
Protein expression was adequate in E. coli grown in L broth at 37 °C in the absence of inducer. 50 ml of L broth supplemented with ampicillin and chloramphenicol were inoculated with a fresh colony, grown for 8 hours, and centrifuged at 5,000 × g for 10 min. The cell pellet was resuspended in water to a total volume of 4 ml and then quick frozen. The thawed suspension was sonicated with 40 bursts from a microtip at 30% duty, using a Bronson sonifier set at output 2. Cellular debris was removed by centrifugation at 12,000 × g for 20 min, and the 3.2 ml of supernatant was combined with 3.2 ml of "bind" buffer and applied to a 2-ml Ni2+ chelate affinity column (Invitrogen). The column was rinsed according to the supplier's instructions, and the recombinant moesin was eluted with an imidazole gradient from 0 to 400 mM imidazole. Fractions were analyzed by SDS-PAGE, and those containing moesin were combined and equilibrated in 10 mM BisTris, pH 6.5, 0.1 mM EDTA, 1 mM 2-mercaptoethanol by passage through a P6DG column (Bio-Rad). Residual phosphatases were removed by passage through a 1-ml CM-cellulose column with a 0-0.3 M NaCl gradient over 10 ml. Fractions of 1 ml were again analyzed by SDS-PAGE, and fractions 5-7 were combined, and glycerol was added to 7%, and the preparation was frozen. Recombinant His6-moesin was judged to be >90% pure by SDS-PAGE and Coomassie Brilliant Blue staining.Immunodepletion of Moesin Kinase Activity-- Isozyme-specific PKC rabbit antibodies were purified from crude antisera by performing a 40% ammonium sulfate precipitation. This step effectively removed phosphatase activity present in serum. The precipitated IgG fraction was dialyzed into TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl), and the total protein was estimated by SDS-PAGE using different amounts of molecular weight standards. An aliquot of the CM-cellulose-purified moesin kinase was incubated with 1 µg each of the isozyme-specific and non-immune IgG in TBS at 4 °C for 60 min, followed by protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz) for an additional 30 min. The beads were collected by centrifugation, and the supernatant was used in the moesin kinase assay described above.
Purification of Moesin-specific Protein Kinase Activity from AML
Cells--
Each step in the purification of protein kinase activity
utilized His6-moesin (10 µg/ml) as a substrate in the
phosphorylation reaction described above. Cells from patients with AML
were obtained by leukapheresis. Cells were pelleted at 500 × g, rinsed with PBS, and stored in BSB (10 mM
Na2HPO4, pH 6.5, 2 mM EDTA, 1 mM EGTA) at 70 °C. Forty ml of packed cells were
thawed, suspended in BSB containing 10 µg/ml each of leupeptin,
aprotinin, and pepstatin A, 0.5 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol, 10 µM NaF, 1 mM Na3VO4, and homogenized in a
Brinkmann Polytron. The homogenate was centrifuged at 20,000 × g for 20 min, and the resulting supernatant was used for
sequential ammonium sulfate fractionation. A 35-60% saturated
ammonium sulfate precipitate was collected by centrifugation, dissolved
in BSB, and then desalted on Sephadex G-50 (50 ml) equilibrated in BSB.
The eluate was applied to a 20-ml CM-cellulose column equilibrated in
BSB and rinsed with 2 column volumes of buffer. A step gradient from
0.05 to 0.3 M NaCl was used to elute protein kinase
activity from the column. Active fractions were pooled and desalted
into DEAE buffer (20 mM Tris-HCl, pH 7.5, 0.1%
2-mercaptoethanol, 10 µM NaF, 1 mM
Na3VO4, 1 mM EDTA, 1 mM
EGTA) using an Amicon ultrafiltration cell. The sample was applied to a
Millipore DEAE 8HR column and eluted with a linear gradient from 0 to
0.5 M NaCl in DEAE buffer. Active fractions were pooled and
desalted into DEAE buffer and then fractionated on heparin-Sepharose.
The heparin-Sepharose column was eluted with a gradient from 0 to 2.0 M NaCl in DEAE buffer containing 0.1 mM EDTA,
0.1 mM EGTA, 0.1% 2-mercaptoethanol, 1 µM
NaF, and 1 mM Na3VO4. Glycerol was
added to each column fraction to obtain a final concentration of 10%
before freezing at
70 °C.
Transfection of Jurkat T-lymphoblastic Cells with
pEF-PKC-His6 and Purification on Ni2+
Affinity Beads--
Jurkat Tag cells are stably
transfected with the SV40 large T antigen, which is used to promote
expression of genes cloned in the pEF vector (16). pEF-PKC-
contains
the complete coding region of PKC-
and an in-frame His6
tag at the C terminus. Cells (2 × 107) were
electroporated with 20 µg of pEF-PKC-
or pEF-neo (empty vector
control) plasmids in a volume of 400 µl of RPMI 1640 medium using a
Bio-Rad Gene Pulser set at 260 V and 960 microfarads. The cells were
transferred to a flask containing 10% fetal calf serum in RPMI medium
and 700 µg/ml G418 and grown for 48 h. The cells were harvested,
rinsed with PBS, and then lysed in 1 ml of binding buffer (1% Nonidet
P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 µg/ml each of leupeptin, aprotinin, pepstatin, and 1 mM
phenylmethylsulfonyl fluoride) for 15 min at 4 °C. Lysates were
centrifuged at 15,000 × g for 15 min, and the
supernatants were used for purification using 0.1 ml of
Ni2+ affinity beads. The beads were mixed with supernatants
for 60 min at 4 °C and then rinsed twice with 1 ml of binding buffer and then three times with 1 ml of binding buffer containing 50 mM imidazole, pH 6.5. The beads were then rinsed once with
TBS to remove detergent, and bound proteins were eluted by incubation of the beads in 0.1 ml of 500 mM imidazole, pH 6.5, in TBS
containing 10 µg/ml bovine serum albumin and 50% glycerol for 15 min
at 4 °C. Aliquots of the eluates from the Ni2+ affinity
beads were subjected to SDS-PAGE and immunoblotting with anti-PKC-
(V3-specific) and anti-His6 antibodies. Both antibodies detected a single immunoreactive band at 80 kDa in the eluate from the
pEF-PKC-
-transfected cells but not pEF neo-transfected cells.
Immunoblotting-- Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes in 62 mM Tris, 190 mM glycine containing 20% methanol at 280 mA for 45 min. The membranes were stained with Ponceau S in 1% acetic acid to identify the molecular weight standards and destained in PBST (PBS containing 0.01% Tween 20). The blots were incubated with 5% non-fat dry milk in PBST for 15 min and then with primary antibodies for 60 min. After several rinses in PBST, the blots were incubated with horseradish peroxidase-conjugated anti-mouse or rabbit IgG for 30 min and then rinsed with PBST before detection with ECL reagent (Amersham).
Phospholipid Binding of PKC--His6--
Chloroform
solutions of lipid were dried with a stream of N2, and the
dried lipid was suspended in water at a concentration of 1 mg/ml with
vigorous vortexing. The solution was sonicated in a 20 °C water bath
for 15 min to give phospholipid vesicles, and buffer was added to give
a stock concentration of 0.5 mg/ml phospholipid, 0.5 M
Tris-HCl, pH 7.5, 0.5 M NaCl. Phospholipid vesicles (100 µg/ml final concentration) were mixed with or without His6-moesin (0.5 µg) and/or expressed
PKC-
-His6 in a buffer containing 3 mM
Mn2+, 20 µM ATP, and 10 µg/ml leupeptin,
100 mM Tris-HCl, pH 7.5, and 100 mM NaCl in a
0.5-ml microcentrifuge tube. The samples were incubated at 37 °C for
10 min and then for an additional 10 min on ice. The tubes were
centrifuged at 12,000 × g for 15 min at 4 °C to
obtain supernatant and pellet fractions, which were subjected to
immunoblot analysis. Prior to the addition of PKC-
-His6
and His6-moesin to vesicles, the solutions were centrifuged at 12,000 × g for 10 min to remove aggregated
protein.
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RESULTS |
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Properties of the Partially Purified Kinase System-- The partial purification of pp73 from 40 ml of human leukemic cells utilized the same ammonium sulfate precipitation and CM-cellulose chromatography as were used previously for identifying pp47, which is phosphorylated under similar conditions (see "Materials and Methods"). The peak CM-cellulose fractions for pp73 purification were obtained at 0.1-0.15 M NaCl and contained both enzyme and substrate but very little phosphatase. We estimated purification at this step at about 100-fold, based upon a 50% yield estimate, which was inexact due to phosphatases in the starting material.
As shown in Fig. 1, the partially purified system exhibits characteristics similar to our previous observations in whole cell extracts, with lane 3 demonstrating optimal phosphorylation of pp73 with Mn2+ and PG (3). The order of phospholipid preference was PG > PI > PS > no lipid, shown in lanes 3, 2, 5, and 1, respectively. We also wished to determine whether the phosphorylation was due to cyclic AMP-dependent protein kinase (PKA) or to classical PKC. In lane 4, addition of rat brain PKC, under optimal conditions for phosphorylation of pp73, i.e. Mn2+ and PG, resulted in a decrease in pp73 phosphorylation compared with the CM-cellulose fraction alone in lane 3. In lane 7, addition of rat brain PKC in the presence of PS/DAG/Ca2+ resulted in the phosphorylation of a 75-kDa protein, not pp73. In contrast to the lack of phosphorylation of pp73, the talin fragment was heavily phosphorylated in lane 7 compared with lane 6, merely by the addition of rat brain PKC. In lane 9, addition of the catalytic subunit of PKA under optimal PKA conditions gave almost no phosphorylation of pp73, whereas a band of about 55 kDa was heavily phosphorylated. Thus the CM-cellulose fraction exhibited the Mn2+ dependence and PG preference in phosphorylation noted previously, and the phosphorylation was not due to PKA or to classical PKC which is stimulated by PS/DAG/Ca2+.
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Purification and Identification of pp73-- Since two-dimensional gels showed that there were no significant amounts of other proteins at this pI (~6.5, data not shown), a preparative purification was conducted using adherence of proteins to PG vesicles under assay conditions as above. This was followed by isoelectric focusing in a sucrose gradient (12), and fractions containing pp73 were identified by SDS-PAGE and autoradiography. These fractions were digested with trypsin, and the resulting peptides were separated by HPLC. Three peptide sequences were obtained and were found to be identical to the ERM protein moesin (Table I). The peptide sequences are inconsistent with those of the closely related proteins ezrin (17), radixin (18), merlin/schwannomin (19, 20), or talin (21).
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Phosphorylation of Bacterially Expressed Moesin-- To confirm the peptide sequencing identification, we tested recombinant His6-moesin for its ability to be phosphorylated by endogenous kinase in the CM-cellulose fraction from leukemic cells. Fig. 2 shows that the phosphorylation of recombinant moesin is PG-stimulable (lane 4 versus lane 3) and that the amount of phosphorylation is roughly comparable to the phosphorylation of endogenous pp73 on a per weight basis (lane 4 versus lane 2). As a control, there is no kinase activity in the purified recombinant moesin fraction alone (lane 5). The cyanogen bromide fragmentation patterns of pp73 and recombinant moesin are identical upon SDS-PAGE (data not shown).
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In Vitro Phosphorylation of Moesin Is on Threonine 558-- Since ezrin has been reported to be multiply phosphorylated, we determined the extent of our in vitro reaction in two ways. First, the maximum phosphate we were able to incorporate was 0.8 mol of phosphate per mol of moesin, which suggests only one phosphorylation. Second, two-dimensional gel electrophoresis was performed on moesin (22), which showed two spots (Fig. 3, top) labeled 1 and 2. Carbamylation of His6-moesin (23) generated a charge train of spots which established that the distance between 1 and 2 corresponded to 1 charge unit (data not shown). It is probable that spot 2 is the deamidation product of spot 1, since moesin contains an asparagine-glycine sequence, which is most permissive for deamidation at mild pH and temperature (24), and we observed that more of spot 2 was produced by longer storage. Phosphorylation to approximately 20% gave two more spots (Fig. 3, middle) labeled 3 and 4; these spots were radioactive (Fig. 3, bottom). We interpret the data to infer that a single phosphorylation of native moesin, spot 1, produces spot 3 and that a single phosphorylation of deamidated moesin, spot 2, gives spot 4. Moesin phosphorylation did not result in additional spots. A similar pattern of spots using recombinant phospho- and dephospho-moesin was observed upon two-dimensional gel analysis (data not shown).
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Moesin Kinase Activity Is Not a Classical PKC-- Moesin kinase was purified from phosphatases using DEAE chromatography (see below), and its activity upon moesin and the purified 47-kDa talin fragment were tested under conditions optimal for moesin phosphorylation and optimal for most forms of PKC. In Fig. 4, lanes 1 and 3, negligible levels of phosphorylation of both moesin and talin fragment are seen in the absence of added lipid. In lane 2, enzyme added under conditions optimal for moesin kinase activity shows heavy labeling of both moesin and talin fragment. In lane 4, enzyme added under conditions optimal for most forms of PKC leads to heavy labeling of talin fragment and a negligible increase in labeling of moesin. Addition of a purified rat brain PKC preparation under optimal PKC conditions showed the same heavy phosphorylation of talin fragment and negligible phosphorylation of moesin (data not shown). Thus, conventional activation conditions for PKC do not account for the PG-stimulable moesin kinase activity. However, we could not discount the possibility that phosphorylation of moesin might be due to a novel or unusual activation of a PKC isozyme.
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The Phosphorylation Site of Moesin Is Homologous to a PKC
Pseudosubstrate Sequence--
As an aid to identifying potential
protein kinases that phosphorylate moesin, we examined the amino acid
sequence of the phosphorylation site to determine whether it had
sequence similarity with consensus protein kinase recognition sequences
in other proteins (26). The phosphorylation site, KYKT*LRQ (where *
indicates the phosphorylation site), was found to resemble the
pseudosubstrate sequence found in the PKC family of enzymes. The
sequence contains the requisite basic residues surrounding the
phosphorylation site threonine with the phosphorylated residue,
Thr558, at the alanine position in the pseudosubstrate
sequences. As shown in Table II, homology
is maximal with the novel PKC isozymes and
(75%) compared with
the conventional isozymes
,
, and
(50%) and the isozymes
,
, and
(38-50%). Although there is a mismatch between
PKC-
or PKC-
and moesin of the two residues immediately
N-terminal of Thr558, one basic residue within this span is
common to all the sequences. This finding suggested that a novel PKC
isozyme may be the moesin kinase we detected.
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Immunological Identification of Moesin Kinase Activity as
PKC---
Due to the high degree of sequence similarity of the
moesin phosphorylation sequence with PKC-
and
, we analyzed the
CM-cellulose fraction for the presence of PKC isozymes by
immunoblotting. As shown in Fig.
5A, the CM-cellulose fraction
contains three PKC isozymes,
,
, and
. The apparent relative
abundance of the
and
isozymes is consistent with their known
expression in hemopoietic cells. We next examined whether antibodies to
specific PKC isozymes could immunodeplete moesin kinase activity from
the CM fraction. As shown in Fig. 5B, antibodies raised
against the V3 region of PKC-
(7) removed >90% of the activity,
whereas antibodies to the other PKC isozymes did not remove or inhibit moesin kinase activity. IgG specific for the C terminus of PKC-
(Fig. 5B, anti-PKC-
, C-Term) is
less efficient at immunodepletion of kinase but was also
correspondingly less efficient at immunoprecipitation of PKC-
protein as assessed by immunoblotting. In Fig. 5C, analysis of the immunoprecipitate formed with anti-PKC-
(V3-specific) by
immunoblotting with anti-PKC-
and
indicated that only PKC-
is
in the immunoprecipitate and that PKC-
is not. Anti-PKC-
IgG does
not cross-react with PKC-
, the next most abundant PKC isozyme in the
CM fraction (Fig. 5, compare A and C). However, we were unable to demonstrate activity in the immunoprecipitates, possibly due to the inactivation of the enzyme bound to IgG and protein
A/G beads or an inability of the bound enzyme to be optimally activated
with phosphatidylglycerol vesicles. Taken together, these data suggest
that the moesin kinase activity in the CM-cellulose purified cell
extracts is attributable to PKC-
.
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Partial Purification of PKC- from AML Cells--
We next
endeavored to purify the moesin kinase activity from human AML cells
using His6-moesin as a substrate in our standard assay (see
"Materials and Methods"). Immunoreactivity of individual fractions
with anti-PKC-
, as well as anti-PKC-
, was also monitored. A
35-65% ammonium sulfate fraction of the cell lysate was
chromatographed over a CM-cellulose column, and aliquots of each
fraction were assayed using PG vesicles (Fig.
6A, upper panel), and in
addition PS/DAG without Ca2+ to assay for novel PKCs (Fig.
6A, middle panel). PG-stimulable moesin kinase activity
elutes from the column at about 0.09-0.18 M NaCl; activity
in the presence of PS/DAG/EGTA is about 10-fold lower than with PG.
Immunoblot analysis of the CM-cellulose fractions indicates that the
fractions displaying moesin kinase activity contain PKC-
(Fig.
6A, lower panel), as well as PKC-
(data not shown). No
PKC-
immunoreactivity was found in side fractions. A pool of the
active CM-cellulose fractions eluting at 0.09-0.18 M NaCl
was fractionated on a DEAE 8HR column. As shown in Fig. 6B,
two pools of moesin kinase activity using PG vesicles are found. A
major peak of activity eluting at a lower [NaCl] in fractions 4-6
contain PKC-
, whereas fractions 8-12, which display lower PG-stimulable moesin kinase activity, contain PKC-
. The peak of
moesin kinase activity and PKC-
immunoreactivity is found in
fraction 5, which has no detectable PKC-
. This chromatographic step
effectively separates PKC-
and PKC-
and enriches the moesin kinase activity in fractions containing PKC-
. Fraction 5 was used
for heparin-Sepharose chromatography because it contained maximal
moesin kinase activity and PKC-
. The moesin kinase activity again
co-fractionates with intact PKC-
as detected by immunoblotting (Fig.
6C). Furthermore, the activity obtained cannot be ascribed to the catalytic fragment of PKC-
in the preparation because no
immunoreactive fragment was detected with a PKC-
antibody directed
to the C terminus. The peak of moesin kinase activity and PKC-
immunoreactivity also coeluted from a Superdex-200 FPLC column at 85 kDa, which is consistent with the molecular mass of PKC-
(data not
shown). Therefore, the purification used here results in the enrichment
of moesin kinase activity corresponding to intact PKC-
and removes
the immunoreactive fragment present at the CM-cellulose step (Fig.
5A). However, attempts to purify the moesin kinase activity
further resulted in a drastic loss of phosphorylating activity, which
precluded direct sequencing of purified kinase. A similar loss of
activity with purification has been observed for some PKC isozymes, for
example PKC-
purified from K562 hematopoietic cells (27) and PKC-
from murine brain (28). All further experiments using PKC-
from AML
cells were done with a kinase preparation containing the full-length
enzyme as determined by SDS-PAGE/immunoblotting and Superdex-200
chromatography.
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Preferential Activation of Moesin Kinase (PKC-) by PG
Vesicles--
We next examined the activation of moesin kinase by PG
and PS in combination with DAG. Previous studies using the [A25S]PKC substrate peptide, which corresponds to the pseudosubstrate sequence of
PKC-
with an alanine to serine substitution, showed that PS at
optimal concentrations gave near maximal activation of PKC-
in the
absence of either phorbol 12-myristate 13-acetate or DAG (Ref. 29, Fig.
5). We examined the activation of moesin kinase with PG and PS vesicles
with added DAG constituting 10% (30) of the total lipid concentration
of either 20 and 200 µg/ml. As shown in Fig.
7, addition of DAG to PG vesicles did not
lead to increased moesin phosphorylation. By using PS and PS/DAG
vesicles, less moesin phosphorylation was obtained compared with PG
vesicles. Addition of 10% DAG to PS vesicles led to a minimal increase
in moesin phosphorylation (Fig. 7, compare 100% PS and 90% PS + 10% DAG) and is more evident using 20 µg/ml total lipid. In addition, we
investigated activation of PKC-
using PC vesicles containing 20%
acidic lipids (PG, PS, or PI), a typical concentration of the inner
leaflet of the plasma membrane (31). PC vesicles alone did not activate
PKC-
. PG, in mixed vesicles with PC, maximally activated PKC-
,
and DAG again had no effect (data not shown).
|
Moesin Kinase (PKC-) Activity toward Different
Substrates--
We next tested whether the moesin kinase activity has
particular substrate and cofactor requirements. By using moesin, MBP, and histone H1, the kinase activity toward each was tested in the
presence of PG, PI, or PS/DAG. As shown in Fig.
8, maximal kinase activity was obtained
using PG and His6-moesin, whereas the kinase activity
toward MBP was lipid-independent, and histone H1 phosphorylation was
minimal. These results are consistent with previous findings of PKC-
using an MBP-derived peptide and histone H1 (29).
|
Recombinant His6-PKC- Phosphorylates Moesin--
To
verify that PKC-
phosphorylates moesin, we transfected eukaryotic
cells with a plasmid bearing PKC-
with a His6 tag at its
C terminus (16, 29). Jurkat (T-lymphoblastic leukemia) cells were
transfected with the His6-PKC-
plasmid and with the neo
vector alone. Cell lysates from each transfection were purified by
Ni2+ affinity chromatography, and aliquots of the eluates
were analyzed by SDS-PAGE. Immunoblotting with anti-His6
probe antibody indicated that cells transfected with pEFPKC-
, but
not pEFneo, contained a single immunoreactive band at 80 kDa (data not
shown). Furthermore, immunoblotting with anti-PKC-
revealed that
only the pEF-PKC-
-transfected cells express recombinant enzyme that
is purified on the Ni2+ affinity beads and that no
endogenous Jurkat cell PKC-
is purified on the beads (Fig.
9A). We next used the purified
His6-PKC-
in the moesin kinase assay to determine
whether the recombinant enzyme could phosphorylate moesin. As shown in
Fig. 9B, recombinant His6-PKC-
phosphorylated
moesin in the presence of PG or PI vesicles, lanes 2 and
4, whereas no phosphorylation was detected using
Ni2+ affinity purified extracts from neo-transfected cells,
lanes 1 and 3.
|
Moesin and PKC- Bind to PG Vesicles--
Next, we investigated
the association of PKC-
with PG vesicles and whether moesin is
required for the binding. PG vesicles were incubated with
Ni2+ affinity purified PKC-
-His6 and
His6-moesin, and vesicles were pelleted by centrifugation.
The vesicle-bound and unbound PKC-
was identified by immunoblotting.
As shown in Fig. 10, PKC-
was found
in the pellet fractions of reactions containing PG vesicles, and
binding was independent of His6-moesin. Furthermore, about 50% of the His6-moesin was pelleted in association with PG
vesicles.
|
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DISCUSSION |
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In this report we present reaction conditions under which PKC-
threonine phosphorylates moesin at its previously identified in
vivo site within its actin-binding domain (1). PKC-
is a
recently discovered novel PKC isozyme, which has not previously been
shown to phosphorylate moesin or to require the unusual conditions defined here nor has it been linked to any other substrate of likely
physiological significance. Although phosphorylation of ERM proteins
has been previously described, the work we present is the first
evidence linking proteins of this class to a specific kinase and
suggests a novel and specific signaling pathway regulating membrane-cytoskeleton linkages.
The reaction conditions described here result in greatly increased
phosphorylation of the prominent 73-kDa protein substrate we originally
described in hemopoietic cells (3-5), which we here identify as moesin
on the basis of protein purification followed by microsequencing and
confirm by phosphorylation of recombinant moesin with partially
purified kinase. Several further observations corroborate this
identification. First, both pp73 and PKC- bind to PG vesicles, and
moesin is known to bind to the plasma membrane (32, 33). Second, in
Fig. 2 we observed four spots of pp73 by two-dimensional gel analysis
with pI ranging from 6.6 to 6.45, which is similar to the four spots of
moesin reported in a comprehensive protein data base (34). Our results
indicate that the latter distribution results from deamidation and
mono-phosphorylation.
By using recombinant moesin as a substrate for in vitro
kinase assays, we have partially purified and identified PKC- as a
protein kinase that phosphorylates moesin at Thr558 in the
actin-binding domain, and we further conclude that it corresponds to
the protein kinase previously described (3). This identification is
based upon immunodepletion experiments and the demonstration of moesin
phosphorylation by recombinant PKC-
. The phosphorylation site of
moesin, KYKTLRQ, containing Thr558, is phosphorylated by a
partially purified PKC-
preparation and is highly homologous with
the pseudosubstrate sequence of PKC-
. The lipid dependence of
PKC-
phosphorylation of moesin displays a preference for PG over PS
vesicles, with minimal contribution to activation by DAG. Furthermore,
both moesin and PKC-
associate with PG phospholipid vesicles, and
PKC-
displays a strict substrate preference for moesin, rather than
MBP or histone H1. PKC-
is thus the major moesin kinase directly
identifiable in the cell extracts we studied. The single
phosphorylation site identified corresponds exactly with that which is
now well established to occur in cells (1, 33). We are currently
extending this work to test further our putative PKC-
/moesin link in
cellular systems and to analyze its control and effects. We cannot,
however, rule out the participation of other moesin kinases in other
cell types or tissues or under other activating conditions. This is
especially a consideration since ERM proteins are ubiquitous, whereas
PKC-
is not. PKC-
can phosphorylate moesin, albeit at an
apparently lower rate, which may be adequate to elicit a physiologic
response.
ERM proteins are involved in a wide variety of cellular functions,
including cell-cell adhesion, cell-substrate adhesion, microvillar
structure, and cytokinesis. Moesin was first isolated by its ability to
bind heparin (36); subsequent cDNA cloning and sequence analysis
showed it to be a 68-kDa protein related to a family of proteins
involved in linking the membrane of a cell to its cytoskeleton (32).
The ERM proteins identified to date have a common domain structure
based upon amino acid sequence homology to the erythrocyte band 4.1 protein. They are comprised of an N-terminal domain which is involved
in membrane binding, an -helical central domain, and a C-terminal
actin-binding domain (37). In vitro work has shown that
native ERM proteins bind to beads displaying the cytoplasmic tail of
the transmembrane protein CD44 at physiological ionic strength in the
presence of phosphatidylinositol-4,5-diphosphate (38). Ezrin has been
shown to bind filamentous
-actin, not
-actin, at physiological
ionic strength (39), whereas such an interaction between moesin and F-actin has not yet been demonstrated. The ezrin/
-actin interaction correlates with the fact that
-actin and ezrin are colocalized to
diverse cellular extensions, whereas
-actins are largely restricted to sarcomeres and stress fibers (40, 41).
Does moesin phosphorylation affect actin binding? Moesin was originally immunolocalized to structurally and functionally diverse cellular extensions, and it was speculated that these "rapidly assembled cell surface structures may be akin to environmental sensors in a very general sense" and that phosphorylation of moesin might be related to these morphological changes (33). The Thr558 site in moesin was phosphorylated in platelets upon thrombin stimulation, which drastically alters platelet shape. Using a phosphorylation state-specific antibody to KYKpT558LR, only a fraction of total moesin molecules found in filopodia and retraction fibers are phosphorylated in RAW macrophages (35). In the presence of the phosphatase inhibitor calyculin A, phosphorylated moesin is increased to nearly 100% and is redistributed from filopodia and retraction fibers into an F-actin containing ring-like structure in the cytoplasm. Thus, this phosphorylation step accompanies actin-based morphological changes (1). We are currently investigating possible interactions of moesin and phosphorylated moesin with various actin isoforms.
ERM proteins have now been shown to interact with several molecules and
signaling pathways in addition to those described here. Tsukita and
co-workers (38) found that the affinity of the moesin and ezrin
interaction with the cytoplasmic tail of CD44 was markedly enhanced by
phosphoinositides. In A431 cells, the redistribution of ezrin into
membrane ruffles after epidermal growth factor stimulation is
concomitant with both serine and tyrosine phosphorylation (42). Studies
using gastric mucosal cells indicate that ezrin associates with the
regulatory (RII) subunit of PKA, suggesting that ezrin
functions as an protein kinase A anchoring protein (AKAP) (43). The
RII-binding site in ezrin, corresponding to residues
417-432, is highly conserved in moesin and corresponds to the
RII-binding site in the C terminus of AKAP79 (44).
Additionally, AKAP79 contains a binding sequence for PKC in the N
terminus, and a peptide corresponding to this sequence inhibits PKC
activity. A similar PKC-binding site has not been identified in either
ezrin or moesin, but several sites in ezrin/moesin have significant
homology with this sequence in AKAP79. We demonstrate that moesin is
not phosphorylated by the catalytic subunit of PKA (Fig. 1) but is
phosphorylated at the KYKTYLRQ site in the actin-binding domain by
PKC-. ERM proteins may participate in a dynamic, macromolecular
signaling complex, analogous to the model proposed by Faux and Scott
(45) in which AKAP79 anchors PKC, PKA, and phosphatase 2B at neuronal
synapses. ERM proteins may similarly function as anchoring proteins
linking the actin cytoskeleton with phospholipid,
phosphatidylinositol-4,5-diphosphate, CD44, PKA, and PKC. Our data
demonstrating that moesin and PKC-
bind to phospholipid vesicles and
that the phosphorylation site sequence in moesin is homologous to the
pseudosubstrate sequence of PKC-
support this hypothesis.
Furthermore, preliminary immunofluorescence studies show significant
colocalization of PKC-
and moesin in punctate structures within
protrusions at the cell periphery in RAW macrophages and HL-60 cells
(data not shown). This pattern appears similar to that recently
described for PKC-
in resting T-cells (46). Thus, PKC-
may
regulate the interaction of ERM proteins with the actin cytoskeleton
and may also interact with other components of such a complex.
The PKC family of serine/threonine kinases consists of multiple
isozymes that are grouped into three categories based on their cofactor
and lipid dependence for activation. The conventional PKCs (,
I,
II, and
) are activated by
Ca2+, phospholipid, and DAG; novel PKCs (
,
,
, and
) are insensitive to Ca2+, and the atypical PKCs (
and
/
) are insensitive to both Ca2+ and DAG (47).
Although some isozymes have widespread tissue and cell type
distribution (
,
, and
), other isozymes such as PKC-
, -
,
and -
are more restricted. PKC-
is the main novel isozyme
expressed in skeletal muscle, testes, platelets, and their megakaryoblastic precursor cells, T-lymphocytes, and neoplastic hemopoietic cells (7, 48, 49). We have found moesin phosphorylation activity in HL-60 cells, spleen cells, and AML cells, which is consistent with the known distribution of PKC-
(29) and in contrast
to the tissue distribution of the µ (50) and the
/
isoforms,
which are not found in spleen (51, 52). We have previously described
modulation of this activity under various conditions affecting cellular
proliferation and differentiation (4, 5). It will be of interest to
determine if PKC-
immunoreactivity is modulated in parallel as
expected. If so, this previous work could be the basis for physiologic
models of PKC-
activation and function.
The unusual lipid activation pattern we have observed in
vitro in the presence of Mn2+ (PG > PI > PS/DAG) is reminiscent of the unusual lipid activation conditions
described for other novel PKCs. Our data demonstrating a minimal
contribution of DAG to the PS activation of PKC- phosphorylation of
moesin is consistent with the findings of Baier et al. (29), who showed a similar minimal effect of phorbol 12-myristate 13-acetate to the PS activation of PKC-
. A comparable pattern has been observed with PKC-
phosphorylation of the
chain of the IgE receptor, where the rate of threonine phosphorylation was increased 2.5-fold using PI compared with PS, whereas the effect of PG was not tested (53). PKC-
displays PG-stimulable activity toward a PKC-
synthetic peptide resembling the PKC-
pseudosubstrate site, with a
serine residue at the alanine position (54). PKC-
was also found to phosphorylate histones poorly, similar to the present findings with
PKC-
.
The activation of classical and novel PKCs by PS and DAG is thought to
reflect an allosteric enzymic effect: DAG generation causes increased
PKC membrane affinity and accompanies release of the pseudosubstrate
sequence from the active site. However, we suspect the mechanism of
PKC- activation and moesin phosphorylation to be more complex. Our
findings indicate first that both moesin and PKC-
bind to PG
vesicles independently of one another; the phospholipid activation
observed may be based upon closely co-concentrating the two components
on a suitable surface. Phospholipid binding could also have a role in
exposing the moesin phosphorylation site which is otherwise masked by
N- and C-terminal domain interactions (58). Moesin phosphorylation is
activated by acidic phospholipids (PG, PS, and PI) but not neutral
lipids, such as PC or DAG. A similar pattern of binding to acidic
phospholipids with subsequent phosphorylation has been described for
the PKC substrates neuromodulin and the myristoylated alanine-rich
protein kinase C substrate protein (55, 56). Although vesicles
containing 20-100% PG support apparently maximal phosphorylation
in vitro, further research is necessary to determine what
in vivo conditions give rise to basal, stimulated, and
possibly inhibited rates. It is likely that membrane binding of moesin
and other ERM proteins within cells is under more complex control
involving other molecules, such as CD44 and
phosphatidylinositol-4,5-diphosphate (36). Other
phosphoinositides such as
phosphatidylinositol-3,4-P2 and phosphatidylinositol-3,4,5-P3, which are known to
activate PKC-
, -
, and -
, may also activate PKC-
and moesin
phosphorylation (57). It is equally probable that other natural
activators of PKC-
phosphorylation of moesin and other ERM proteins
remain to be elucidated.
The in vivo function of PKC- has mainly been studied in
cells of the hemopoietic system. The chromosomal location of the human
PKC-
gene was mapped to the short arm of chromosome 10 (10p15), a
region that is frequently deleted or subject to translocations in
T-cell leukemia, lymphoma, and T-cell immunodeficiency (59). In
T-lymphocytes, PKC-
in conjunction with 14-3-3 may be a constituent of the signaling cascade leading to T-cell activation by activating the
AP-1 transcriptional complex (60). Recent evidence clearly shows that
antigen stimulation results in the translocation of PKC-
, along with
talin, to the site of contact between T-cells and antigen-presenting
cells, whereas in unstimulated cells PKC-
is found in a punctate
distribution (46). The HIV protein Nef, whose association with PKC-
results in a loss of enzyme, is known to down-regulate CD4 in infected
T-cells and blocks interleukin-2 production in Jurkat cells, possibly
by inhibiting NF
B and AP-1 activation (61). The identification of
moesin, ezrin, actin and cofilin inside HIV-1 virions provides evidence
for a role of the cytoskeleton in virus budding from microvillar-like
pseudopods of infected T-cells (62). Taken together, these findings
raise the intriguing possibility that the pathway described here may play a role in virus production from infected T-cells as well as normal
T-cell functions.
In summary, this laboratory first characterized the surprisingly
specific phosphorylation of two proteins in crude extracts under
somewhat unusual conditions: in the presence of Mn+2, the
addition of PG or PI vesicles resulted in 10-fold stimulation of
phosphorylation (3). We previously identified one of these substrates
as the 47-kDa calpain fragment of talin (6), and we here identify the
second substrate as moesin. These two proteins are structurally related
and are proteins linking the plasma membrane to the actin cytoskeleton.
We provide further evidence that the moesin kinase activity corresponds
to PKC-, using both purified kinase from human leukemic cells and
recombinant PKC-
. The assay requirement for vesicles correlates with
their cellular localization on the cytoplasmic surface of the plasma
membrane. The in vitro phosphorylation site of moesin
Thr558 corresponds to that occurring in activated platelet
membranes and in macrophages (1, 35). We are currently investigating such interactions of moesin and PKC-
within cells to address the
physiologic significance of this system.
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ACKNOWLEDGEMENTS |
---|
We thank Heinz Furthmayr (Stanford University, Palo Alto, CA) for providing the human moesin cDNA clone and Anthony Bretscher (Cornell University, Ithaca, NY) for providing human placental ezrin. Oligonucleotides used in this study were synthesized by the Protein Chemistry Core Facility at the University of New Mexico School of Medicine. We thank Ken Williams and Kathy Stone at the W. M. Keck Biotechnology Resource Laboratory at Yale University (New Haven, CT) for the amino acid sequencing and mass spectroscopic analyses.
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FOOTNOTES |
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* This work was supported by Grant CB-158 from the American Cancer Society (to L. E.) and National Institutes of Health Grants CA42520 (to L. E.) and CA35299 (to A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: University of New Mexico Cancer Center, 900 Camino de Salud NE, Albuquerque, NM 87131. Tel.: 505-272-5837; Fax: 505-272-2841; E-mail: lelias{at}cobra.unm.edu.
1 The abbreviations used are: ERM, ezrin-radixin-moesin; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; PC, phosphatidylcholine; AML, acute myelogenous leukemia; CM, carboxymethyl; MBP, myelin basic protein; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)- propane-1,3-diol; HPLC, high pressure liquid chromatography; DAG, diacylglycerol; AKAP, protein kinase A anchoring protein; HIV, human immunodeficiency virus.
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REFERENCES |
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