Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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Abstract |
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Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to
the fast-growing ends of actin filaments. The basic
MARCKS-related domain, present in ,
, and
adducin subunits, binds calmodulin and contains the major
phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation
of the MARCKS-related domain modifies in vitro and
in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in
vivo substrate for PKC or other phorbol 12-myristate
13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping
measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine,
which is the major PKC phosphorylation site in the
MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in
human embryonal kidney (HEK 293) cells, threefold in
MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo
phosphorylation site for PKC or other PMA-activated
kinases but not for cAMP-dependent protein kinase in
a variety of cell types. Physiological consequences of
the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting
MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant
adducin. The mutant
adducin was no longer concentrated at the cell membrane at sites of cell-cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover,
the cells expressing the mutant
adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant
adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of
adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were
detected in the dendritic spines of cultured hippocampal
neurons. Spectrin also was a component of dendritic
spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin
is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are
believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.
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Introduction |
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ADDUCIN is a ubiquitously expressed calmodulin-binding protein (Gardner and Bennett, 1986; Bennett
et al., 1988
) and substrate for protein kinase C
(PKC)1 (Palfrey and Waseem, 1985
; Cohen and Foley,
1986
; Ling et al., 1986
; Kaiser et al., 1989
). Adducin was
first purified from human erythrocyte membrane skeletons
(Gardner and Bennett, 1986
) and from brain membranes
(Bennett et al., 1988
). Adducin is localized at spectrin-actin
junctions in erythrocyte membrane skeletons (Derick et
al. 1992
) and colocalizes with spectrin at sites of cell-cell
contact in epithelial cells (Kaiser et al., 1989
; Hu et al.,
1995
) and in dendritic spines of hippocampal neurons
(Seidel et al., 1995
). Adducin exhibits in vitro activities of
promoting association of spectrin with actin (Gardner and
Bennett, 1987
; Bennett et al., 1988
), association with sides
of actin filaments (Mische et al., 1987
; Taylor and Taylor,
1994
), and capping the fast-growing ends of actin filaments
(Kuhlman et al., 1996
). Recently, adducin has been demonstrated to preferentially cap and recruit spectrin to the
fast-growing ends of actin filaments (Li et al., 1998
).
Adducin is a heteromeric protein comprised of either and
or
and
subunits (Gardner and Bennett, 1986
;
Bennett et al., 1988
; Dong et al., 1995
; Hughes and Bennett, 1995
).
adducin is expressed in most tissues, while
adducin has a more restricted pattern of expression (Joshi
et al., 1991
).
adducin, which is similar in sequence to
and
adducin, is a likely companion for
adducin in cells
lacking the
subunit (Dong et al., 1995
). Adducin subunits are closely related in amino acid sequence and domain organization (Joshi and Bennett, 1990
; Joshi et al.,
1991
; Dong et al., 1995
). Each adducin subunit has three
distinct domains: a 39-kD NH2-terminal globular protease-resistant head domain, connected by a 9-kD "neck" domain to a COOH-terminal protease-sensitive tail domain
(Joshi and Bennett, 1990
; Joshi et al., 1991
; Dong et al.,
1995
). Adducin isolated from erythrocytes is a mixture of
heterodimers and tetramers with NH3-terminal head domains in contact to form a globular core and with interacting tails extended away from the core (Hughes and Bennett, 1995
).
COOH termini of all three subunits of adducin contain a
highly basic stretch of 22 amino acids with sequence similarity to a domain in the myristoylated alanine-rich C kinase substrate (MARCKS protein) (Joshi et al., 1991;
Dong et al., 1995
). The MARCKS-related domain is required for interactions of adducin with actin and spectrin,
which is consistent with the possibility that the MARCKS-related domain mediates contact with actin (Li et al.,
1998
). The MARCKS-related domain also has a major
phosphorylation site, the RTPS-serine, for PKC as well as
cAMP-dependent protein kinase (PKA), and contains the
primary calmodulin-binding site of adducin (Matsuoka
et al., 1996
). Ca2+/calmodulin inhibits actin-capping and
spectrin recruitment activities of adducin (Gardner and
Bennett, 1987
; Kuhlman et al., 1996
). Although phosphorylation of the MARCKS-related domain by PKC inhibits calmodulin binding (Matsuoka et al., 1996
), effects of
phosphorylation on the other activities of adducin have
not been resolved.
The MARCKS family of proteins has been studied extensively as in vivo substrates of PKC (Aderem, 1992;
Blackshear, 1993
). A 25-amino acid basic domain is the
site for both PKC phosphorylation and calmodulin binding of the MARCKS protein and is similar in sequence to
the MARCKS-related domain of adducin (Aderem, 1992
; Blackshear, 1993
). The MARCKS protein binds and cross-links actin in in vitro assays, and its capacity to cross-link
actin is regulated by PKC phosphorylation and calmodulin-binding to the phosphorylation site domain (Hartwig
et al., 1992
). A COOH-terminal (C1) region in the NR1
subunit of N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor of neuron, has sequence similarity to
the PKC phosphorylation/calmodulin-binding domain of
the MARCKS protein (Tingley et al., 1993
; Ehlers et al.,
1996
). The C1 region also contains PKC phosphorylation
sites and is a high-affinity binding site for calmodulin (Tingley et al., 1993
; Ehlers et al., 1996
). Moreover, phosphorylation and calmodulin-binding to the C1 region may regulate interaction of the NR1 subunit of NMDA receptor
with the actin cytoskeleton (Ehlers et al., 1995
, 1996
). The
PKC phosphorylation site domain of the MARCKS protein is also involved in its cell membrane binding through
the interaction between a cluster of basic residues and
acidic phospholipids, such as phosphatidylserine (Kim et al.,
1994
; McLaughlin and Aderem, 1995
). Phosphorylation of
the serine residues in the poly-basic domain reduces its
electrostatic interaction with the phospholipids and has
been proposed to provide an electrostatic switch mechanism for the reversible binding of the MARCKS protein to
the cell membrane.
This report presents evidence that adducin is an in vivo
substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases, and that activities of adducin in capping and recruiting spectrin to actin filaments are
regulated by PKC phosphorylation of the MARCKS-
related domain. Mutation of the two PKC phosphorylation sites in the MARCKS-related domain of adducin
produced striking effects on distribution of the mutant adducin as well as of spectrin in MDCK cells. Adducin phosphorylated at the major PKC site in the MARCKS-related
domain was specifically localized in dendritic spines of cultured neurons. Adducin is a candidate molecule to mediate downstream consequences of PKC activation in postsynaptic sites in neurons as well as other dynamic cellular
domains.
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Materials and Methods |
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Proteins
Actin was purified from acetone powder of rabbit skeletal muscle (Pardee
and Spudich, 1982) with a modification (Li and Bennett, 1996
). Brain
spectrin was isolated by high salt extraction from bovine brain membranes
(Davis and Bennett, 1983
; Li and Bennett, 1996
). Erythrocyte adducin and
a recombinant
adducin (
335-726) were purified as previously described
(Hughes and Bennett, 1995
; Li et al., 1998
). The catalytic domain of PKC
was purchased from Calbiochem (San Diego, CA).
Antibodies
Phosphoadducin-specific rabbit polyclonal antibody was raised against a
synthetic phosphopeptide (FRTPphosphoSFLKK) (Andrews et al., 1991)
corresponding to the major PKC phosphorylation site of adducin (Matsuoka et al., 1996
) and affinity-purified as described (Matsuoka et al., 1992
).
This antibody was preincubated with the unphosphorylated form of the
peptide and used in this study. Polyclonal antibodies against
adducin and
the MARCKS-related domain were generated using a recombinant human
adducin (residues 536-737) and a synthetic peptide corresponding to the
residues 696-726 of human
adducin, respectively, as antigens. Affinity-
purified rabbit polyclonal antibody against brain spectrin was reported previously (Davis and Bennett, 1983
). An anti-spectrin
G monoclonal antibody
was raised against a synthetic peptide corresponding to the residues 2096-
2122 of human
spectrin. Monoclonal anti-hemagglutinin (HA) epitope antibody (HA.11) was purchased from Berkeley Antibody Co. (Richmond,
CA), monoclonal antisynaptophysin antibody was from Boehringer Mannheim (Indianapolis, IN), anti-GluR2/4 antibody (clone 3A11) was from
PharMingen (San Diego, CA), and FITC- and TRITC-conjugated goat
secondary antibodies were from Pierce Chemical Co. (Rockford, IL).
Phosphorylation of Adducin and Protease Digestion
Erythrocyte adducin (2.5 µM monomer) and 335-726 (5 µM monomer)
were phosphorylated by incubation with 0.4 µg/ml catalytic domain of
PKC (PKM), 0.1 mM ATP, 5 mM MgCl2, 2 mM sodium EGTA, 25 mM
Tris-HCl, pH 7.5 at 15°C for 12-14 h. The reaction was terminated by adding 50 nM (final) bisindolylmaleimide (Calbiochem). ATP was added to
the unphosphorylated adducin sample after quenching the reaction. To
measure stoichiometry of the phosphorylation, adducin was phosphorylated in parallel using 0.1 mM [
-32P]ATP. The sample was processed as
described previously (Matsuoka et al., 1996
). In some experiments,
[32P]phosphoadducin (50 µg) was digested with Staphylococcus aureus V8
protease (1:50 wt/wt; Pierce Chemical Co.) for 3 h at 30°C as described
previously (Matsuoka et al., 1996
). After quenching the digestion by adding 2 mM PMSF, the sample was applied to an S-Sepharose column (0.5 ml bed volume; Pharmacia Biotech, Piscataway, NJ) equilibrated with 20 mM Tris-HCl, 2 mM sodium EGTA, 1 mM DTT, 1 mM PMSF, pH 8.0 (column buffer). Fractions (0.2 ml) were collected by eluting the column
with a step gradient of NaBr (0.1, 0.2, 0.3, and 0.5 M in the column buffer)
after wash with the column buffer and subjected to scintillation counting
and SDS-PAGE (3.5-17%).
Actin Polymerization and Depolymerization Assays
Pyrene-labeled actin was prepared according to the method modified by
Weber et al. (1987). G buffer (5 mM Tris-HCl, 0.2 mM Na2ATP, 0.5 mM
2-mercaptoethanol, 0.2 mM CaCl2, 0.005% sodium azide, pH 8.0, at 25°C)
was used in all assays. The assay used to quantitate inhibition of actin polymerization at the barbed ends used the method of Pollard (1983)
, in
which rapid polymerization was initiated using F-actin nuclei.
Spectrin Recruitment Assay
A full description of this assay system is presented in Li et al. (1998).
Immunoblotting
Hippocampal slices (220 µm) were prepared from 5-6-wk-old Sprague-Dawley rats according to Garver et al. (1995). Slices were immediately immersed in an ice-cold preincubation buffer (Garver et al., 1995
) and incubated with 40 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris
Cookson, Ballwin, MO) in the buffer at 37°C for 30 min. Buffer was replaced with the fresh one without CNQX before further manipulations.
Human erythrocytes were separated and resuspended in the modified
Hepes-Tyrode buffer (Matsuoka et al., 1994
) at a final concentration of
20%. Human embryonal kidney cell line (HEK 293; American Type Culture Collection, Rockville, MD) was maintained in 5% FCS/DME and serum-starved overnight before the following treatments. Human erythrocytes (1 ml suspension), HEK 293 cells (
6-cm dish), and rat hippocampal
slices (5-10 slices) were treated either with 50 µM 3-isobutyl-1-methylxanthine (IBMX; Calbiochem)/50 µM forskolin (Calbiochem) or 1 µM PMA
(Calbiochem) at 37°C for 15 min. After the treatments, erythrocytes were
lysed, and the membrane fractions were collected. Erythrocyte membranes, HEK 293 cells, and rat hippocampal slices were solubilized in a
minimum volume of 50 mM Tris-HCl, pH 7.5, 7 M urea, 1% SDS, 5 mM
sodium EDTA, 1 mM DTT, 10 µg/ml leupeptin, 1 mM PMSF with brief
sonication. Proteins (10 µg) were separated by SDS gel electrophoresis with buffers of Fairbanks et al. (1971)
on 5% gels in 0.2% SDS. The
amounts of phosphoadducin and
adducin were determined from scans
of the autoradiographs using a densitometer as described elsewhere (Matsuoka et al., 1994
). The amount of phosphoadducin in each lane was normalized by the amount of
adducin loaded and was used to calculate the
change of phosphoadducin level after treatment.
Construct Generation and Transfection of MDCK Cells
An HA epitope-tagged wild-type adducin cDNA in pGEMEX vector
was used as a template to mutate the PKC phosphorylation sites in
MARCKS-related domain (Ser716 and Ser726) to alanine residues. Sense
(5'-CCGGCCTTTCTTAAGAAGAGCAAGAAGAAGAGTGACTCC-3') and antisense (5'-GGTACGAAACTTCTTCTTCTTTTTGGCTGGGGACTTGC-3') primers were used to mutate the sites with the Exsite
PCR-based mutagenesis kit (Stratagene, La Jolla, CA). Mutation of the
construct was confirmed by DNA sequencing. Both HA epitope-tagged
wild-type and PKC sites-mutated
-adducin cDNA were subcloned into
pCMV vector (Garver et al., 1997
) at NheI-XhoI sites. Transfection of
MDCK cells was performed using lipofectamine (GIBCO-BRL, Gaithersburg, MD) as described elsewhere (Garver et al., 1997
).
Rat Hippocampal Cell Culture
Isolation of hippocampal formations from newborn (P2) Sprague-Dawley
rats and the dissociation of neurons was performed as described (Brewer
et al., 1993) with a modification. Briefly, hippocampi from newborn rats
were first incubated with 0.027% trypsin in HBSS without Ca2+ and Mg2+,
0.035% sodium bicarbonate, 1 mM pyruvate, 10 mM Hepes, pH 7.4 (HBSS), in a 95% O2, 5% CO2 incubator for 20 min at 35°C. Hippocampi were washed with HBSS and triturated through a 1-ml plastic pipette tip
in HBSS. 1 ml of cell suspension was diluted with 2 ml HBSS with Ca2+,
Mg2+. The cells were collected by centrifugation for 1 min at 200 g, resuspended in Neurobasal medium (GIBCO-BRL) containing 0.5 mM
glutamine, 25 µM glutamate, and 1% B-27 supplement (GIBCO-BRL), and plated at a concentration of 160 cells/mm2. Half of the culture medium was replaced by the same medium without glutamate every 4 d. Primary culture of hippocampal neuron was maintained for at least 7 d before the following treatment. Neurons were incubated with fresh
Neurobasal medium (no glutamate) containing 15 mM MgCl2 without
B-27 supplement for 90 min, and with 40 µM CNQX for a further 30 min
before activation. The medium was changed to the new one (0.8 mM
MgCl2), and either 10 µM glutamate or 1 µM PMA was applied to the
neurons at 37°C for 6 min.
Immunofluorescence
P15 rat brain was fixed and processed as described elsewhere (Lambert
and Bennett, 1993). Cells were fixed with 4% formaldehyde (freshly made
from paraformaldehyde) in 0.1 M phosphate buffer, pH 7.4, containing
0.32 M sucrose for 10 min at room temperature. After fixation, cells were
permeabilized and blocked with 0.05% Triton X-100 (1% for MDCK cells)
in a blocking buffer (PBS with 10% goat serum, 1% BSA, and 5% sucrose)
for 15 min at room temperature. Cells were then incubated with primary antibodies diluted in the blocking buffer followed by FITC- or TRITC-conjugated goat secondary antibodies (1.5 µg/ml; Pierce Chemical Co.).
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Results |
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PKC Phosphorylation of the MARCKS-related Domain of Adducin Modulates Activities of Actin Capping and Promoting Spectrin-Actin Complexes
In a previous study, we did not observe an effect of PKC
phosphorylation on the activity of adducin in promoting
binding of spectrin to F-actin (Matsuoka et al., 1996). In
these experiments, phosphatidylserine and a phorbol ester
were included to activate PKC isolated from rat brain,
which was used at a concentration of 125 nM (Matsuoka
et al., 1996
). It is possible that either PKC itself, a contaminant in the PKC preparation, or phosphatidylserine obscured effect of the phosphorylation in the assay, even
though in some experiments the phosphoadducin was isolated by cation exchange chromatography. In the present
study, PKM, which lacks the regulatory domain and does
not require phosphatidylserine or PMA for its activity, was
used at 15-fold lower concentrations than used previously to
phosphorylate adducin. Kinase activity was quenched with
bisindolylmaleimide, and adducin samples in the phosphorylation mixture were directly used for actin capping and
spectrin recruitment assays in this study. Controls included the kinase and inhibitor, with ATP added after quenching
the phosphorylation reaction.
The MARCKS-related domain of adducin was also the
major phosphorylation site for PKM. 75% of the radioactivity in V8 protease digests of 32P-labeled adducin phosphorylated by PKM (0.9 mol phosphate/mol subunit) associated with fragments migrating at the same position as the
MARCKS-related domain of adducin (Fig. 1 A). In addition, these fragments were highly positively charged,
since the polypeptides tightly bound to a cation-exchange
resin at pH 8 (>0.3 M NaBr was required to elute the fragments) (Fig. 1 A), as did the polybasic MARCKS-related
domain (Matsuoka et al., 1996
). Therefore, the V8 fragments most likely are the MARCKS-related domains of
and
adducin. PKM also phosphorylated the major PKC-site in the MARCKS-related domain based on immunoreactivity with phosphoadducin-specific antibody (see below) (Fig. 3 C). Similar results were obtained using the
neck-tail domain construct of human
adducin (
335-726) (data not shown). Taken together, these results support
the conclusion that the MARCKS-related domain is the
major phosphorylation site of adducin for PKM as well as
for PKC.
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In a recent study, we have demonstrated that recombinant 335-726 adducin has comparable activity with that of
erythrocyte adducin (Li et al., 1998
). Therefore,
335-726
adducin was first used to investigate phosphorylation effects on its actin capping activity. PKM phosphorylation of
the MARCKS-related domain (1.9 mol Pi/mol
335-726)
greatly reduced the capping activity of
335-726 adducin
from 68 to 12% at 0.5 µM of the construct (Fig. 1 B). Similar results were obtained at up to 1 µM of the construct
(data not shown). The ability of PKM phosphorylation to
eliminate the barbed end capping activity of adducin was
also seen in actin depolymerization experiments, as evidenced by the return of the depolymerization rate in the
presence of adducin phosphorylated by PKM to that of pure
actin (Fig. 1 C). PKM phosphorylation of the MARCKS-
related domain (0.9 mol Pi/mol subunit) also greatly reduced the capping activity of native adducin (Fig. 1 D).
The average reduction of capping activity by phosphorylation was 64 ± 14%. Similar results were obtained using
335-726 (data not shown).
Spectrin recruiting activity of adducin was also found to
be a target of regulation through phosphorylation of the
MARCKS-related domain. Phosphorylation of adducin by
PKM almost completely abolished spectrin recruiting activity at up to 600 nM of the phosphorylated adducin (Fig.
2). Since adducin promotes binding of spectrin to the fast-growing ends of actin filaments in addition to filament sides (Li et al., 1998), recruitment of spectrin to both the
ends and sides of actin filaments is inhibited by PKM
phosphorylation. PKM phosphorylation showed a stronger effect on spectrin recruitment activity than actin capping, although the basis for this difference is not known.
This is the first demonstration that actin filament capping
and spectrin recruiting activities of adducin can be regulated by phosphorylation of the MARCKS-related domain. The MARCKS-related domain is thus a site of regulation for actin filament capping and spectrin recruiting
activities of adducin as well as the primary functional domain for these activities.
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Phosphorylation of the MARCKS-related Domain of Adducin Occurs In Vivo
Antibodies specific to phosphorylated sites of a substrate
are particularly useful tools to evaluate the extent of in
vivo phosphorylation and to determine the cellular localization of phosphorylated proteins (Nishizawa et al., 1991;
Dent and Meiri, 1992
; Matsuoka et al., 1992
; Liao et al.,
1995
). We produced an antibody against a peptide, FRTPphosphoSFLKK, containing the phosphorylated form of
the RTPS-serine, which is the major site of phosphorylation by PKC of adducin (Matsuoka et al., 1996
). The
RTPS-serine is also phosphorylated by PKA, although the
Km of PKA for adducin is 10-fold higher than that of PKC
(Matsuoka et al., 1996
).
Immunoblots revealed that the RTPS-phosphoserine-
specific antibody has negligible reactivity towards adducin
in unstimulated human erythrocytes (Fig. 3 A, a), but at
least a 10-fold increase in signal occurred after activation
of PKC by addition of phorbol ester (PMA) (Fig. 3 A, c).
The phosphoadducin antibody was specific for adducin
based on lack of reaction with other proteins in crude homogenates or with recombinant human MARCKS protein
(data not shown). Moreover, the antibody reaction was
displaced completely by a 10-fold molar excess of the
phosphopeptide (data not shown). An eight- to ninefold
increase in reactivity with the phosphoadducin-specific antibody occurred after PMA treatment of human embryonal kidney (HEK 293) cells (Fig. 3 A, d and f), and a twofold increase occurred in rat hippocampal slices (Fig. 3 A,
g and i). In contrast to PMA, forskolin (50 µM), an adenylyl cyclase activator, even in combination with a phosphodiesterase inhibitor (IBMX, 50 µM), resulted in only a
1.4-fold increase of the phosphorylation in HEK 293 cells
(Fig. 3 A, b, e, and h). Thus, the RTPS-serine in the
MARCKS-related domain of each adducin subunit is an in
vivo phosphorylation site for PKC or other PMA-activated kinases but not PKA. A 90-kD adducin polypeptide
detected in HEK 293 cells may correspond to the human
counterpart of rat adducin (Dong et al., 1995
) (Fig. 3 A, f).
Recombinant human adducin (residues 536-737) was
used to produce an anti-
adducin antibody. The affinity-purified antibody specifically recognized
adducin in human erythrocytes (Fig. 3 B, a-c). The amount of
adducin
was used, as a representative of adducin subunits, to normalize the amount of phosphoadducin in each sample.
and
adducin in HEK 293 cells and rat hippocampal lysates migrated at the same position as human erythrocyte
adducin, as confirmed by antibodies specific to human
(Fig. 3 B, d-f and g-i) and
(data not shown) adducin.
Specificity of the phosphoadducin antibody was further analyzed using adducin mutated at Ser716Ala/
Ser726Ala and resistant to phosphorylation by PKC (referred to as
adducin
pkc). The HA epitope-tagged
adducin and
adducin
pkc expressed in MDCK cells were
immunoprecipitated by an anti-HA epitope antibody followed by immunoblot with the phosphoadducin-specific antibody. Reactivity of the phosphoadducin antibody for
the HA epitope-tagged
adducin was increased about
threefold after PMA treatment (0.1 µM, 15 min) of the
cells, whereas the antibody showed no reactivity toward
adducin
pkc (Fig. 3 C). Lack of reactivity of the phosphoadducin antibody with
adducin
pkc provides additional
support for its specificity toward the RTPS-phosphoserine of adducin.
The RTPS-Phosphoserine Adducin and Spectrin Accumulate in the Cytoplasm in MDCK Cells after PMA Treatment
adducin and spectrin were concentrated at the cell membrane of cell-cell contact sites in serum-starved MDCK
cells as reported previously (Fig. 4 A and Kaiser et al.,
1989
). The anti-
adducin antibody also consistently
showed weak nuclear staining (Fig. 4 A, b and e). Very low
levels of the RTPS-serine phosphorylation was detected in
the cytoplasm, while none was detected at the cell-cell
contact sites of the cells using the phosphoadducin antibody
(Fig. 4 B, b). When the cells were stimulated with PMA, levels of
adducin slightly but significantly increased in the cytoplasm (Fig. 4 A, e). Increase of the RTPS-phosphoserine
adducin was clearly observed in the cytoplasm (Fig. 4 B,
e). The increases of
adducin and the phosphoadducin
were accompanied by redistribution of spectrin from the
cell-cell contact sites into the cytoplasm (Fig. 4, A and B).
Phosphoadducin and spectrin were distributed in a punctate pattern in the cytoplasm after PMA treatment (Fig. 4
B, d and e). Redistribution of adducin and spectrin from
the cell membrane into the cytoplasm has been reported in
several types of epithelial cells treated with PMA (Kaiser
et al., 1989
; Dong et al., 1995
). Similar results were also obtained using HEK 293 cells (data not shown). Therefore,
phosphorylation of the RTPS-serine in adducin appears to
correlate closely with redistribution of the protein and
spectrin from the cell membrane into the cytoplasm. This
is consistent with the biochemical data showing that phosphorylation of the MARCKS-related domain by PKC
interferes with interactions of adducin with spectrin and
actin.
|
Phosphorylation State of the MARCKS-related Domain Determines Adducin Distribution in MDCK Cells
Roles of the phosphorylation state of the MARCKS-
related domain in determining distribution of adducin and
spectrin were evaluated by transfecting cells with adducin
pkc, which was resistant to phosphorylation by PKC
at the MARCKS-related domain. MDCK cell lines were
generated that stably expressed the HA epitope-tagged
human
adducin (MDCK/
add) and the mutant human
adducin (MDCK/
add
pkc) (see Materials and Methods).
The HA epitope-tagged human adducin and spectrin
in MDCK/
add cells were concentrated at the cell membrane at sites of cell-cell contact (Fig. 5, a-c) as observed
for the native proteins in nontransfected cells (Fig. 4 A).
We assumed that a PKC-unphosphorylatable version of
adducin would stay at the cell membrane even after PMA
treatment if phosphorylation of the PKC sites in
MARCKS-related domain was responsible for the redistribution of adducin. Surprisingly, the mutant
adducin
was not localized at the cell-cell contact sites of the PMA-untreated cells, but instead exhibited a punctate distribution in the cytoplasm (Fig. 5, d and f). Moreover, cells expressing the mutant
adducin exhibited increased levels
of cytoplasmic spectrin in a punctate pattern, which was in
some cases colocalized with the mutant
adducin (Fig. 5, e
and f). This result clearly showed involvement of the PKC
phosphorylation sites in controlling subcellular distribution of adducin as well as spectrin. The nature of the punctate structures containing the mutant adducin and spectrin
has not been resolved, but they could represent vesicles en
route to or returning from the plasma membrane.
|
Localization of the RTPS-Phosphoserine Adducin in the Hippocampus
High levels of the phosphoadducin were detected in the
dendritic fields, i.e., stratum radiatum and stratum oriens,
of CA1 region in P15 rat hippocampus (Fig. 6 a). In the
dentate gyrus, the staining intensity for phosphoadducin
was higher in the hilus region than in the molecular layer
(Fig. 6 a). In the dendritic fields of CA3 region, phosphoadducin staining was slightly weaker than CA1 region
(Fig. 6 b). Confocal microscopy for stratum radiatum of
CA1 region at high magnification revealed phosphoadducin
antibody staining of round and elliptical structures with diameters <1 µm (Fig. 6 c). Double labeling with antibodies
against synaptophysin and the phosphoadducin revealed
that some of the phosphoadducin-stained structures were
immediately adjacent to presynaptic endings defined by
synaptophysin staining. (Fig. 6 c, arrows). Synaptophysin is a marker for neurosecretory vesicles, which are concentrated at presynaptic endings (Wiedenmann and Franke,
1985). Therefore, at least a subpopulation of the phosphoadducin-positive structures may correspond to dendritic
spines, which are specialized postsynaptic structures.
|
Localization of the Phosphoadducin in Dendritic Spines of Cultured Hippocampal Neurons
Resolution of dendritic spines is difficult in brain tissue sections, but these structures can be visualized in cultured hippocampal neurons. Distributions of the RTPS-phosphoserine adducin and total adducin were compared in cultured hippocampal neurons using the phosphoadducin-specific and the MARCKS-related domain-specific antibodies, respectively. Phosphoadducin was highly concentrated in spots flanking dendrites, which we interpret to be dendritic spines based on colocalization with AMPA receptors (a non-NMDA glutamate receptor) detected using antibody against the GluR2/4 subunits (Fig. 7, a-c, arrowheads). At least 80% of the spots contained both proteins, although in some cases the phosphoadducin-positive spots did not contain GluR2/4 and vice versa (Fig. 7, compare a and b). This result strongly supports the interpretation that the small structures containing high levels of the phosphoadducin observed in brain sections (Fig. 6 c) are dendritic spines.
|
Neither glutamate nor PMA treatment caused further increase in the level of adducin phosphorylation in spines (the ratio of the signals, phosphoadducin/glutamate receptor, was used to assess changes of adducin phosphorylation, n = 20 each) (Fig. 7, d-i). The cultures were preincubated with glutamate-free medium in the presence of high Mg2+ (15 mM) and an AMPA receptor antagonist (CNQX) to block NMDA and non-NMDA receptors, respectively (see Materials and Methods). Adducin therefore already was fully phosphorylated in the dendritic spines of hippocampal neurons under resting conditions in culture. However, PMA treatment did strongly enhance levels of the phosphoadducin located at the plasma membrane of cell body, the cytoplasm and dendrites (Fig. 7 h). The membrane-associated phosphoadducin was unanticipated. This result suggests involvement of additional domains, such as the head domain, for adducin to associate with the cell membrane.
Total adducin staining using antibody against the MARCKS-related domain, in contrast to the phosphoadducin, was observed along the entire cell membrane and in the cytoplasm of cell body and dendrites as well as in the dendritic spines (Fig. 8, a-c). No significant changes of adducin distribution were observed after glutamate and PMA treatments of the neurons (Fig. 8, d-f and g-i). However, the MARCKS-related domain-specific antibody has weak reactivity with the RTPS-phosphoserine adducin and underrepresents phosphoadducin (data not shown). These results combined with Fig. 7 demonstrate that adducin is uniformly distributed along the plasma membrane and that dendritic spines are the most active sites for adducin phosphorylation in hippocampal neurons.
|
Spectrin G was located mostly in a punctate pattern associated with dendrites that were not labeled with the
phosphoadducin antibody (Fig. 9). These findings suggest
that spectrin and phosphoadducin exist in distinct sites in
neurons.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study presents the first evidence that phosphorylation
of the RTPS-serine in the MARCKS-related domain of adducin occurs in vivo and has an important role in regulating
distribution of adducin in cells. Furthermore, phosphorylation by PKC of adducin at the MARCKS-related domain
inhibits adducin activities of promoting spectrin-actin interactions and capping the fast-growing ends of actin filaments.
PMA-activated phosphorylation of the RTPS-serine was
demonstrated, using a phosphoadducin-specific antibody,
in erythrocytes, HEK 293 cells (human embryonic kidney
origin), brain slices, and MDCK cells. A PKC-unphosphorylatable S716A/S726A mutant adducin expressed in
MDCK cells was distributed in a cytoplasmic punctate pattern and was no longer concentrated at the cell membrane at sites of cell-cell contact. Moreover, cells expressing the mutant
adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant adducin in a punctate pattern. Finally, high levels of the RTPS-serine phosphorylation were visualized in sections of rat
hippocampus and in the dendritic spines of cultured hippocampal neurons.
Dendritic spines are dynamic structures that receive the
majority of exitatory synaptic connections in the mammalian central nervous system (Harris et al., 1992; Papa et al.,
1995
; Ziv and Smith, 1996
). Dendritic spines change in
shape during neuronal development as well as concurrently
with long-term potentiation, a widely studied experimental
model of learning (Fifkova and Van Harreveld, 1977
;
Schuster et al., 1990
; Geinisman et al., 1991
; Hosokawa et al.,
1995
). Actin and actin-binding proteins, including spectrin,
adducin, and myosin, are the major cytoskeletal components in dendritic spines, where microtubules and neurofilaments
are virtually absent (Westrum et al., 1980
; Landis and Reese,
1983
; Morales and Fifkova, 1989
; Seidel et al., 1995
). The
actin filaments of the spine neck are longitudinally situated, whereas those in the head are organized into a lattice
(Fifkova and Delay, 1982
). This organization suggests that
actin filaments provide the basic structural scaffolding of
the spine (Harris and Kater, 1994
). Thus, changes in the
spine actin network through modulation of the activities of
actin-regulating proteins have been proposed as a basis for
activity-dependent structural changes in spine morphology
(Coss and Perkel, 1985
; Fifkova and Morales, 1991
).
Synaptic stimulation of dendritic spines induces an increase
in Ca2+ up to micromolar levels (Holmes, 1990; Gold and
Bear, 1994
). Probable targets for Ca2+ in dendritic spines
include calmodulin (Malenka et al., 1989
; Ehlers et al., 1996
;
Wyszynski et al., 1997
), PKC (Malinow et al., 1989
; Abeliovich et al., 1993
; Hrabetova and Sacktor, 1996
), Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Silva et al., 1992
;
Kennedy, 1993
; Lledo et al., 1995
), and Ca2+/calmodulin-
dependent protein phosphatase 2B or calcineurin (Mulkey et
al., 1994
). Among substrates for these enzymes are several cytoskeletal proteins, including microtubule-associated protein 2, (Quinlan and Halpain, 1996
), myosin (Kawamoto et
al., 1989
), and adducin. However, adducin is the first cytoskeletal protein directly demonstrated to be phosphorylated
in dendritic spines of living hippocampal neurons. Given the
biochemical activities of adducin in promoting assembly of
spectrin-actin complexes and that these activities of adducin
are regulated by PKC and calmodulin, adducin is a logical
candidate to participate in activity-dependent shape changes
of dendritic spines. In addition, the striking localization of
phosphoadducin in the spines further supports the idea that dendritic spines are distinct biochemical and Ca2+ compartments (Guthrie et al., 1991
; Müller and Connor, 1991
; Koch
and Zador, 1993
; Yuste and Denk, 1995
).
The identity of adducin kinase(s) and phosphatases responsible for regulating the phosphorylation state of adducin in dendritic spines remains to be determined. Since
CaMKII does not phosphorylate or
adducin in vitro
(Matsuoka, Y., unpublished results) and PKA does not in
vivo (Fig. 1 A), an isoform of PKC is the most likely candidate. It is noteworthy that at least one isoform of PKC,
PKC
, is localized in dendritic spines by immunoelectron microscopy (Tsujino et al., 1990
; Saito et al., 1994
). Phosphoadducin is likely to be a substrate for protein phosphatase(s) in dendritic spines, which could represent an
important aspect of activity-dependent regulation of adducin functions. Calcineurin, a Ca2+/calmodulin-dependent
protein phosphatase, is present at high levels in hippocampal neurons (Steiner et al., 1992
) and is a good candidate
for an adducin phosphatase.
Kaibuchi and colleagues have found that adducin is an
in vivo substrate for Rho-associated kinase (Rho-kinase)
and myosin phosphatase, which are involved in the regulation of actin cytoskeleton in the cells (Amano et al., 1997;
Kimura et al., 1998
). Phosphorylation by Rho-kinase occurs at a different site from PKC and increases the affinity
of
adducin for F-actin (Kimura et al., 1998
). It will be of
interest to determine localization of Rho-kinase-phosphorylated adducin in neurons and possibly dendritic spines.
The basic MARCKS-related domain of adducin has recently been demonstrated to be required for association of
adducin with spectrin and actin (Li et al., 1998). Moreover,
activity of adducin in promoting association of spectrin
with actin is inhibited by high ionic strength, consistent
with an electrostatic origin of binding energy between adducin and actin (data not shown). These findings considered together with the evidence in this study that phosphorylation of a site in the MARCKS-related domain inhibits
actin interactions suggest that this domain provides a direct contact with actin. Incorporation of phosphate with its
bulk and negative charge into the basic MARCKS-related
domain would be anticipated to interfere with electrostatic
interactions dependent on positively charged residues.
These considerations suggest that the actin contact sites
for adducin will include negatively charged residues. Negatively charged residues (aspartate or glutamate) are exposed on the lateral surface of rabbit skeletal actin in positions 1-4, 24, 25, 99, 100, 360, 361, 363, and 364, while
residues 167, 288, and 292 are located at the fast-growing
ends of actin filaments (Holmes et al., 1990
; Kabsch et al.,
1990
). The negatively charged residues exposed on the lateral surface of actin have been implicated in association
with a positively charged lysine-rich loop in the head domain of myosin (Rayment et al., 1993
; Schröder et al., 1993
)
and make up a possible actin-myosin interface (Holmes et al., 1990
; Kabsch et al., 1990
; Johara et al., 1993
). It will be
of interest to determine if adducin can inhibit actin-activated Mg2+-ATPase activity of myosin and if such effects
are controlled by phosphorylation of adducin.
Nonphosphorylatable mutant adducin ( adducin
pkc)
and spectrin were colocalized as a punctate pattern in the
cytoplasm of MDCK/
add
pkc cells (Fig. 5 f). The identity
of structures containing the mutant adducin and spectrin is
not known but could represent vesicles in transit between
the Golgi and plasma membrane. It is interesting in this regard that adducin and spectrin associate with dynactin, which is a multiprotein complex including an actin-related
protein Arp1 or centractin and promotes dynein-mediated
vesicle motility along microtubules (Schafer et al., 1994
;
Holleran et al., 1996
). It will be of interest to determine if
the structures with the mutant adducin contain other
members of the dynactin complex.
We propose adducin as a candidate for a Ca2+/calmodulin- and phosphorylation-sensitive modulator of the organization of spectrin-actin complexes in a variety of dynamic cellular domains. PKC has been demonstrated to be
involved in reorganization of the actin cytoskeletons coupling with membrane ruffling in neutrophils (Downey et al., 1992), spreading of platelets (Haimovich et al., 1996
), and
integrin clustering in lymphocytes (Pardi et al., 1992
). Adducin, based on results of this study, is a candidate downstream
effector of PKC in these cells and possibly in dendritic
spines. Since homologues of mammalian adducin have been
identified in Caenorhabditis elegans (Moorthy, S., L. Chen,
and V. Bennett. 1997. Mol. Biol. Cell. 8:274a), adducin may
have a fundamental role conserved during evolution in
regulation of assembly of spectrin-actin complexes in neurons as well as other types of cells.
![]() |
Footnotes |
---|
Received for publication 6 November 1997 and in revised form 10 June 1998.
Address all correspondence to Yoichiro Matsuoka, Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, NC 27710. Tel.: (919) 684-3105. Fax: (919) 684-3590.We are grateful to Dr. P.J. Blackshear for providing recombinant human
MARCKS protein and Dr. T. Garver for technical assistance for rat hippocampal slice experiments. We also thank Suraj Moorthy for preparing
and providing an HA-tag adducin construct and
and
adducin-specific antibodies, and Mildred McAdams and Judith Phelps for peptide synthesis and analysis.
![]() |
Abbreviations used in this paper |
---|
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;
HA, hemagglutinin;
IBMX, 3-isobutyl-1-methylxanthine;
MARCKS, myristoylated alanine-rich C kinase substrate;
NMDA, N-methyl-D-aspartate;
PKA, cAMP-dependent protein kinase;
PKC, protein kinase C;
PKM, the catalytic domain of protein kinase C;
PMA, phorbol 12-myristate
13-acetate.
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