From the Department of Hematology-Oncology, Istituto
Superiore di Sanità, 00161 Rome, Italy, the
Department of
Experimental Medicine, University of L'Aquila, 67100 L'Aquila, Italy,
the § Department of Experimental Medicine and Pathology,
University "La Sapienza," 00161 Rome, Italy, the
Department of Molecular Pharmacology and
the Albert Einstein Cancer Center, Albert Einstein College of
Medicine, Bronx, New York 10461, and the ¶¶ Kimmel
Cancer Center, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Recent studies have highlighted the
existence of discrete microdomains at the cell surface that are
distinct from caveolae. The function of these microdomains remains
unknown. However, recent evidence suggests that they may participate in
a subset of transmembrane signaling events. In hematopoietic cells,
these low density Triton-insoluble (LDTI) microdomains (also called
caveolae-related domains) are dramatically enriched in signaling
molecules, such as cell surface receptors (CD4 and CD55), Src family
tyrosine kinases (Lyn, Lck, Hck, and Fyn), heterotrimeric G proteins,
and gangliosides (GM1 and GM3). Human T
lymphocytes have become a well established model system for studying
the process of phorbol ester-induced down-regulation of CD4. Here, we
present evidence that phorbol 12-myristate 13-acetate (PMA)-induced
down-regulation of the cell surface pool of CD4 occurs within the LDTI
microdomains of T cells. Localization of CD4 in LDTI microdomains was
confirmed by immunoelectron microscopy. PMA-induced disruption of the
CD4-Lck complex was rapid (within 5 min), and this disruption occurred
within LDTI microdomains. Because PMA is an activator of protein kinase
C (PKC), we next evaluated the possible roles of different PKC isoforms
in this process. Our results indicate that PMA induced the rapid
translocation of cytosolic PKCs to LDTI microdomains. We identified
PKC Recent studies have highlighted that the plasma membrane is not
homogenous but instead consists of a variety of discrete microdomains (1-4). CD4 is an ~ 55-59-kDa membrane glycoprotein expressed on the surface of T helper cells and to a lesser extent on
monocytes/macrophages. It is the human receptor for
HIV,1 whose binding allows
the entrance of HIV to target cells (5).
CD4 is considered to be the TCR co-receptor in T-cell activation and
thymic selection (6). In this regard, it binds to major
histocompatibility complex class II epitopes, thereby strengthening cell-to-cell contact and TCR-major histocompatibility complex formation
(7). In addition, CD4 itself mediates intracellular signals that
influence TCR-CD3 complex formation and augments the cellular response
(8). It is well established that this effect is due to CD4 interaction
with Lck (9), a member of the Src family of tyrosine kinases. Lck is
anchored to the cytoplasmic side of the membrane via lipid
modifications and is involved in CD3 Lck also plays a role in regulating the endocytic properties of CD4,
thereby controlling the cellular distribution of this co-receptor (11).
Treatment of T cells with phorbol esters such as PMA is one of the
methods used to mimic modulation of CD4 that occurs during the antigen
encounter (12). Upon activation, cytoplasmatic serine residues of CD4
are phosphorylated most likely via an isoform or isoforms of PKC (13,
14).
PKCs are a family of at least 12 isoenzymes, whose 8 isotypes ( PKCs differ in substrate specificity, cofactor requirements, tissue and
cellular distributions, subcellular localizations (21), and regulatory
mechanisms (22) that lead to their differential translocation in the
cell following stimulation (23). In T lymphocytes, PMA activation
induces PKC Little is known about the specific role played by each of the multiple
isoforms present in a given cell type, although the involvement of
PKC The subcellular localization of a given PKC isoform may represent an
important clue in determining the specific function of a given PKC
isoform. Electron microscopy and plasma membrane fractionation in the
absence of detergent have demonstrated that the PKC We recently purified and characterized these low density
Triton-insoluble (LDTI) microdomains from hematopoietic cells. These LDTI microdomains morphologically and biochemically resemble raft domains and were highly enriched in signal transducing molecules such
as a subset of cell surface receptors, Src family tyrosine kinases,
heterotrimeric G proteins, and gangliosides (GM3) (2, 4).
New insights into the dynamic clustering of raft domains have
highlighted their potential role as a starting point for many membrane-linked processes, including certain transmembrane signaling events (30). However, in the hematopoietic system, the exact function
of these domains has not yet been established. To address this issue,
we have analyzed the well described process of CD4 internalization in
human T cells that is induced by activation with PMA.
Here, we show that this process takes place within LDTI microdomains.
More specifically, we demonstrate that upon PMA treatment (i) the
CD4-Lck complex is disrupted within LDTI domains; (ii) CD4 shifts from
LDTI domains to a Triton-soluble particulate fraction in a
time-dependent manner, whereas Lck remains within the LDTI domain; and (iii) many PKC isoforms are activated and translocated from
the cytosol to LDTI domains but at different rates. In addition, PKC Materials--
Anti-CD4 monoclonal antibody used for Western
blotting was purchased from Novocastra (Newcastle-upon-Tyne, UK).
Anti-CD4 monoclonal antibody used for immunoprecipitation was obtained
from Santa Cruz Biotechnologies (Santa Cruz, CA). For FACS analysis and
immunoelectron microscopy anti-CD4, OKT4 was purchased from Ortho
Diagnostic System (Raritan, NJ). Anti-Lck, anti-PKC Isolation of Cell Membranes (M) and Detection of Surface
Proteins--
An established protocol was followed to prepare total
cell membranes (or particulate), enriched in plasma membrane, from
human peripheral lymphocytes, with some modifications (65). Briefly, 1 × 109 lymphocytes were surface labeled by
incubation with biotin-NHS (0.5 mg/ml) for 30 min at 4 °C. After
washing with ice-cold serum-free Dulbecco's modified Eagle's medium
and then with PBS, cells were incubated with PMA (100 ng/ml) at
37 °C for the indicated times in warm RPMI medium. After three
washes with ice-cold PBS, cells were Dounce-homogenized with 2 ml
of lysis buffer (20 mM Tris, pH 8,0, 2 mM EGTA
containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 2 µg/ml
aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A). The homogenate
was first centrifuged at 1,000 × g for 5 min at
4 °C, and then the supernatant was centrifuged at 2,000 × g for 5 min to remove nuclear debris. The resulting
supernatant was centrifuged at 60,000 rpm for 30 min to produce a
nucleus-free membrane fraction (pellet) and a cytosol fraction
(supernatant). Proteins were quantified by Peterson method (33) and
then resolved by SDS-PAGE, transferred to a 0.22-µm nitrocellulose
filter (Amersham Life Science, Buckinghamshire, UK), and blocked with
4% nonfat milk and 1% BSA in TBST (10 mM Tris-HCl, pH
8.0, 150 mM NaCl, 0,05% Tween 20). The membrane was then
probed with streptavidin-horseradish peroxidase conjugated at 1:30,000
dilution in TBST for 1 h at room temperature. The membrane was
washed six times with TBST and incubated with SuperSignal chemiluminescence ULTRA (Pierce) according to the manufacturer's instructions. Reactive proteins were detected by autoradiography on
Kodak T-Mat G/RA film (Eastman Kodak, Rochester, NY).
Isolation of LDTI from Total Cells and Membrane
Fractions--
LDTI complexes were isolated as described previously
(2, 34). Briefly, 1 × 109 lymphocytes were washed and
lysed in 1 ml of MBS (25 mM MES, pH 6, 5, 150 mM NaCl) or were treated as described to recover membrane
fractions (M). Next both preparations were Dounce homogenized in buffer
containing 0, 0.02, 0.1, or 1% Triton X-100 and 0.1 mg/ml
phenylmethylsulfonyl fluoride, adjusted to 40% sucrose, and placed at
the bottom of four different ultracentrifuge tubes. A 5-30% linear
sucrose gradient was then placed above the lysate, and the mixture was
centrifuged at 45,000 RPM, 16 h, 4 °C in a SW60 rotor (Beckman
Instruments, Palo Alto, CA). In both cases LDTI, visible as a band
migrating at approximately 20% sucrose, was harvested and washed twice
with MBS at 14,000 RPM for 30 min at 4 °C and then protein
quantitated. 1 × 109 lymphocytes consisting of 50 mg
of total protein, yielded a mean of 7 mg of particulate fractions, and
110 µg of LDTI fractions. Thus LDTI represents 0.2% of the initial
homogenate and 1.5% of membrane fraction, whereas membrane represents
14% of the initial homogenate.
Western Blotting Analysis and Immunoprecipitation--
LDTI,
total cell lysates or total membrane proteins were resolved by 8%
SDS-PAGE under reducing conditions and transferred to nitrocellulose
filter. The blots were blocked using 5% nonfat milk in TBST for 1 h at room temperature, followed by incubation with anti-Lck polyclonal
(dilution 1:100), anti-Fyn monoclonal (dilution 1:400),
anti-Gi polyclonal (dilution 1:2000), anti CD4 monoclonal
(dilution 1:200) or anti-PKC-specific antibodies in TBST for 1 h
at room temperature. PKC Kinase Assay--
1 µg of LDTI complexes isolated from
PMA-treated cells were resuspended in 20 µl of kinase reaction buffer
(20 mM Hepes, pH 7.4, 5 mM MgCl2,
and 1 mM MnCl2) supplemented with Gö 6976 (10 Immunofluorescence Staining--
CD4+ lymphocytes
(1 × 106 in 1 ml of PBS) were incubated with PMA
as reported above. PMA-treated and untreated cells were then fixed with
acetone/methanol 1:1 (v/v) for 10 min at 4 °C. Cells were soaked in
Hank's balanced salt solution for 30 min at 25 °C and incubated for
20 min at 25 °C in the blocking buffer of 2% BSA in PBS containing
5% glycerol and 0.2% Tween 20. Cells were then labeled with
anti-PKC Flow Cytometry Analysis--
CD4 expression on untreated and
PMA-treated lymphocytes was investigated using monoclonal antibody CD4
(OKT4) fluorescein conjugated (1:20 in PBS, 1% BSA for 30 min at
4 °C). After washing with PBS/BSA cells were fixed with 1%
formaldehyde in PBS. Green fluorescence intensity was analyzed with
FACS scan cytometer (Becton Dickinson). For every histogram 5000 cells
were counted to evaluate the percentage of CD4+ cells. The
percentage of surface expression at different times of incubation with
PMA was calculated using the mean fluorescence divided by the mean
fluorescence at time 0 minus the background fluorescence.
Immunoelectron Microscopy--
To assess CD4 localization at the
cell plasma membrane of Triton-treated cells we followed a previously
reported protocol with some modifications (35). Briefly,
CD4+ cells were isolated from peripheral lymphocytes using
the IsoCellTM human CD4 Isolation Kit (Pierce) according to
the manufacturer's instruction. CD4+ cells were then
incubated with monoclonal antibody (OKT4) at 1:5 dilution in 0.5 ml of
PBS/BSA for 1 h at 4 °C, followed by two washes with ice-cold
PBS. Cells were resuspended with 1 ml of paraformaldehyde 3% in PBS,
pH 7.2 for 30 min at 4 °C. After washing, cells were left untreated
or 1% Triton X-100-treated for 30 min at 4 °C, followed by a second
incubation with monoclonal antibody (OKT4) for 1 h at 4 °C.
After incubation with rabbit anti-mouse IgG (Sigma) (1:10 in PBS for
1 h at 4 °C), cells were fixed with glutaraldehyde (1% in PBS
for 1 h at 4 °C), extensively washed, and then labeled with
colloidal gold (18 nm, prepared by the citrate method) conjugated with
protein A (Amersham Pharmacia Biotech) for 3 h at 4 °C. Control
experiments were performed omitting the incubation with OKT4 monoclonal
antibody in both untreated and 1% Triton X-100-treated lymphocytes.
All samples were postfixed in 1% osmium tetroxide in Veronal acetate
buffer, pH 7.4, for 2 h at 4 °C, stained with uranyl acetate (5 mg/ml), dehydrated in acetone, and embedded in Epon 812.
Morphometry--
Mophometric analysis of the length of plasma
membrane in Triton X-100-treated cells and in untreated controls as
well as quantification of colloidal gold granules were performed on 30 micrographs printed at the same magnification. The results were
expressed as the means ± S.D.
Characterization of CD4 in LDTI Domains of Resting
Lymphocytes--
The detergent insolubility of caveolae and
caveolae-related domains is based on their high content of cholesterol
and sphingolipids; many distinct classes of lipid-modified signaling
molecules are retained within these detergent-resistant membrane
domains. However, certain caveolae- and caveolin-1-associated proteins
are dissociated from these domains as a consequence of detergent
treatment; these include receptor tyrosine kinases (such as epidermal
growth factor receptor) and a variety of prenylated proteins (such as
Ha-Ras) (36). In addition, a small amount of caveolin-1 is found in the
Triton-soluble fraction, and this fraction of caveolin-1 is associated
with the Golgi complex (37). More interestingly, recent evidence
indicates that proteins can move in and out of caveolae-related
domains, depending on their activations state and that this dynamic
movement can be monitored by changes in Triton solubility and
partitioning into low density Triton-insoluble domains (LDTI, a
biochemically descriptive term for caveolae-related domains) (38).
In a previous report, we demonstrated that CD4 is dramatically enriched
within these LDTI domains in human lymphocytes (4). Here, we analyze
the changes in CD4 localization after cell activation by phorbol esters
(PMA). To study changes in CD4 localization following phorbol ester
cell activation, we first analyzed the Triton sensitivity of CD4 in
resting lymphocytes.
Thus, we experimentally defined the optimal Triton X-100 concentration
that is required for the isolation of Triton-insoluble CD4 from the
bulk of soluble plasma membrane proteins (Fig.
1a). To this end, ~ 1 × 109 lymphocytes were surface labeled with
sulfo-NHS-biotin. Two mg of the cell membrane fraction (M) were
prepared from these labeled lymphocytes and homogenized with increasing
amounts of Triton X-100, followed by equilibrium density gradient
centrifugation. Each tube was then divided into twelve 375-µl
fractions, and 10 µl/fraction was subjected to SDS-PAGE and blotting
with streptavidin-horseradish peroxidase. Only biotin-labeled cell
surface proteins were detected by this procedure (Fig. 1a,
left).
Treatment of the membrane fraction with increasing concentrations of
Triton X-100 resulted in differential solubilization of the plasma
membrane. As expected, in the absence of detergent, all of the
biotin-labeled cell surface proteins partitioned exclusively at the
bottom of the gradient (fractions 7-12); none of the labeled proteins
attained buoyancy. In contrast, increasing concentrations of Triton
X-100 (0.02 or 0.1%) led to differential protein solubilization resulting in bands migrating at many different gradient densities. Note
that a concentration of 1% Triton X-100 allowed optimal isolation of
the LDTI microdomains (fractions 4-6), which migrated to the upper
20% sucrose region of the gradient. (Fig. 1a,
left).
In parallel experiments, each fraction was diluted, and the
sedimentable material was collected by centrifugation and used to assay
the distribution of CD4 by immunoblotting (Fig. 1a,
right). Analysis of the distribution of CD4 demonstrated
selective partitioning of CD4 into the LDTI fraction at a concentration
of 1% Triton. We identified a 55-kDa biotin-labeled cell surface
protein as CD4 by immunoprecipitation with CD4-specific antibodies
(Fig. 1a, inset).
In addition, the protein profile of the sucrose gradient fractions
indicated that ~99% of total cellular protein was recovered in
fractions 8-12, whereas only ~1% of the total protein was recovered as LDTI domains (Fig. 1b). Densitometric analysis of the
distribution of CD4 indicated that it quantitatively co-distributed
with the LDTI domains (Fig. 1b).
The enrichment of CD4 in LDTI domains was estimated by comparing the
amount of CD4 at different steps during the fractionation procedure
(total lysate, total membrane fraction, and the LDTI fraction) (Fig. 1,
c and d). Our results indicate that CD4 is ~66-fold enriched in total membranes (M) and ~1000-fold enriched in
LDTI domains, relative to the total cell lysate. A representative blot
is shown in Fig. 1d.
Detergent-resistant Plasma Membrane Microdomains Are Present in the
Native Plasma Membrane of Lymphocytes--
To assess the native
distribution of molecules found associated with detergent-insoluble
membrane domains, other laboratories have used an electron microscopy
approach. In one such study, cells were first detergent extracted,
fixed, and analyzed by standard transmission electron microscopy. These
authors elegantly demonstrated that caveolae and caveolae-related
domains exist in intact cells (35).
Here, we have applied this type of approach to localize CD4 in
lymphocytes. Our ultrastructural observations reveal for the first time
that in Triton-treated lymphocytes, although most of the plasma and
intracellular membranes were almost completely dissolved, small
portions of lipid bilayer were still present (Fig.
2a). These microdomains of the
plasmalemma provide morphological evidence for the existence of
intrinsically detergent-insoluble membrane domains characterized by the
typical trilaminar unit membrane appearance (Fig. 2a,
arrowheads).
To verify the presence of CD4 molecules localized on these
Triton-insoluble membrane microdomains, we applied an
immunocytochemical approach to Triton-treated lymphocytes. Briefly,
after detergent treatment, cells were incubated with the OKT4
monoclonal antibody followed by anti-IgG antibodies and protein
A-colloidal gold conjugates. Our results show that most of the gold
particles corresponding to immunolabeled CD4 molecules are steadily
associated within these Triton-insoluble microdomains (Fig.
2b). Gold particles were observed both on the nonvillous
portion of the retained membrane (Fig. 2b) and on microvilli
(not shown). Similarly, in cell surface biotinylated lymphocytes the
immunolabeling corresponding to Triton-insoluble biotinylated membrane
proteins showed that gold particles were restricted to domains
retaining the bilayer unit membrane appearance (Fig. 2, c
and d). In further support of these observations, in lymphocytes that were not treated with detergent, the distribution of
CD4 was clearly uneven over the plasma membrane (Fig. 2e). This uneven distribution is in accordance with previous reports (39).
Quantitative morphometric analysis revealed that the portions of
unextracted membrane retaining the trilaminar unit membrane appearance
measured approximately 3.5 ± 0.3 µm/cell corresponding to
17 ± 2% of the total plasma membrane length detected.
Moreover, in these Triton-insoluble membrane portion we detected
7.2 ± 0.7 immunogold-labeled CD4 particles/µm. This density is
significantly higher as compared with that observed in the total plasma
membrane of untreated lymphocyte (5 ± 0.5 gold particles/µm).
However, this is clearly an underestimate of enrichment, as the
efficiency of immunolabeling is generally accepted to be quite low.
In support of our current observations, other laboratories have
demonstrated that low density microdomains exist in the absence of
detergent solubilization in neuronal cells lacking caveolae and that
they display biochemical and morphological characteristics similar to
LDTI (31). To verify the existence of LDTI domains per se in
plasma membrane in our system, we used an independent and well
established detergent-free method based on cell fractionation and
sucrose density gradient centrifugation (40). Using this detergent-free
method, we found that CD4 was exclusively localized in the low density
fractions in the absence of detergent, as predicted (data not shown).
PMA Treatment Rapidly Induces Disruption of the CD4-Lck Complex
within LDTI Microdomains--
It is well established that PMA induces
phosphorylation of the cytoplasmic tail of CD4 and disruption of the
CD4-Lck complex. This is followed by the aggregation and the
internalization of CD4 via the activation and recruitment of PKC to
plasma membrane (41). Moreover, it is generally accepted that CD4
internalization occurs through clathrin-coated pits (42, 43) that were
found to be Triton-soluble (44). Thus, we next investigated whether any
of these events occurred within LDTI microdomains.
For this purpose, we monitored the amount of CD4 present in total
plasma membrane fractions and LDTI domains, before and after acute
treatment with PMA (0-60 min). In this regard, note that the LDTI
domains would be considered a subfraction of the total plasma membrane.
Briefly, lymphocytes were biotinylated to label cell surface CD4, and
processed to obtain the particulate/plasma membrane fraction
(designated M, representing Total CD4). The M fraction was then used as
the starting material to isolate LDTI domains.
The amount of CD4 in total plasma membrane (M) and detergent-resistant
membranes (LDTI) was directly compared by Western blotting (Fig.
3a) and immunoprecipitation
(Fig. 3b). In addition, we used FACS analysis to
independently monitor the extent of CD4 down-modulation. Note that our
results obtained with FACS analysis were almost identical to those
obtained with using total plasma membrane fractions, further
demonstrating the validity of this approach (Fig. 3, a and
b).
Quantitation revealed that the amount of CD4 in LDTI domains was
reduced by ~60-70% within 5 min of PMA treatment. In contrast, in
total plasma membrane fractions, the amount of CD4 was only reduced by
~10-15% within 5 min. Virtually identical results were obtained by
both Western blotting and immunoprecipitation of biotinylated cell
surface CD4. We reasoned that this difference may be a result of a
molecular modification of CD4 following Lck detachment.
It is well known that Lck is localized at the cytoplasmic face of the
plasma membrane (10). High levels of Lck membrane attachment are
achieved through dual acylation (myristoylation and palmitoylation) of
its N-terminal domain (45, 46). Moreover, the interaction of Lck with
CD4 is mediated via a reciprocal interaction between the cytoplasmic
domain of CD4 and the N-terminal domain of Lck. A cysteine within the
N-terminal domain of Lck is critical for this interaction, and this
cysteine residue is distinct from the cysteine that undergoes
palmitoylation (47). Thus, we would predict that disruption of the
CD4-Lck complex would not affect the localization of Lck, because dual
acylation of other Src family kinases is sufficient to mediate caveolar
localization in other cell systems. In this sense, Lck would serve as
an internal control for these studies and a stable marker for the LDTI fraction.
Thus, we next analyzed the amount of Lck in the LDTI fraction before
and after PMA treatment. The same protein amounts were separated by
SDS-PAGE and subjected to immunoblotting for Lck. As shown in Fig.
3a (left) no differences in the distribution of
Lck were observed before or after PMA treatment.
Disruption of the CD4-Lck complex was also monitored by performing a
series of co-immunoprecipitation experiments. Antibodies directed
against CD4 were used to recover the CD4-Lck complex, and the presence
of Lck in this complex was then visualized by Western blotting. Note
that Lck is only present in the CD4 immunoprecipitates in untreated
cells, as expected (Fig. 3b) (17).
During PMA time course experiments no appreciable differences in LDTI
total protein (data not shown) or the Lck content of the LDTI fraction
were observed. In addition, two other dually acylated LDTI marker
molecules, Fyn and Gi (2, 48, 49), were analyzed and found
unchanged after short PMA incubations (5 min) (Fig. 3c).
These critical control experiments clearly indicate that the
down-modulation of CD4 with LDTI microdomains is a highly selective event.
The acquired detergent solubility of plasma membrane CD4 at 5 min is
confirmed by the persistence of CD4 at this time point in total plasma
membrane (Fig. 3, a and b, see M fraction). The delayed disappearance of CD4 from the plasma membrane indicates a shift
of CD4 from its initial location within LDTI microdomains to the
detergent-sensitive areas of the plasma membrane. This PMA-induced
shift was also observed in an independent experiment in which the
amount of CD4 in LDTI (fraction 4-6) and soluble plasma membrane
(fractions 8-12) was monitored by sucrose density gradient
centrifugation (Fig. 3d).
PMA-induced Recruitment of PKC Isoforms to LDTI
Microdomains--
In lymphocytes, PMA activates PKCs and induces their
translocation from the soluble to particulate fraction consisting of the plasma membrane proper and other subdomains of the plasma membrane
(for review see Refs. 23 and 50).
To establish whether a particular PKC isoform was involved in the CD4
internalization event, we analyzed the distribution of PKC isoforms by
immunoblotting before and after PMA treatment. We found that all seven
PKC isoenzymes (
In line with previous studies showing an immediate PKC redistribution
to plasma membrane following activation (50), our data with PKCs (
Surprisingly PKC
In an attempt to better quantitate this translocation event, we
compared the amount of PKC immunoreactivity present in total membranes
(M, particulate) and within LDTI microdomains, before and after
stimulation with PMA (Fig. 5). Thus, 1 µg of protein of both preparations was resolved by SDS-PAGE and
subjected to immunoblotting with PKC isoform-specific antibodies.
Results are expressed in arbitrary units as a ratio between the amount
within LDTI domains and the amount within total membranes
(LDTI/particulate ratio). The results from densitometric analysis of
three independent experiments are shown.
When we evaluated the translocation event as a ratio of LDTI to total
membrane levels PKC
In the total membrane fraction, PKC isoforms
Since PKC isoforms may have an intrinsically different Triton
solubility, we separately analyzed the co-localization of PKC
Double-labeling studies with Lck and PKC
Parallel experiments were performed to verify whether, after treatment
with PMA, PKC
In agreement with our previous observations (4), the GM3
signal appeared uneven and punctate over the plasma membrane, indicating a clustered distribution of GM3 molecules (Fig.
6b, panel 2). Anti-PKC
These observations independently demonstrate that PMA treatment induces
a preferential translocation of PKC PMA Treatment Up-regulates the Kinase Activity Associated with LDTI
Domains--
To further elucidate the activation state of PKC
To investigate whether this enhanced kinase activity might be due to
the presence of activated PKCs, we tested the effect of a new PKC
inhibitor (Gö 6976) that selectively affects only Ca2+-dependent isoforms of PKC (
Finally, to evaluate the proposed role of PKC
Treatment with either PKC inhibitor greatly reduced (to 30-35%; with
Gö 6976) or completely abolished (with Gö 6850) CD4 internalization. Virtually identical results were obtained by FACS
analysis and immunoprecipitation of LDTI domains.
Taken together, these data suggest a preferential involvement of
PKC The existence of plasma membrane microdomains distinct from
caveolae is now generally accepted. A biochemical definition of these
membrane compartments has been derived from their unusually high
glycolipid and cholesterol content, whose interaction creates "the
detergent-resistant platform" (1, 57, 58). Receptors (31), Src family
tyrosine kinases (2, 31, 44), heterotrimeric G proteins (2, 31), and
Ha-Ras (31) have all been found associated with these microdomains.
However, the precise function of these membrane rafts or
caveolae-related domains still remains to be defined.
We recently described that the CD4-Lck complex is localized within LDTI
microdomains isolated from lymphoid cells (2). Lck, among the PTKs,
represents the best characterized lymphocyte-specific tyrosine kinase,
whose unique N-terminal binding region is responsible for CD4 anchoring
and surface expression on the plasma membrane (59). At steady state, it
has been estimated that only ~5-7% of cell surface CD4 is
endocytosed by coated pits in T cells (11).
Conversely, in phorbol ester-activated T cells, disruption of CD4-Lck
complex occurs following serine phosphorylation of CD4 (12, 18) and Lck
(60, 61). This sequence of events initiates the rapid cell surface
down-modulation of CD4 that occurs through an increased uptake via
coated pits (17).
We report here data on the molecular events leading to PMA-induced
down-regulation of CD4 in lymphocytes. We find that (i) plasma membrane
CD4 is steadily confined to and enriched within LDTI microdomains
(15-fold versus plasma membranes); (ii) in agreement with
previous studies, PMA-induced disruption of the CD4-Lck complex occurs
rapidly within 5 min; and (iii) analysis of total membrane fractions
revealed that CD4 was down-modulated by 20% at 5 min and by 90% at 60 min (17, 41). Interestingly, CD4 levels within LDTI microdomains
decreased by 60% within 5 min (Fig. 3). This time lag indicates that
the disappearance of CD4 from LDTI microdomains occurs more rapidly
than from plasma membrane.
One possible explanation for this difference in the CD4 content of LDTI
microdomains and total plasma membrane is that down-modulation of CD4
requires it to move from LDTI microdomains to another region of the
plasma membrane that is Triton-soluble. This movement may be initiated
by a conformation change in CD4 that is due to PMA-induced serine
phosphorylation of CD4. Release of CD4 from the confines of LDTI
microdomains and Lck may then expose a putative endocytosis signal that
mediates its uptake via clathrin-coated pits and targets it for
lysosomal degradation (41).
Additionally, we demonstrate that Lck, which at steady state in T cells
is 60-70% complexed with CD4 (62), and other LDTI marker proteins
(Fyn and Gi) (2, 48, 49), remain within the LDTI
microdomains during PMA treatment. Thus, Lck is firmly restricted to
these LDTI microdomains, and its localization within LDTI microdomains
is independent of CD4. This may be due to the observation that Lck,
Fyn, and Gi all undergo N-terminal dual acylation (62). It
has been proposed that dual acylation targets Src family kinase to
detergent-resistant membrane domains (63).
A dissection of the novel CD4-Lck molecular events observed
within the LDTI microdomains of activated T cells has been hindered by
a lack of methods to separate distinct microdomains of the plasma
membrane. Moreover, the analysis of these LDTI microdomains provide a
tool to evaluate the specific role of PKC in this process by the
elucidation of kinase selected substrates. Here, we have used LDTI
microdomains to assess the translocation and compartmentalization of
the PKC isoforms ( Involvement of PKCs in the PMA-induced phosphorylation of CD4 has been
hypothesized based on (i) in vitro experiments suggesting that CD4 serves as substrate for PKC (18) and (ii) the observation that
prolonged PMA treatment exhausts cytoplasmic reserves of PKC and
inhibits down-modulation of CD4 (20). However, evidence for the
involvement of a specific PKC isoform in this process has been lacking.
Here, we provide evidence that PKC As seen by FACS analysis, other PKCs partially contribute to the
phenomenon (Fig. 8a). Future studies with PKC selective
inhibitors will elucidate the specific involvement of a given PKC isoform.
Similarly, in the present study we observed that CD4 is enriched within
low density membranes that were purified in the absence of detergent.
These membranes had the same buoyant density as LDTI microdomains.
Furthermore, we show by immunogold labeling and transmission electron
microscopy that CD4 is localized within detergent-resistant areas of
the plasma membrane of intact cells. Taken together, these data provide
strong evidence that purified LDTI domains correspond to native plasma
membrane compartments where signals involving CD4-Lck complex may be
transduced. In addition, while this paper was under review, another
paper appeared demonstrating that the entire T-cell receptor complex is
recruited after its engagement to plasma membrane domains resembling
LDTI domains (38).
These observations highlight the importance of these caveolae-related
microdomains in lymphocyte signal transduction. Moreover, this system
provides a new tool to assess the translocation of PKC and other signal
molecules in response to a variety of stimuli presented to the
hematopoietic cell system.
as the major isoform involved in this translocation event. Taken together, our results support the hypothesis that LDTI microdomains represent a functionally important plasma membrane compartment in T cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
chain phosphorylation
(10).
,
1,
2,
,
,
,
, and
) are
expressed in T cells (15, 16) and are responsible for CD4
down-modulation by endocytosis through clathrin-coated pits (17). In
this regard, CD4 lacking intracellular serine residues (a possible
target for PKC phosphorylation) is not down-regulated by phorbol esters
(18, 19). CD4 expression returns to normal only upon prolonged PMA
stimulation, which exhausts cytoplasmic stores of PKC (20).
,
1, and
2 redistribution
from a diffusely cytoplasmic localization to a discrete focal
distribution around the plasma membrane and nucleus (24).
in T cell activation following stimulation by antigen presenting
cells has been recently described. In this regard PKC
was spatially
restricted to the site of contact, where receptors on the T cells
encounter their counterparts on antigen presenting cells (25).
isoform is
enriched with caveolae (26, 27). The co-existence in the same cell of
caveolae and membrane "rafts" enriched in glycolipids has been
described (28-30) as well as membrane rafts in cells devoid of
morphologically recognizable caveolae such as neuronal and hematopoietic cells (2, 3, 31, 32). These rafts have also been termed
LDTI or "caveolae-related domains."
appears to be the most abundant isoform within LDTI domains, suggesting
that it plays an important role in this process.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,
2,
,
, and
polyclonal antibodies were
purchased from Santa Cruz Biotechnologies; anti-Fyn, anti-PKC
, and
anti-PKC
monoclonal antibodies were purchased from Transduction
Laboratories, Inc. (Lexington, KY); anti-PKC
was purchased from
Calbiochem (La Jolla, CA). Anti-monoclonal or -polyclonal secondary
antibodies horseradish peroxidase-conjugated were purchased from
Bio-Rad. Biotin-NHS was purchased from Calbiochem, and
streptavidin-horseradish peroxidase conjugated was from Pierce. Gö 6976 and Gö 6850 were obtained from Calbiochem. PKC
purified enzyme was purchased from Upstate Biotechnology Inc. (Lake
Placid, NY). PMA and Histone H1, Type IIIS, were obtained from Sigma. Anti-Gi antibody was a generous gift of Dr. Tommaso Costa.
was 1:4000 diluted, PKC
was 1:250
diluted, and the other PKC isoforms were 1:100 diluted. After washing
with TBST, each filter was incubated with the appropriate secondary
antibody-horseradish peroxidase conjugated at 1:3000 dilution for
1 h at room temperature. Reactive proteins were detected as
described above. For immunoprecipitation experiments LDTI domains or
membrane fraction from biotin-labeled lymphocytes were prepared as
described above. 1-5 µg of protein were precleared with 30 µl of a
50% slurry protein A/G-agarose (Pierce) and 1 µg of nonimmune serum
in 0.5 ml of lysis buffer (150 mM NaCl, 10 mM
Tris-HCl, pH 7.4, 1% Nonidet P-40, 10% glycerol, 0.1 mg/ml
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml
leupeptin, 1 µg/ml pepstatin A) for 1 h at 4 °C. CD4 antibody
(0.5 µg/ml) or PKC
(1 µg/ml) or PKC
(1 µg/ml) were then
added to the sample and kept overnight at 4 °C, followed by
incubation with prewashed beads (40 µl), 1 h at 4 °C. The
beads were spun down and washed four times with lysis buffer,
resuspended in 30 µl of SDS-PAGE sample buffer under reducing
conditions, boiled, and spun down. The supernatant was loaded on a 8%
SDS-PAGE. Cell surface CD4 was detected by blotting the filter with
streptavidin-horseradish peroxidase conjugated under the conditions
described for detection of surface proteins. Using this procedure only
one band corresponding to 55-kDa CD4 was detectable.
7 M) and 5 µCi of
[
-32P]ATP for 10 min at room temperature. The reaction
was stopped by addition of 20 µl of Laemmli sample buffer (2×) under
reducing conditions. The mixture was boiled, separated on 10% SDS-PAGE that was then dried, and exposed to Kodak XAR film. In other
experiments, to test the activity of PKC
, 5 µg of Histone type
IIIS and 25 ng of PKC
purified enzyme were incubated with 10 µg of
phospatidylserine in ADB buffer (1 mM sodium ortovanadate,
25 mM
-glycerophosphate, 20 mM MOPS, pH 7.2, 1 mM dithiothreitol, 1 mM CaCl2, 5 mM MgCl2), prior to the addition of 5 µCi of
[
-32P]ATP.
,
, or
polyclonal antibodies (Santa Cruz
Biotechnology) for 1 h at 4 °C. After three washes in PBS,
cells were incubated with FITC-conjugated goat anti-rabbit IgG (Sigma)
for 30 min at 4 °C. After washing three times in PBS, pH 7.4, cells
were then incubated for 1 h at 4 °C with anti-Lck monoclonal
antibody, followed by 3 washes in PBS and the addition (30 min at
4 °C) of goat anti-mouse IgG (
-chain-specific) conjugated with
Texas Red (Calbiochem Biochem). Cells were finally washed three times
in PBS and then mounted upside down onto a glass slide in 5 ml of
glycerol/Tris-HCl, pH 9.2. The coverslips were sealed with nail varnish
to prevent evaporation and stored at 4 °C before imaging. The images
were acquired through a confocal laser scanning microscope (Sarastro
2000, Molecular Dynamics) equipped with a NIKON OPTIPHOT microscope
(objective 60/1.4 oil) and an Argon Ion Laser (25 mW output).
Simultaneously, the green (FITC) and the red (Texas Red, which reduces
greatly overlapping) fluorophores were excited at 488 and 518 nm.
Acquisition of single FITC-stained samples in dual fluorescence
scanning configuration did not show contribution of green signal in
red. Images were collected at 512 × 512 pixels (0.08 µm/pixel
lateral dimension, 0.48 µm/pixel axial dimension). Serial optical
sections were assembled in Depth-Coding (Molecular Dynamics) mode.
Acquisition and processing were carried out using Image Space software
(Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization and enrichment of CD4 in
LDTI of resting lymphocytes. a, a total membrane
fraction obtained from 1 × 109 surface biotinylated
lymphocytes was homogenized in a buffer containing: 0, 0.02, 0.1, or
1% Triton X-100 as indicated and subjected to sucrose gradient density
centrifugation. An aliquot of each fraction (10 µl) was resolved by
SDS-PAGE and transferred to nitrocellulose. Fraction 1 corresponds to
the top of the gradient. Biotinylated proteins were visualized by
blotting with streptavidin-horseradish peroxidase. Particulate material
from each fraction was also collected by centrifugation, resuspend in
30 µl, and analyzed by SDS-PAGE/Western blotting using a monoclonal
antibody direct against CD4. Inset, 3 µg of fraction 5 obtained from 1% Triton gradient was immunoprecipitated with 0.5 µg
of anti-CD4 IgG and subjected to SDS-PAGE and streptavidin-horseradish
peroxidase blotting. For comparison, 10 µl from fraction 5 of the
same gradient was analyzed as in parallel. b, 1 × 109 lymphocytes were used to prepare a particulate total
membrane fraction, homogenized in a buffer containing 1% Triton X-100,
and subjected to sucrose density gradient centrifugation. Twelve
fractions of equal volume were collected, and their protein content was
determined. The protein profile is expressed as a percentage of the
total particulate loaded (2 mg). The distribution of CD4 was obtained
by densitometric analysis of CD4 immunoblots. c and
d, CD4 analysis in cell fractionation representing
sequential steps of CD4 enrichment, starting from total cell lysate.
T, total cell lysate; M, total membrane fraction
(or particulate). c, values were derived from densitometry
of band intensity relative to the total cell lysate (total lysate
value = 1). CD4 fold enrichment was determined by the amounts used
for normalized CD4 expression/relative fraction protein content (which
is expressed as a percentage of the total protein). M and
LDTI represent 15 and 0.2% of total protein, respectively.
d, same CD4 band intensity was obtained with 50 µg of
total lysate, 5 µg of total membranes, and 1 µg of the LDTI
microdomains. A representative blot is shown.
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Fig. 2.
CD4 labeling is associated within
Triton-insoluble plasma membrane microdomains in
vivo. CD4+ lymphocytes were prefixed,
treated with 1% Triton X-100 for 30 min at 4 °C, and processed for
immunogold labeling to detect CD4. a, a large portion of the
plasma membrane (indicated by arrowheads) appeared resistant
to Triton X-100 treatment. b, immunogold labeling of CD4
after Triton X-100 treatment was selectively associated with
detergent-insoluble plasma membrane portions. Gold particles were
localized over the nonvillous portion of the retained membrane.
c and d, the distribution of immunogold-labeled
biotin corresponded to areas of detergent-resistant membrane domains.
e, native distribution of CD4 molecules on lymphocytes where
Triton treatment was omitted; gold particles were preferentially
localized over the microvilli. a and b,
200,000×; c and d, 180,000×; e,
50,000×.
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Fig. 3.
Kinetics of PMA-induced CD4 down-regulation
at the plasma membrane and within LDTI microdomains. Lymphocytes
(1 × 109) were cell surface labeled with biotin and
incubated with 100 ng/ml PMA for 0, 5, 30, and 60 min at 37 °C.
Total particulate plasma membrane fractions (M) were then
prepared and either analyzed directly or used to purify LDTI
microdomains. a, CD4 was monitored by immunoblotting. LDTI
(0.5 µg) and M (10 µg) were separated on 8% SDS-PAGE and
transferred to nitrocellulose. b, cell surface CD4 was
detected by immunoprecipitation with antibodies directed against CD4
and streptavidin blotting. LDTI (3 µg) and M (2 µg) were
immunoprecipitated with 1 µg/ml anti-CD4 IgG. A representative blot
is shown for each condition. For Lck detection, LDTI (2 µg) were
analyzed by 10% SDS-PAGE and immunoblotted with anti-Lck. LDTI (5 µg) were immunoprecipitated with anti-CD4 and assayed for Lck by
immunoblotting with anti-Lck antibody. FACS analysis was also performed
as an independent measure of CD4 cell surface expression. For the
graphs in panels a and b, the results shown are
the means ± S.E. of three separate experiments. c, LDTI (1 µg) from untreated
or 5 min PMA-treated cells were separated on a 8% SDS-PAGE,
transferred to nitrocellulose and blotted with anti-Fyn or
anti-Gi antibodies. d, total particulate
membrane fraction from 1 × 109 untreated or 5 min
PMA-treated lymphocytes were homogenized in 1% Triton X-100 and
subjected to sucrose density gradient centrifugation. 0.5 µg/fraction
was then analyzed by CD4 immunoblotting.
,
1,
2,
,
,
,
and
) shifted, although in variable amounts, from Triton-soluble fractions (8-12) to the LDTI microdomains (fractions 4-6) (Fig. 4a).
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Fig. 4.
PMA induces recruitment of PKC isoforms to
LDTI microdomains. a, lymphocytes were treated with PMA
for 1 h (+) or left untreated ( ) and then used to prepare LDTI
microdomains by sucrose density gradient centrifugation. 5 µg of each
fraction were analyzed by 8% SDS-PAGE and immunoblotted with
antibodies directed against PKC isoforms. Note that PMA induces a
significant recruitment of PKCs to fractions 4-6 that represent LDTI
microdomains. Results shown are representative of three independent
experiments. b, kinetics of the recruitment of PKC isoforms
to LDTI microdomains. Total lymphocytes (1 × 109)
were incubated with 100 ng/ml PMA for 0, 5, 30, or 60 min at 37 °C
and used to prepare LDTI microdomains by sucrose density gradient
centrifugation. LDTI microdomains (2 µg of protein for each
condition) were analyzed by 8% SDS-PAGE and immunoblotted with
antibodies directed against PKC isoforms (
,
, or
).
Alternatively, LDTI microdomains (5 µg for each condition) were
immunoprecipitated with 1 µg of anti-CD4, -PKC
, or -PKC
, and
blotted with the same antibodies. WB, Western blot;
IP, immunoprecipitation; N.D., not done.
and
isoforms) show a complete recruitment of PKC
and
isoforms to LDTI microdomains within 5 min of PMA addition (Fig.
4b).
, which lacks the phorbol ester binding region, was
also recruited to LDTI microdomains, although at later times, reaching
maximum recruitment between 5-30 min (Fig. 4b). In Jurkat
and peripheral blood T cells translocation of PKC
from cytosol to
membrane fraction after 15 min of PMA stimulation has been described
(51, 52). As has been suggested by other laboratories using
Jurkat cells, the delayed recruitment of PKC
is likely due to
an indirect effect of PMA activation (51).
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Fig. 5.
Relative LDTI/particulate ratio of the PKC
isoforms. Total particulate membrane fractions or LDTI
microdomains (purified using the total particulate membrane fraction as
the starting material) were prepared from lymphocytes that were left
untreated (a) or treated with PMA for 30 min (b).
Immunoblots containing 1 µg of protein of either particulate membrane
fractions or LDTI microdomains were quantitated by densitometry and
values expressed as a ratio (LDTI/particulate) for each isoform. Values
are expressed as the means ± S.E. of three independent
experiments.
showed a remarkably high translocation ratio to
LDTI microdomains. Its relative enrichment was 6-32 times greater
compared with the other PKC isoforms analyzed (Fig. 5b).
1,
2,
,
, and
were poorly represented at steady
state (
and
were completely absent) (data not shown), in
agreement with results obtained by others (51, 53, 54). However, most
of these PKC isoforms underwent translocation after PMA-induced
activation, resulting in a noticeable increase of 0.5-4-fold within
total membranes (data not shown).
and
two lesser involved isoforms PKC
and
with Lck in
CD4+ intact cells, by scanning confocal fluorescence
microscopy (Fig. 6a). PKCs
appeared in an uneven and punctate distribution at the plasma membrane
after PMA addition (Fig. 6a, panels 4 and
5), whereas in untreated cells PKC isozymes were mostly
diffuse in the cytoplasm (Fig. 6a, panels 1 and
2). The clustered distribution of the PKCs indicates that
these enzymes translocate mostly to specific microdomains of the plasma
membrane. Membrane distribution of Lck in untreated cells (Fig.
6a, panel 3) was confirmed to be unaffected by
PMA treatment (Fig. 6a, panel 6).
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Fig. 6.
Immunolocalization of PKC isoforms by
scanning confocal microscopy. a, scanning confocal
microscopic analysis of PKC redistribution after PMA treatment of
CD4+ human peripheral blood lymphocytes. Cells were fixed
with acetone/methanol and then labeled with anti-PKC ,
, or
polyclonal antibodies followed by incubation with FITC-conjugated goat
anti-rabbit IgG. After washing three times in PBS, cells were then
incubated with an anti-Lck monoclonal antibody followed by the addition
of goat anti-mouse IgG conjugated with Texas Red. Panel 1,
untreated cell stained with anti-PKC
revealed a diffuse cytoplasmic
immunolabeling. Panel 2, untreated cell stained with
anti-PKC
revealed a diffuse cytoplasmic immunolabeling. Panel
3, untreated cell stained with anti-Lck revealed an uneven
immunolabeling on the plasma membrane. Panel 4, PMA-treated
cells stained with anti-PKC
showed a clustered distribution of
immunolabeling on the plasma membrane. Panel 5, PMA-treated
cells stained with anti-PKC
presented a clustered distribution of
immunolabeling on the plasma membrane. Panel 6, PMA-treated
cells stained with anti-Lck revealed an uneven immunolabeling on the
plasma membrane. Panel 7, dual immunolabeling of anti-PKC
and anti-Lck revealed nearly complete co-localization
yellow-stained portions of the membrane. Panel 8,
dual immunolabeling of anti-PKC
and anti-Lck revealed a few
co-localization yellow areas of the plasmamembrane.
Panel 9, dual immunolabeling of anti-PKC
and anti-Lck
revealed a few co-localization yellow areas of the plasma
membrane. b, after PMA treatment, the possible association
of PKC
and GM3 on the plasma membrane of CD4+ human
peripheral blood lymphocytes was analyzed by scanning confocal
microscopy. Cells were fixed with acetone/methanol and then labeled
with anti-PKC
, followed by incubation with FITC-conjugated goat
anti-rabbit IgG. After washing three times in PBS, cells were then
incubated with anti-GM3 monoclonal antibody (gmr6) followed by the
addition of goat anti-mouse IgM conjugated with Texas Red. Panel
1, PMA-treated cells stained with anti-PKC
. Panel 2,
PMA-treated cells stained with anti-GM3 monoclonal antibody (gmr6).
Panel 3, dual immunolabeling of anti-PKC
and anti-GM3
revealed nearly complete co-localization, as indicated by the
yellow portions of the plasma membrane. Magnification,
1000×; bar, 1 µm.
revealed nearly complete
co-localization of the two proteins at the plasma membrane, appearing
as yellow-stained membrane microdomains (Fig. 6a,
panel 7). This finding indicates that after treatment with
PMA a large fraction of translocated PKC
effectively co-localized
and was associated with Lck. In striking contrast, only a few areas of co-localization were evident with Lck and PKC
or Lck and PKC
in
PMA-treated lymphocytes (Fig. 6a, panels 8 and
9).
became associated with monosialoganglioside GM3, which
represents the main ganglioside constituent of human peripheral blood
lymphocytes (55) and is selectively recovered in LDTI microdomains
of cell plasma membrane (4).
and
anti-GM3 double labeling revealed yellow areas,
corresponding to nearly complete co-localization, indicating that
PKC
molecules were localized in membrane microdomains enriched in
GM3.
to discrete microdomains of the
plasma membrane where glycosphingolipids and Lck molecules are highly enriched.
, we
examined the in vitro kinase activity associated with LDTI
microdomains before and after stimulation of intact cells with PMA for
1 h. Our results indicate that the amount of kinase activity
associated with LDTI domains was increased, as assessed by the
increased phosphorylation of endogenous substrates (Fig.
7a) or an exogenously added
substrate (Histone III) (Fig. 7b).
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Fig. 7.
PMA treatment up-regulates the kinase
activity associated with LDTI microdomains. a, LDTI
microdomains were obtained from lymphocytes treated with PMA for 1 h or left untreated. These LDTI microdomains (1 µg for each
condition) were then preincubated with a PKC inhibitor (1 µM Gö 6976 for 15 min at 37 °C) and assayed for
in vitro kinase activity. b, as in panel
a, except an exogenous PKC substrate (Histone III) was added.
LDTI domains were incubated with 5 µg of histone and 5 µCi of
[
-32P[ATP as described under "Experimental
Procedures." In the first lane, as positive control only
exogenous PKC
was incubated with Histone and
[
-32P]ATP.
,
1,
2, and
) (56). Treatment of LDTI
domains with Gö 6976 (10
6 M for 1 h at 37 °C) blocked the enhanced proteins phosphorylation (Fig.
7a). These results support the hypothesis that enhanced kinase activity associated with LDTI domains is due to PKC translocation.
in down-modulation of
CD4, we incubated CD4+ cells with PKC inhibitors (Gö
6850 or Gö 6976), followed by PMA stimulation. We then examined
CD4 expression in intact cells by FACS analysis (Fig.
8a) and within LDTI
microdomains by immunoprecipitation of surface-labeled cells (Fig.
8b).
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Fig. 8.
PKC inhibitors block the down-modulation of
CD4 in vivo. a, CD4+
lymphocytes were preincubated with PKC inhibitors (1 µM
Gö 6850 or 1 µM Gö 6976) for 15 min at
37 °C prior to treatment with PMA (100 ng/ml) for 1 h at
37 °C. Cells were then labeled with fluorescein-conjugated anti-CD4
and assayed for CD4 cell surface expression by FACS analysis. Virtually
identical results were obtained with peripheral blood lymphocytes (data
not shown). b, lymphocytes were surfaced labeled with biotin
and treated as described in panel a. These cells were then
used to prepare LDTI microdomains. These LDTI microdomains (2 µg for
each condition) were immunoprecipitated with 0.5 µg/ml of anti-CD4.
Immunoprecipitates were separated by 8% SDS-PAGE and analyzed by
streptavidin-horseradish peroxidase blotting to detect the cell surface
pool of CD4.
, and in to lesser extent of PKC
, in this process of CD4 internalization.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
1,
2,
,
,
,
, and
) known to be expressed in T cells (64).
is the most abundant isoform
recovered within LDTI microdomains upon PMA treatment (5 min to 1 h) This specificity was independently confirmed by (i) in
vivo inhibition of CD4 down-modulation by a PKC
selective inhibitor (Gö 6976); (ii) a series of in vitro
phosphorylation experiments using purified LDTI microdomains; and (iii)
immunofluorescence data showing that PKC
, GM3, and Lck share
significant co-localization at the level of the plasma membrane after
PMA treatment.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Maria Giammatteo (Centro Interdipartimentale di Microscopia Elettronica Università di L'Aquila) for precious help on image acquisition with confocal microscope. We thank Dr. T. Costa for the helpful discussion, M. Teragnoli for graphics, and D. Marinelli and A. Rocca for editorial help.
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FOOTNOTES |
---|
* 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.
¶ Supported by a grant from Consiglio Nazionale delle Ricerche, Italy.
** Supported by grants from Ministero dell'Università e della Ricerca Scientifica (ex 60% e ex 40%).
§§ Supported by National Institutes of Health Grant R01-CA-80250 from the National Cancer Institute and by grants from the Charles E. Culpeper Foundation, the G. Harold and Leila Y. Mathers Charitable Foundation, and the Sidney Kimmel Foundation for Cancer Research.
|| To whom correspondence should be addressed. Tel.: 11-39-06-4990-2633; Fax: 11-39-06-4938-7086; E-mail: m.sargiacomo{at}ema.net.iss.it.
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ABBREVIATIONS |
---|
The abbreviations used are: HIV, human immunodeficiency virus; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; LDTI, low density Triton-insoluble; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; FITC, fluorescein isothiocyanate.
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REFERENCES |
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