From the Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, March 9, 2001, and in revised form, April 25, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Translocation of conventional protein
kinases C (PKCs) to the plasma membrane leads to their specific
association with transmembrane-4 superfamily (TM4SF; tetraspanin)
proteins (CD9, CD53, CD81, CD82, and CD151), as demonstrated by
reciprocal co-immunoprecipitation and covalent cross-linking
experiments. Although formation and maintenance of TM4SF-PKC
complexes are not dependent on integrins, TM4SF proteins can act as
linker molecules, recruiting PKC into proximity with specific
integrins. Previous studies showed that the extracellular large loop of
TM4SF proteins determines integrin associations. In contrast,
specificity for PKC association probably resides within cytoplasmic
tails or the first two transmembrane domains of TM4SF proteins, as seen
from studies with chimeric CD9 molecules. Consistent with a TM4SF
linker function, only those integrins
( Integrin-dependent cell adhesion, through integration
of cell signaling pathways and cytoskeletal reorganization, markedly influences cell growth, death, and differentiation (1-3). Signaling through many different integrins causes similar calcium fluxes, pH
changes, and activation of focal adhesion kinase. However, specific integrins may also differ markedly from each other in support
of cell cycle progression, cell survival, or gene induction (4-6).
Consistent with signaling differences, different integrin cytoplasmic
domains may interact with a number of specific integrin-associated proteins (7).
Integrin signaling may not only involve cytoplasmic domain associations
but also may utilize lateral interactions through integrin
transmembrane and ectodomains (4, 8). In this regard, some integrins
(e.g. The PKC family of phospholipid-dependent serine and
threonine kinases participates in a wide spectrum of biological
activities (18-20). Activation of cytosolic PKC by phorbol ester or
diacylglycerol occurs in parallel with PKC translocation to cellular
membranes. Membrane association is largely attributed to specific PKC
interactions with phosphatidylserine. Various PKC isoforms also
associate with a number of specific binding proteins (20). However,
aside from PKC interaction with the transmembrane proteoglycan
syndecan-4 (21), a role for specific transmembrane proteins during PKC translocation has not previously been suggested.
Conventional PKC isoforms participate in the "inside-out"
activation of cell adhesion mediated by Here we demonstrate that upon activation and translocation,
conventional PKCs associate closely with several different
TM4SF/tetraspanin proteins. Upon PKC activation, those integrins
( Antibodies--
Anti-integrin antibodies used were as follows:
anti- Immunoprecipitation and Reimmunoprecipitation--
Cells were
surface-labeled with Na125I (PerkinElmer Life
Sciences) using lactoperoxidase by an established protocol,
or cells were 32P-labeled by growth in sodium phosphate
deficient medium supplemented with
[32P]orthophosphate (PerkinElmer Life Sciences) for 3-6
h. In all experiments involving PMA stimulation, cells were treated
with 100 nM PMA for 20-30 min at 37 °C prior to lysis.
Cells were lysed in immunoprecipitation buffer (1% Brij 96 or 1% Brij
99, 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, 20 mg/ml aprotinin, and 10 mg/ml leupeptin) for 1 h at
4 °C. For 32P-labeled cells, immunoprecipitation buffer
was supplemented with phosphatase inhibitors (1 mM sodium
orthovanadate, 1 mM NaF, and 10 mM
Western Blot Analysis--
For Western blot analysis,
immunoprecipitated samples were subjected to SDS-PAGE under reducing
conditions and then electrophoretically transferred to nitrocellulose
membrane. After blocking with 5% nonfat milk in PBS-Tween 20 buffer at
room temperature for 1 h, nitrocellulose membranes were
sequentially blotted at room temperature for 1 h with specific
antibody and then horseradish peroxidase-conjugated goat anti-mouse
IgG. Each step was followed by four 15-min washes with PBS-Tween
20 buffer. Membranes were then developed using chemiluminescence
(Renaissance; PerkinElmer Life Sciences).
Immunofluorescence--
Circular glass coverslips (12 mm;
Fisher) were coated with fibronectin (10 µg/ml) in 0.1 M
NaHCO3 at 4 °C overnight. HT1080 cells were harvested in
PBS with 2 mM EDTA, washed once in serum-free Dulbecco's
minimal essential medium, and plated on coverslips for 1-2 h at
37 °C. Some HT1080 cells were treated with 100 nM PMA
for the last 20 min prior to rinsing in PBS and fixing in PBS
containing 3% paraformaldehyde for 10 min. Permeabilization was with
0.05% Triton X-100 in PBS for 2 min at room temperature. Nonspecific
binding sites were blocked with 20% goat serum in PBS for 1 h at
room temperature. Primary mAbs (~1 µg/ml final concentration) were
diluted in 20% goat serum/PBS and incubated with cells for 1 h at
room temperature. Coverslips were washed four times with PBS and then
incubated for 30 min with rhodamine-conjugated secondary antibodies.
Finally, coverslips were washed four times with PBS, mounted on glass
slides in FluroSave reagent (Calbiochem), and analyzed using an
Axioskop fluorescent microscope (Zeiss).
Cells, Transfectants, and Mutants--
HT1080 fibrosarcoma,
Jurkat T leukemia, and K562 erythroleukemia cells were cultured in RPMI
medium containing 10% fetal bovine serum. K562 cells transfected with
mutant and wild type integrin
Chimeric integrin TM4SF proteins were produced by the overlapping
oligonucleotide polymerase chain reaction technique. In the reciprocal
CD9-il.A15 and A15-il.CD9 chimeras, the intracellular loops from CD9
(QESQC) and A15 (RGSPW) were swapped. The A15-lel34.CD9 chimera was
produced by replacing, in A15, the large extracellular loop and
flanking TM3 and TM4 domains with the corresponding region from CD9.
The sequence (with CD9 underlined) becomes ...
GSPWM/LGLFF ... MILCC/FITAN ... The entire
polymerase chain reaction regions of the chimeric constructs were
sequenced to confirm fidelity. Chimeric cDNA was subsequently
cloned into the expression plasmid pCR3.1-uni (Invitrogen, Carlsbad,
CA) and stably transfected into K562 cells via electroporation at 960 microfarads and 280 V using a gene pulser. Transfectants were selected
with 1 mg/ml G418 (Life Technologies, Inc.) and subcloned by limiting
dilution. Positive subclones stably expressing chimeric integrin
subunits were assessed, pooled, and sorted by flow cytometry, using
monoclonal antibodies specific for the large extracellular loops.
PKC Forms Complexes with Specific Tetraspanin Proteins--
During
our studies of tetraspanin protein association with intracellular PI
4-K (16, 17), we noticed that another enzyme, PKC, showed an even
stronger tetraspanin association. From a series of K562 erythroleukemia
cell transfectants, the tetraspanin protein CD81 was
immunoprecipitated, and then PKC
To confirm results in Fig. 1B, PKC
Because PKC and tetraspanins may both associate with integrins, we
considered that TM4SF-PKC associations are mediated through integrins.
In 1% Brij 96 conditions, CD81 in K562 cells associates readily with
Another means of inducing PKC activation is through second antibody
cross-linking of the CD3-T cell receptor complex on the surface of T
cells (47). Antibody/second antibody-induced cross-linking of the CD3-T
cell receptor on 125I-surface-labeled Jurkat T cells
triggered association of PKC
From human peripheral blood mononuclear cells stimulated with either
PMA or anti-CD3 antibody cross-linking, PKC TM4SF-PKC Interactions Are Stabilized by Covalent
Cross-linking--
To characterize TM4SF-PKC interactions further,
intact K562 cells were treated with dithiobis(succinimidyl propionate)
(DSP), a homobifunctional cross-linking agent with a span of 12 Å.
Without cross-linker, and under relatively mild detergent conditions, immunoprecipitation of CD81 from PMA-stimulated K562 cells yielded associated PKC PKC Specificity--
Conventional PKCs, including PKC TM4SF Specificity--
As seen in Fig. 1, PKC PKC Isoforms Are Induced to Form Complexes with Specific
Integrins--
Because TM4SF proteins associate with specific
integrins (see Introduction) and with activated PKC, we predicted that
TM4SF proteins may link activated PKC to integrins. In this regard, antibodies to either CD81 (from HT1080 cells) or PKC
To confirm co-immunoprecipitation of specific integrins with PKCs, we
analyzed another conventional isoform (PKC
Whereas Figs. 6 and 7A show integrins co-precipitated with
PKC
As seen by immunofluorescent staining of HT1080 cells spread on
fibronectin (Fig. 8), localization of
integrin Specificity for Intracellular PKC Determined by Integrin
Extracellular Domain--
Specificity for TM4SF-integrin association
resides within the ectodomains of both TM4SF proteins and integrins
(42). Thus, if PKC is linked to integrins via a TM4SF linker protein,
an extracellular integrin site should be needed for
recruitment of intracellular PKC. In this regard, deletion
of the cytoplasmic tail of Potential Relevance of PKC Recruitment to TM4SF-PKC Association--
Activation and translocation of PKC
promoted a relatively robust association of conventional PKC isoforms
with multiple TM4SF proteins. TM4SF-PKC complexes were seen in multiple
cell lines (including both adherent and nonadherent cells), were seen
for several different tetraspanins (CD9, CD53, CD81, CD82, and CD151), and were promoted by multiple PKC activating stimuli (phorbol ester,
bryostatin 1, or CD3-T cell receptor triggering). Analysis of TM4SF-PKC
complexes in Jurkat T cells suggests a reasonable stoichiometry.
Although each TM4SF protein might only associate with ~3-5% of the
total PKC, the presence of about five or more different TM4SF proteins
in a given cell type would engage a substantially greater fraction of
the total PKC. Four kinds of biochemical evidence support the presence
of TM4SF-PKC complexes. (i) Immunoprecipitation of TM4SF proteins
yielded PKC; (ii) immunoprecipitation of PKC yielded multiple TM4SF
proteins; (iii) immunoprecipitation of either TM4SF protein
(i.e. CD81) or PKC yielded essentially identical protein
complexes (e.g. Fig. 2); and (iv) TM4SF-PKC complexes were
stabilized by covalent cross-linking. The covalent cross-linking results provide perhaps the most compelling evidence for TM4SF-PKC association. The span of the cross-linking agent (12 Å) is such that
only highly proximal interactions would be captured. Furthermore, the
membrane-permeable cross-linking agent was added to intact cells and
thus captured the native complexes before exposure to any detergents.
Finally, the cross-linked complexes were solubilized using relatively
stringent detergent conditions, such that uncross-linked proteins are
largely removed.
The occurrence of TM4SF-PKC complexes was specific, with respect to
both TM4SF proteins and PKC. Considering the tendency of TM4SF proteins
to associate with each other, it was reassuring to find that at least
one prominently expressed TM4SF protein (A15/Talla1) did not associate
with PKC in Jurkat cells. Among PKC isozymes, conventional PKCs
Thus far, nearly all tetraspanin sites tested have mapped to the large
extracellular loop (42, 50-52). The crystal structure of CD81
indicates that the large extracellular loop is also involved in protein
multimerization (55). Thus, if PKC were being recruited indirectly, by
means of TM4SF protein interactions with each other or with other
surface proteins, then the large extracellular loop might be required.
However, our CD9/A15 chimeras allowed us to map the PKC interaction
site away from the large extracellular loop. This provides further
evidence in support of a more direct interaction of TM4SF proteins with
intracellular PKC. The intracellular large loop and transmembrane
domains 3 and 4 were also ruled out, thus leaving the cytoplasmic tails
and transmembrane domains 1 and 2 as remaining candidate sites for
determining PKC specificity.
Compared with tetraspanin interactions with other intracellular
signaling proteins (PI 4-kinase, phosphatase, and GTP binding proteins)
the PKC interactions described here are quite distinct and perhaps more
robust. Those other interactions have not been demonstrated using
covalent cross-linking. Furthermore, tetraspanin association with PI
4-K was observed, not in Brij 96, but in the less stringent Brij 99 conditions. Also, whereas tetraspanin-PKC associations are induced,
association with PI 4-K is constitutive (8, 16). Finally, PI 4-K
associates well with TM4SF protein A15 but not with CD82 and CD53 (17),
whereas PKC associates well with CD82 and CD53, but not with A15. Thus,
TM4SF proteins may have distinct sites for recruitment of these two key
signaling enzymes (PI 4-K and PKC). The CD53 and CD63 tetraspanins may
associate with a low level of an unidentified phosphatase activity
(56). Again, this appears to be distinct from PKC association, since CD63, compared with other tetraspanins, showed relatively little PKC
association. Elsewhere, association of CD9 with unidentified GTP-binding proteins was demonstrated (57), but due to the rather nonstringent conditions utilized, this association is probably part of
a large complex.
Tetraspanins Link PKC to Integrins (PKC-TM4SF-Integrin
Model)--
Reciprocal co-immunoprecipitation experiments showed that
specific integrins (
An alternative model would involve integrins providing a link between
PKC and tetraspanins (PKC-integrin-TM4SF model). However, this model
does not explain how integrin association with PKC, an intracellular
enzyme, would be specified by the extracellular domain of the integrin
Do Other Proteins Contribute to the Linking of PKC to
Integrins?--
Because integrins, PKC
In 1% Brij 96 lysate conditions, the majority of tetraspanin complexes
appeared in the dense fractions of sucrose gradients, indicating that
associations with other proteins (including
PKC may interact with a number of substrate proteins (20) and other
intracellular proteins termed receptors for activated C kinase (RACKs)
(61). However, none of these are transmembrane proteins. A protein
associating with PKC Functional Role of PKC-TM4SF-integrin Complexes--
The
The formation of integrin-TM4SF-PKC complexes may allow PKC
localization to be closely coordinated with cell adhesion involving particular integrins. Thereby PKC may become optimally positioned to
regulate a host of downstream events involving cytoskeletal organization and signaling. For example, PKC regulates the interaction of cellular membranes with several cytoskeletal proteins (including the
myristoylated alanine-rich C kinase substrate protein) that are also
PKC substrates (20, 64). Notably, the myristoylated alanine-rich C
kinase substrate protein also colocalizes with TM4SF proteins at the
periphery of spread cells (63). Integrin-TM4SF-PKC complexes may be
particularly important during cell migration, since TM4SF proteins
(13-15), TM4SF-integrin complexes (8, 65), PKC (66), and PKC-integrin
complexes (28) have each been linked to cell migration.
The role of PKC in the context of integrin-TM4SF-PKC complexes is
distinct from previously described PKC-dependent modulation of integrin adhesion (23, 24), cell spreading (26), and focal adhesion
formation (67). For example, some integrins (e.g.
In conclusion, studies of integrin signaling can now be expanded to
include not only integrin cytoplasmic domains but also membrane-proximal 3
1,
6
1, and a chimeric "X3TC5"
3 mutant) that associated strongly with tetraspanins
were found in association with PKC. We propose that PKC-TM4SF-integrin
structures represent a novel type of signaling complex. The
simultaneous binding of TM4SF proteins to the extracellular domains of
the integrin
3 subunit and to intracellular PKC helps to
explain why the integrin
3 extracellular domain is needed for both
intracellular PKC recruitment and PKC-dependent
phosphorylation of the
3 integrin cytoplasmic tail.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
1,
4
1,
6
1, and
IIb
3) interact specifically with various
cell surface
TM4SF1/tetraspanin proteins
(9-12). The TM4SF proteins share 20-30% sequence similarity, and
contain four highly conserved transmembrane domains, flanked by short N
and C termini. The TM4SF proteins, including CD9, CD53, CD63, CD81, and
CD82, may regulate cell signaling, motility, and tumor cell metastasis
(13-15). TM4SF proteins tend to assemble into protein complexes at the
plasma membrane (14, 15), where they may recruit other molecules (such
as growth factor ligands and phosphatidylinositol 4-kinase) into
proximity with integrins (11, 16, 17). As shown here, TM4SF proteins may also link specific integrins to protein kinase C (PKC).
1,
2, and
3 integrins (22-24). PKC not only
appears in focal adhesion complexes, as seen in well spread cells (25),
but also is required for cell spreading (26, 27). In addition, PKC
may associate with
1 integrins and regulate their
trafficking (28). A subset of integrins
(
3
1,
6 integrins) becomes
phosphorylated in a PKC-dependent manner (29-32).
Phosphorylation of the
3 integrin may regulate cell
signaling, morphology, migration, and cytoskeletal organization
(31).
3
1,
6 integrins) already
constitutively associated with TM4SF proteins then become linked to
PKC. Within these PKC-TM4SF-integrin complexes, integrin
3 and
6 tails are phosphorylated in a
PKC-dependent manner. The presence of TM4SF linker proteins
helps to explain how association of intracellular PKC may be determined
by integrin extracellular domains.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2, A2-IIE10 (33); anti-
3, A3-X8,
A3-IVA5, and A3-IIF5 (34); anti-
4, A4-B5G10 (35) and
A4-PUJ1 (36); anti-
5, A5-PUJ2 (36);
anti-
6, A6-ELE (37); anti-
1, A-1A5 (38)
and TS2/16 (39). Anti-TM4SF mAbs used were as follows: anti-CD9,
DU-ALL-1 (Sigma); anti-CD53, HD77 (40); anti-CD81, M38 (41); anti-CD82,
M104 (41); anti-CD151, 5C11 (42); and anti-A15/TALLA1, B2D (43). mAbs
to PKC
, PKC
, and phosphatidylinositol 3-kinase (PI 3-K) were
obtained from Transduction Laboratories (Lexington, KY). Control rabbit
IgG and polyclonal antibody to PKC
II were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA), and anti-PLC
mAb was from
Upstate Biotechnology, Inc. (Lake Placid, NY). Other mAbs were as
follows: anti-CD3, OKT3 (American Type Cell Culture, Manassas, VA);
anti-CD28, 4B10 (Dr. C. Rudd, Dana-Farber Cancer Institute); anti-CD71,
DF1513 (Sigma); anti-CD98,
6B122; anti-MHC class I,
W6/32. Rhodamine-conjugated goat anti-mouse secondary antibodies (for
immunofluorescence staining) were from BIOSOURCE
(Camarillo, CA). Goat anti-mouse IgG antibody (for cell surface
antibody cross-linking) was from Roche Molecular Biochemicals. For most
experiments involving reimmunoprecipitation or Western blotting, mAbs
used for initial immunoprecipitation were covalently conjugated to
CNBr-activated Sepharose 4B beads.
-glycerophosphate). Immunoprecipitations and reimmunoprecipitations were then carried out as described (10, 44). Immune complexes collected
on beads were then washed three times with immunoprecipitation buffer
and analyzed by SDS-PAGE under nonreducing conditions, and radiolabeled
proteins were visualized by autoradiography.
2,
3,
4, and
6 subunits were prepared as
previously described (10, 45). Mutant subunits include X3C0, in which
the
3 cytoplasmic tail is deleted; X3TC5, in which the
transmembrane portion and cytoplasmic tail of
3 are
replaced by those of
5; and X2C3, in which the
2 integrin cytoplasmic tail is replaced by that of
3 (46). Transfected integrins were expressed at
comparable levels (i.e. varied by less than a factor of 2)
on the surface of K562 cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II (a conventional PKC isoform) was
detected by immunoblotting, provided that that the K562 cells had been
activated with PMA (Fig. 1A,
compare upper and lower panels).
Immunoprecipitates of CD98 (right lanes) yielded no associated PKC. To expand these results, we next immunoprecipitated multiple tetraspanin proteins from Jurkat T cells, and immunoblotted to
detect PKC
(another conventional PKC isoform). As indicated, the
TM4SF proteins CD9, CD81, and CD82 each showed a clear association with
PKC
(Fig. 1B, upper panel). In
contrast, PKC
was not present in immunoprecipitates of A15 (another
TM4SF protein), CD71 (transferrin receptor), CD98 (another prominent
transmembrane protein), or MHC class 1. Cell surface levels of A15,
CD71, CD98, and MHC class 1 were each greater than that of CD9, and
were comparable with CD81 and CD82 (not shown). Association of PKC
with TM4SF proteins was not observed unless Jurkat cells were
pretreated with PMA (Fig. 1B, upper
panel). PMA had no effect on the levels of TM4SF proteins
(CD9, CD81, CD82, and A15) or control proteins (CD71, CD98, and MHC
class I) directly immunoprecipitated using appropriate mAbs (not
shown). The TM4SF protein CD151 also co-precipitated with PKC (see
below). Compared with total cell lysate samples in Fig. 1, A
and B, comparable levels of PKC were detected in CD9 and
CD81 immunoprecipitates derived from 20-30-fold more cell equivalents.
Thus, ~3-5% of the total PKC may be associated with each of these
TM4SF proteins.
View larger version (40K):
[in a new window]
Fig. 1.
PKC association with TM4SF proteins.
A, K562 transfectants treated with or without PMA were lysed
in 1% Brij 96, and then immunoprecipitates of CD81 (a representative
TM4SF protein) and CD98 mAb (negative control) were prepared. After
SDS-PAGE and electrophoretic transfer, PKC II was detected by
immunoblotting. For integrin expression in the various K562
transfectants, see Fig. 7A. B, Jurkat cells
treated with or without PMA were lysed in 1% Brij 99 and then
immunoprecipitated (IP) with mAb to TM4 proteins (CD9, CD81,
CD82, and A15) or other cell surface molecules (CD71, CD98, and MHC-1).
After SDS-PAGE and electrophoretic transfer, whole cell lysate and
immunoprecipitated proteins were blotted using anti-PKC
mAb.
C, PMA-treated, 125I-labeled Jurkat cells were
lysed in 1% Brij 99. PKC
(lanes a-e) and PI
3-K (lanes f-j) complexes were
immunoprecipitated, dissociated, and then reprecipitated with
antibodies to TM4SF proteins (CD9, CD53, CD81, and CD82) or to MHC
class I as indicated. Also, MHC-1 was directly immunoprecipitated
(lane k). Note that mAb to PKC and PI 3-K yielded
comparable amounts of appropriate target proteins from metabolically
labeled lysate (not shown).
was immunoprecipitated
from a lysate of Jurkat cells that had been PMA-treated and
125I-labeled. Reimmunoprecipitations were then carried out,
showing that CD9, CD53, CD81, and CD82 were associated with PKC
(Fig. 1A, lanes a-d). In control
experiments, a nontetraspanin protein (MHC-1) was not recovered from
PKC
immunoprecipitates (lane e) although it
could be directly immunoprecipitated (lane k).
Furthermore, no tetraspanins or other proteins were recovered from
immunoprecipitations of PI 3-K (lanes f-j) or
PKA (not shown).
3 and
6 integrins but hardly at all with
2,
4, or
5 integrins (12).
However, native K562 cells (containing only endogenous
5
1), and transfected cells expressing
abundant levels of
2,
3,
4, or
6 integrins showed minimal
differences in the levels of CD81-associated PKC
II (Fig.
1A). Thus, CD81-PKC
II association appears to be
independent of CD81-
3
1 or
CD81-
6
1 complex formation. Likewise,
TM4SF-PKC
complexes could be isolated from Jurkat cells under
detergent conditions (1% Brij 96) in which no TM4SF-integrin complexes
were present (not shown).
with CD81 and a few other unknown cell
surface proteins (Fig. 2, lane
b). In contrast, antibody/second antibody cross-linking of
MHC class I, CD28, CD81, and CD98 (all abundant on Jurkat T cells)
failed to stimulate PKC
association with CD81 protein complexes
(lanes a and c-e). In another control
experiment, CD81 and associated proteins were not associated with PKA
following CD3 antibody cross-linking (lane f).
The identity of CD81 was verified in Fig. 2B. After antibody
cross-linking of CD3 on Jurkat cells, a PKC
immunoprecipitation was
carried out, and from the resulting protein complex, CD81 could be
reimmunoprecipitated (Fig. 2B, lane
i). In contrast,
5
1 integrin,
CD98, and MHC-1 proteins could not be reimmunoprecipitated
(lanes h, j, and k). Likewise, CD81 could not be reimmunoprecipitated from a PI 3-K immunoprecipitate after CD3 antibody cross-linking of Jurkat cells (lane l). A direct immunoprecipitation of CD81
and associated proteins is shown in lane g. The
pattern of surface-labeled proteins looks remarkably similar to that
immunoprecipitated with an anti-PKC
antibody (lane
b), thus providing further evidence for the presence of
PKC
in CD81 complexes.
View larger version (72K):
[in a new window]
Fig. 2.
Stimulation of CD3 promotes PKC-TM4SF protein
complex formation. A, 125I-labeled Jurkat
cells were incubated for 1 h with mAb to the indicated cell
surface proteins, and then a secondary rabbit anti-mouse antibody was
added to cross-link the primary mAb. Following cell lysis (in 1% Brij
99), PKCa (lanes a-e) or PKA (lane
f) was immunoprecipitated (I.P.), and proteins
were resolved by SDS-PAGE and then visualized by autoradiography.
B, 125I-labeled Jurkat cells were incubated for
1 h with mAb to CD3, and then second antibody cross-linking was
carried out as in A. Following cell lysis (in 1% Brij 99),
PKCa (lanes h-k) or PI 3-K (lane
l) was immunoprecipitated. Complexes were then dissociated,
and reprecipitations were carried out as indicated. In a control
experiment, CD81 was directly immunoprecipitated (lane
g) from cells untreated with antibodies.
again formed complexes
with TM4SF proteins (not shown). Another agent that stimulates PKC
translocation (bryostatin 1 (48)) also induced association of PKC with
TM4SF proteins (not shown). In other experiments, PKC inhibitors
(chelerythrine D, Go6976, calphostin C, and staurosporine) did not
inhibit PMA-induced association of PKC with TM4SF proteins (data not
shown). Thus, although PKC translocation is required for TM4SF protein
association, PKC activity is not required.
II that was readily visualized by Western blotting (Fig. 3A, lane
a), in agreement with results in Fig. 1A. Without cross-linker, association with PKC
II was lost when 0.2% SDS was included in the Brij 96 lysis buffer (Fig. 3A,
lane c). However, after treatment of intact cells
with DSP cross-linker, association with PKC
II persisted despite the
stringent detergent conditions (lane e). The
PKC
II protein was not present in control Ig immunoprecipitations under any conditions (lanes b, d, and
f). In a second experiment (Fig. 3B), another
TM4SF protein (CD151) maintained association with PKC
II under
relatively harsh detergent conditions (1% Brij 96 plus 0.2% SDS) only
when cells were treated with both PMA (to activate PKC) and DSP
cross-linker. Although CD71 (transferrin receptor) was present on the
cell surface at a high level, it did not cross-link to PKC
II
regardless of PMA or DSP treatment.
View larger version (49K):
[in a new window]
Fig. 3.
Biochemical cross-linking of TM4SF-PKC
complexes. A, intact K562 cells were treated with PMA
(100 nM at 37 °C, 20 min) and then (4 °C for 60 min)
with or without 1 mM DSP (a membrane-permeable
cross-linker; Pierce). Cells were next lysed in either 1% Brij 96 or
1% Brij 96 plus 0.2% SDS (as indicated), and then antibody to CD81 or
control Ig (cIg) was used for immunoprecipitation
(I.P.). Samples were then reduced (boiled in 5%
-mercaptoethanol), fractionated by SDS-PAGE, and immunoblotted with
anti-PKC
II antibody. B, intact K562 cells were treated
with or without PMA and with or without DSP as indicated. Cells were
lysed in 1% Brij 96 plus 0.2% SDS, and then CD71 (transferrin
receptor) and CD151 (TM4SF protein) were immunoprecipitated, and
PKC
II was immunoblotted.
and
PKC
II, undergo calpain-dependent proteolytic conversion
to PKM, a form lacking the N-terminal C1 and C2 regulatory domains
(49). The addition of calcium and the omission of the protease
inhibitor leupeptin caused the partial conversion of intact PKC
II in
K562 cells (Fig. 4A, lane a) into two closely migrating fragments of
~50 kDa characteristic of PKM (lane b).
However, CD81 immunoprecipitation from PMA-treated K562 cells yielded
only the intact PKC
II fragment and no PKM (lane
g). No PKM was observed even upon overexposure of
lane g, such that the PKC intensity was
comparable with that in lanes a-d (not shown).
Also, no PKC was co-precipitated with CD81 in the absence of PMA
treatment (lane e) or with control Ig
(lanes f and h). In conclusion, the
PKM form of PKC
II, lacking N-terminal regulatory domains, does not
associate with CD81. In addition, we failed to recover PKC
, PKC
,
or PKCµ from TM4SF immunoprecipitates, despite testing multiple cell
lines and 4-6 different TM4SF proteins for each isozyme (Fig.
4B).
View larger version (29K):
[in a new window]
Fig. 4.
Specificity for TM4SF determined by PKC
regulatory domains. A, K562 cells treated with
(lanes d, g, and h) or
without (lanes a-c, e, and
f) PMA were lysed either in standard lysis buffer
(lane a) or in lysis buffer lacking leupeptin and
supplemented with 5 mM CaCl2 (lanes
b-h). From samples of whole cell lysate or
immunoprecipitates of CD81 or control Ig (cIg) (as
indicated), PKC II was detected by Western blotting. B,
summary of TM4SF association results for PKC isoforms and PKM (from
PKC
II).
interacted with
four different TM4SF proteins (CD9, CD53, CD81, and CD82) but not
another TM4SF protein (A15/Talla1). Showing a similar specificity,
PKC
II in K562 cells interacted again with CD9 but not A15 (Fig.
5). With A15 being negative for PKC
association, an opportunity for a chimeric mapping approach was
provided. So far, nearly all TM4SF associations and functions have
mapped to the large extracellular loop (42, 50-52). However, the
A15-lel34.CD9 chimera (A15 with the large extracellular loop and
flanking transmembrane domains of CD9) failed to interact with PKC
11
in K562 cells (Fig. 5). Thus, the CD9 large extracellular loop and
flanking transmembrane domains are not sufficient for PKC association.
Because the short inner loop of A15 (RGSPW sequence) is quite distinct
from most other known tetraspanins (53), we hypothesized that it could
selectively prevent PKC association. However, replacement of the short
inner loop QESQC of CD9 with the loop RGSPW from A15 (CD9-il.A15) did
not abolish PKC interaction. Conversely, replacement of the A15 RGSPW
with the QESQC from CD9 (A15-il.CD9) did not confer PKC interaction.
For additional mutants, in which N-terminal or C-terminal domains of
CD9 were replaced by corresponding A15 domains, results regarding PKC
association were inconclusive (not shown). At present, we conclude that
TM4SF specificity is determined by regions other than the short inner loop, the large outer loop, or transmembrane 3 or 4.
View larger version (19K):
[in a new window]
Fig. 5.
TM4SF domains needed for PKC
association. K562 cells expressing CD9/A15 chimeras were treated
with PMA, lysed in 1% Brij 96, and immunoprecipitated
(I.P.) using mAb to CD9 (first three
panels) or A15 (last two
panels) or control antibodies (to CD98 or MHC-1). After
SDS-PAGE, proteins were blotted using anti-PKC II antibody. By flow
cytometry, CD9, A15, and chimeras were at comparable levels (210-290
mean fluorescence intensity units), compared with negative
control antibody (30-40 mean fluorescence intensity units).
Direct immunoprecipitation of CD9, A15, and chimeras also yielded
comparable protein levels (not shown).
(from activated HT1080 cells) co-immunoprecipitated similar surface-labeled proteins (Fig. 6A, lanes
b and c) that precisely comigrate with
3
1 integrin (lane
a). Upon longer exposure of lanes b
and c (see lanes k and l),
a small amount of surface-labeled CD81 was also obvious in both CD81
and PKC
immunoprecipitations. Notably, integrin-like proteins were
not obtained using anti-PKA, anti-PLC
, or anti-PI 3-K mAbs
(lanes d-f) and were not seen if HT1080 cells
were not stimulated with PMA (lanes g-j). Upon
long exposure (lane m), immunoprecipitation of
another conventional PKC (PKC
) also yielded integrin-like protein,
whereas control PLC
(lane n) did not. As seen
previously (12), PMA pretreatment was not required for CD81-integrin
association (lanes b and k). Anti-PKC,
anti-PKA, anti-PI 3-K, and anti-PLC
mAbs immunoprecipitated
comparable amounts of appropriate target proteins from
35S-labeled cells (not shown). Considering that the
immunoprecipitations are from 125I-labeled HT1080 cell
total lysate, the patterns of proteins associated with CD81 and
activated PKC are remarkably simple. Thus, few other cell surface
proteins may be present in PKC
-CD81-
3
1
complexes under these conditions. To identify specific
1
integrins co-immunoprecipitated with PKC
, complexes derived from
125I-labeled HT1080 cells were dissociated and subjected to
reimmunoprecipitation (Fig. 6B). As indicated, anti-integrin
antibodies yielded integrin
1,
3, and
6 subunits (lanes f, h,
and j) but not
2 or
5 subunits (lanes g and i). Each of these
subunits was expressed in HT1080 cells at moderate to high levels as
seen by direct immunoprecipitation (lanes a-e)
and by flow cytometry (not shown). Complexes obtained using anti-PI 3-K
failed to yield reprecipitated integrin subunits (lanes
k-o).
View larger version (49K):
[in a new window]
Fig. 6.
PKC association with integrins.
A, HT1080 cells were treated with or without PMA (100 nM at 37 °C, 20 min), surface-labeled with
125I, and then lysed in 1% Brij 96. mAbs to the indicated
proteins were used for immunoprecipitations. B, HT1080 cell
lysates (prepared as in A) were immunoprecipitated
(I.P.) (lanes a-e), using mAbs
TS2/16, A2-IIE10, A3-IVA5, A5-PUJ2, and A6-ELE, respectively. Also,
protein complexes initially precipitated (from PMA-treated cells) using
anti-PKC or PI 3-K antibodies were dissociated and then
reimmunoprecipitated using mAb to the indicated integrin subunits
(lanes f-o).
II) and another cell line
(K562). From cell surface 125I-labeled
K562-
3 and K562-
6 Brij 96 lysates,
anti-PKC
II antibody co-immunoprecipitated abundant integrins (Fig.
7A, lanes
g and j). However, little if any integrin
was co-precipitated from K562-
2, K562-
4,
or K562 mock transfectants containing substantial endogenous
5 (lanes f, h, and
i). Labeled proteins of ~120 kDa in these latter lanes do
not resemble heterodimeric integrins and appear to be background
proteins. All integrins tested were well expressed in their respective
K562 transfectants (lanes a-e). No integrins were detected from normal rabbit IgG control immunoprecipitations (lanes k-o) or from PKC
II
immunoprecipitations from cells not treated with PMA (not
shown).
View larger version (53K):
[in a new window]
Fig. 7.
PKC II association
with integrins in K562 cells. A, K562 cells transfected
with integrin
subunits were surface-labeled with 125I
and lysed in 1% Brij 96, and integrins were immunoprecipitated
(IP) (lanes a-e), using mAbs
A2-IIE10, A3-IVA5, A4-PUJ1, A5-PUJ2, and A6-ELE, respectively. Also,
lysates from PMA-treated cells were precipitated using rabbit
anti-PKC
II sera (lanes f-j) or control rabbit
sera (lanes k-o). B, lysates (in 1%
Brij 96) of K562 transfectants (with or without PMA treatment) were
immunoprecipitated using mAb against indicated integrin
subunits.
Immunoprecipitated proteins were separated by SDS-PAGE and then blotted
using anti-PKC
II antisera.
and PKC
II, the reciprocal result (PKC co-precipitated with specific integrins) is shown in Fig. 7B. Integrin
3 and
6 immunoprecipitations, from Brij
96 lysates of K562-
3 and K562-
6 cells,
respectively, yielded prominent PKC
II proteins as detected by
Western blotting (lower panel, +PMA).
In contrast, no PKC
II was detected in
2,
4, or
5 immunoprecipitates (Fig.
7B, lower panel), although those
proteins are well represented in the respective K562 transfectants (Fig. 7A, lanes a-e). Furthermore, in
the absence of PMA treatment of K562 cells, no PKC
II was detected
from integrin immunoprecipitations, although it was present in the
whole cell lysate (Fig. 7B, upper panel). While PMA greatly stimulated PKC
and PKC
II
association with integrins, PMA had no effect on the solubilization and
direct immunoprecipitation of any of the integrins tested. Besides PMA, bryostatin 1 also induced PKC-integrin association, consistent with its
ability to induce PKC-TM4SF association (as mentioned above). As seen
for TM4SF association (see above), PKC inhibitors did not prevent
integrin association (not shown). Thus again, PKC translocation was
required for integrin association, but PKC catalytic activity was not needed.
3 and TM4SF protein CD81 to lamellipodia was
substantially increased following PMA treatment. Additionally, PKC
was translocated from the cytoplasm to lamellipodia, whereas PI 3-K
distribution was relatively unaffected upon PMA treatment. These
results are again consistent with PKC-CD81-
3 integrin
complex formation.
View larger version (106K):
[in a new window]
Fig. 8.
PMA-induced co-distribution of
PKC ,
3
1
integrin, and CD81. HT1080 cells plated on fibronectin were
untreated (left panels) or treated
(right panels) with PMA and then stained with
primary antibodies against either integrin
3 (A3-X8),
CD81 (M38), PKC
or PI 3-K, followed by rhodamine-conjugated
secondary antibody as described under "Experimental
Procedures."
3 (K562-X3C0 transfectant,
Fig. 9A, lane
c) or exchange of the
3 transmembrane and
tail regions with those from
5 (K562-X3TC5 transfectant, lane d) did not diminish association with
PKC
II, relative to that seen for wild type
3 integrin
(K562-
3, lane b). Conversely, an
2 integrin bearing an
3 tail
(K562-X2C3) failed to associate with PKC
II (lane
e). Also, anti-PKC
II antibody did not co-precipitate integrin from cells bearing predominately
5
1 (mock-transfected K562 cells,
lane f), and none of the K562 transfectants
yielded integrins with normal rabbit IgG control antibody
(lanes g-h). Thus, the integrin
3
chain extracellular domain determines specificity for
interaction of an intracellular protein (PKC) with integrin. These results are consistent with PKC-integrin association requiring a
transmembrane linker protein such as a tetraspanin. Similar to results
seen in Fig. 6A, anti-PKC
II antibody
co-immunoprecipitation of
3 integrin was remarkably
devoid of other surface-labeled proteins (Fig. 9A,
lanes b-d).
View larger version (31K):
[in a new window]
Fig. 9.
Integrin
3 ectodomain determines PKC
recruitment. A, PMA-stimulated K562 transfectants were
125I-labeled, lysed in 1% Brij 96, and then
immunoprecipitated (IP) using anti-PKC
II
(lanes b-f) or control rabbit Ig
(lanes g-k). Co-precipitated proteins
(lanes b-d) align with control
3
1 (directly immunoprecipitated,
lane a). As indicated by flow cytometry, the
X3C0, X3TC5, X2C3, and
3 proteins were all expressed at
comparable levels (not shown). B, K562 transfectants, with
or without PMA stimulation, were labeled with 32P, lysed in
1% Triton X-100, and then immunoprecipitated using the relevant mAb to
integrin
2 or
3 or control mAb to
CD98.
3 and
6 Integrins--
Cytoplasmic domains from integrin
3A and
6A but not
2,
4, or
5 are phosphorylated by a mechanism
that involves activated PKC (29, 31, 54). Furthermore,
3A phosphorylation may regulate integrin-dependent cell motility, signaling, and
cytoskeletal organization (31). If TM4SF proteins are required to link
PKC to integrin, then an integrin unable to associate with TM4SF
proteins should not be phosphorylated even if the correct
tail is
present. Consistent with this prediction, the
3 tail
present within an X2C3 integrin chimera was not phosphorylated in
PMA-treated K562-X2C3 cells (Fig. 9B, lane
i), although the
3 tail was phosphorylated in
K562-
3 cells (lane k). Also, no
phosphorylation was observed if the
3 tail was deleted
(lane m) or if the tail and transmembrane regions
were replaced with those of
5 (lane
o). In addition, no phosphorylation was observed in CD98
control immunoprecipitations or if PMA treatment was omitted
(lanes a-h).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
II, and
associated with TM4SF proteins, whereas representative
other PKC types (
,
, and µ) did not. Likewise, PKM (PKC
II
lacking regulatory domains (49)) failed to associate. Thus, unique
features within the regulatory regions (e.g. C1 or C2
domains) of conventional PKCs are critical for TM4SF association. The
C1 and C2 regulatory domains interact with membrane diacylglycerol and
phosphatidylserine, respectively, and play key roles in PKC activation
and translocation (19). We now suggest that C1 and/or C2 domains of PKC
also could directly interact with TM4SF proteins, thus facilitating
membrane targeting of conventional PKC isoforms. Alternatively, the C1 and/or C2 domains could act indirectly. They may only be required insofar as they bring activated PKC into proximity with the membrane, while other PKC domains then associate with TM4SF proteins. While the
C2 domain of PKC looks promising, it remains to be demonstrated which
particular PKC regulatory domains are especially important for enabling
and/or mediating TM4SF protein interactions.
3
1,
6
1) form complexes with conventional PKCs. Our evidence suggests that tetraspanin proteins provide a key
linker function between PKC and integrins. First, we found PKC, tetraspanins, and integrins all within the same complexes. For
example, in many experiments (e.g. Fig. 6A and
not shown), antibodies to PKC and tetraspanins both yielded the same
pattern of co-immunoprecipitated integrins. Also, all experiments
showing PKC-integrin complexes were carried out under conditions (1%
Brij 99, 1% Brij 96) in which TM4SF-integrin complexes are maintained (12, 44). Second, immunofluorescence staining revealed a
similar localization pattern for
3
1
integrin, CD81, and activated PKC
at the periphery of spread cells.
Third, only tetraspanin proteins have been shown (by
covalent cross-linking) to have high proximity to both PKC (as shown
here) and relevant integrins (42). Fourth, linkage through
tetraspanin proteins explains how an extracellular integrin site could
determine specificity for an associated intracellular enzyme such as
PKC. Importantly, it is the extracellular domains of both
integrin
chains and TM4SF proteins that determine specificity for
TM4SF-integrin association (8, 10, 44), whereas it is
intracellular and/or transmembrane domains of tetraspanin
proteins that most likely determine tetraspanin-PKC association. In
this regard, the TM4SF protein CD151 may similarly link the integrin
3
1 extracellular domain to another
intracellular enzyme, PI 4-K (8). Fifth, those
integrins (e.g.
3
1 and
6
1) that associate strongly with
tetraspanins were seen in association with PKC, whereas integrins not
well associated with tetraspanins (
2
1 and
5
1) did not associate with PKC. Perhaps
most importantly, experiments with chimeric integrins showed that
chain mutants lacking capability for tetraspanin association also lost
PKC association, whereas
chain tail and transmembrane mutants that
retain tetraspanin association also retained PKC association. Together,
these results strongly support a PKC-TM4SF-integrin arrangement.
chain. Likewise, the model does not account for the formation of
PKC-TM4SF complexes in the absence of associated integrins
(e.g. as seen in K562 cells). Finally, we have not yet observed PKC-integrin complexes in the absence of TM4SF proteins. In
another report describing PKC
-
1 integrin complexes
(28), the potential presence of TM4SF proteins was not addressed. At present, we prefer a model (PKC-TM4SF-integrin) in which, upon activation and translocation, PKC is brought into direct association with either TM4SF proteins or preexisting TM4SF-integrin complexes in
cellular membranes.
, and tetraspanin proteins
have all been found in organized lipid microdomains (58-60), we
considered that our PKC-TM4SF-integrin complexes may occur in the
context of large, incompletely solubilized membrane aggregates.
However, in direct co-immunoprecipitation experiments (such as shown in Figs. 2, 5A, and 8A), antibodies to PKC and/or
tetraspanins yielded (especially in HT1080 and K562 cells) a
remarkably clean pattern of 125I-labeled surface-labeled
proteins. In some experiments, we observed no prominent surface-labeled
proteins, aside from surface-labeled integrin, and a low amount of
labeled TM4SF protein.
3 integrin)
typically do not depend on a low density lipid microdomain (60).
Furthermore, in 1% Brij 96, the majority of CD81 and CD9 complexes
were included well within Sepharose 6B gel filtration columns,
again indicating that they are well solubilized and of a reasonable
size (<2 × 106 Da). Even in the less stringent
(i.e. less hydrophobic) Brij 99 detergent,
tetraspanin-integrin complexes appeared to be well solubilized and of
reasonable size (60). These prior results, coupled with our
co-immunoprecipitation and biochemical cross-linking results, suggest
that few other proteins, besides tetraspanins, may be needed to
facilitate PKC-integrin complex formation.
II, termed RACK1, may bind to the integrin
1 cytoplasmic domain. Furthermore, association of
integrins with intact RACK1 was promoted upon stimulation with PMA
(62). However, both the RACK1 integrin specificity (
1
and
2 cytoplasmic domains) and the PKC specificity
(restricted to PKC
) are distinct from the specificities seen here
for integrin-TM4SF-PKC complexes. PKC
may also interact with
syndecan-4, a transmembrane proteoglycan regulating localization of PKC
to focal adhesions (21). However, syndecan complexes may be distinct,
since TM4SF proteins are not usually in focal adhesions (63).
3 and
6 integrins undergo serine
phosphorylation, dependent on both activated PKC and an unidentified
serine kinase (31). Mutation of the
3 phosphorylation
site caused alterations in cell morphology,
3 integrin
distribution and signaling, actin distribution, and cell migration
(31). Our evidence suggests that TM4SF proteins may play a role during
PKC-dependent integrin phosphorylation. First,
as indicated above, TM4SF proteins are closely associated with both
integrins and activated PKC. Second, only those integrins
(
3 and
6 integrins) able to associate
with TM4SF proteins become phosphorylated. In the most informative example, the chimeric X2C3 integrin failed to associate with TM4SF proteins and was not phosphorylated, although it contained the
3 cytoplasmic tail phosphorylation site and was well
expressed at the cell surface. Third, the same agents that
promoted integrin
3 or
6 phosphorylation
(PMA, bryostatin 1) also promoted activated PKC-TM4SF complex formation.
L
2,
5
1, and
2
1) that show PKC-dependent
adhesion and/or spreading functions are not those typically found in
integrin-TM4SF-PKC complexes under moderately stringent detergent
conditions. In addition, PKC-dependent triggering of cell
adhesion, for example through CD28 on T cells (68), can occur in the
absence of integrin-TM4SF-PKC complex formation (not shown). Also, in
contrast to cell migration, cell adhesion is typically not regulated by
TM4SF proteins (8, 44).
chain extracellular domains. These latter domains
provide specificity for the formation of integrin-TM4SF-PKC signaling
complexes. Results shown here contribute to an emerging paradigm
whereby association and activity of intracellular signaling enzymes can
be determined through integrin extracellular domains. Also, for the
first time we have demonstrated a close and possibly direct association
of PKC with a class of widely expressed transmembrane proteins (TM4SF
proteins) that probably play a role in PKC activation, translocation,
subcellular distribution, and signaling.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA86712 (to M. E. H.).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.
Present address: Vascular Biology Center, University of Tennessee
Health Science Center, Memphis, TN 38163.
§ To whom correspondence should be addressed: Dana-Farber Cancer Institute, Rm. D-1430, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3410; Fax: 617-632-2662; E-mail: Martin_Hemler@DFCI.Harvard.EDU.
Published, JBC Papers in Press, April 26, 2001, DOI 10.1074/jbc.M102156200
2 B. Mannion, F. Berditchevsky, J. Bodorova, and M. E. Hemler, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TM4SF, transmembrane-4 superfamily; DSP, dithiobis(succinimidyl propionate); PI 4-K, phosphatidylinositol 4-kinase; PI 3-K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PKM, protein kinase M; PLC, phospholipase C; PMA, phorbol myristate acetate; mAb, monoclonal antibody; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; RACK, receptor for activated C kinase; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Clark, E. A., and Brugge, J. S. (1995) Science 268, 233-239[Medline] [Order article via Infotrieve] |
2. | Hynes, R. O. (1996) Dev. Biol. 180, 402-412[CrossRef][Medline] [Order article via Infotrieve] |
3. | Miyamoto, S., Teramoto, H., Coso, O. A., Gutkind, J. S., Burbelo, P. D., Akiyama, S. K., and Yamada, K. M. (1995) J. Cell Biol. 131, 791-805[Abstract] |
4. | Wary, K. K., Mainiero, F., Isakoff, S. J., Marcantonio, E. E., and Giancotti, F. G. (1996) Cell 87, 733-743[Medline] [Order article via Infotrieve] |
5. |
Zhang, Z. H.,
Vuori, K.,
Reed, J. C.,
and Ruoslahti, E.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
6161-6165 |
6. | Huhtala, P., Humphries, M. J., McCarthy, J. B., Tremble, P. M., Werb, Z., and Damsky, C. H. (1995) J. Cell Biol. 129, 867-879[Abstract] |
7. |
Liu, S.,
Calderwood, D. A.,
and Ginsberg, M. H.
(2000)
J. Cell Sci.
113,
3563-3571 |
8. |
Yauch, R. L.,
Berditchevski, F.,
Harler, M. B.,
Reichner, J.,
and Hemler, M. E.
(1998)
Mol. Biol. Cell
9,
2751-2765 |
9. | Rubinstein, E., Le Naour, F., Billard, M., Prenant, M., and Boucheix, C. (1994) Eur. J. Immunol. 24, 3005-3013[Medline] [Order article via Infotrieve] |
10. |
Berditchevski, F.,
Bazzoni, G.,
and Hemler, M. E.
(1995)
J. Biol. Chem.
270,
17784-17790 |
11. | Nakamura, K., Iwamoto, R., and Mekada, E. (1995) J. Cell Biol. 129, 1691-1705[Abstract] |
12. | Berditchevski, F., Zutter, M. M., and Hemler, M. E. (1996) Mol. Biol. Cell 7, 193-207[Abstract] |
13. | Wright, M. D., and Tomlinson, M. G. (1994) Immunol. Today 15, 588-594[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Maecker, H. T.,
Todd, S. C.,
and Levy, S.
(1997)
FASEB J.
11,
428-442 |
15. | Hemler, M. E., Mannion, B. A., and Berditchevski, F. (1996) Biochim. Biophys. Acta 1287, 67-71[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Berditchevski, F.,
Tolias, K. F.,
Wong, K.,
Carpenter, C. L.,
and Hemler, M. E.
(1997)
J. Biol. Chem.
272,
2595-2598 |
17. | Yauch, R. L., and Hemler, M. E. (2000) Biochem. J. 351, 629-637[CrossRef][Medline] [Order article via Infotrieve] |
18. | Nishizuka, Y. (1992) Science 258, 607-614[Medline] [Order article via Infotrieve] |
19. | Newton, A. C. (1997) Curr. Opin. Cell Biol. 9, 161-167[CrossRef][Medline] [Order article via Infotrieve] |
20. | Jaken, S., and Parker, P. J. (2000) Bioessays 22, 245-254[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Oh, E. S.,
Woods, A.,
and Couchman, J. R.
(1997)
J. Biol. Chem.
272,
8133-8136 |
22. |
Shattil, S. J.,
and Brass, L. F.
(1987)
J. Biol. Chem.
262,
992-1000 |
23. | Dustin, M. L., and Springer, T. A. (1989) Nature 341, 619-624[CrossRef][Medline] [Order article via Infotrieve] |
24. | Shimizu, Y., Van Seventer, G. A., Horgan, K. J., and Shaw, S. (1990) Nature 345, 250-253[CrossRef][Medline] [Order article via Infotrieve] |
25. | Jaken, S., Leach, K., and Klauck, T. (1989) J. Cell Biol. 109, 697-704[Abstract] |
26. |
Vuori, K.,
and Ruoslahti, E.
(1993)
J. Biol. Chem.
268,
21459-21462 |
27. | Chun, J.-S., and Jacobson, B. S. (1993) Mol. Biol. Cell 3, 1863-1871 |
28. |
Ng, T.,
Shima, D.,
Squire, A.,
Bastiaens, P. I.,
Gschmeissner, S.,
Humphries, M. J.,
and Parker, P. J.
(1999)
EMBO J.
18,
3909-3923 |
29. | Shaw, L. M., Messier, J. M., and Mercurio, A. M. (1990) J. Cell Biol. 110, 2167-2174[Abstract] |
30. |
Hogervorst, F.,
Kuikman, I.,
Noteboom, E.,
and Sonnenberg, A.
(1993)
J. Biol. Chem.
268,
18427-18430 |
31. |
Zhang, X. A.,
Bontrager, A. L.,
Stipp, C. S.,
Kraeft, S.-K.,
Bazzoni, G.,
Chen, L. B.,
and Hemler, M. E.
(2001)
Mol. Biol. Cell
12,
351-365 |
32. | Dumont, J. A., and Bitonti, A. J. (1994) Biochem. Biophys. Res. Commun. 204, 264-272[CrossRef][Medline] [Order article via Infotrieve] |
33. | Bergelson, J. M., St. John, N. F., Kawaguchi, S., Pasqualini, R., Berdichevsky, F., Hemler, M. E., and Finberg, R. W. (1994) Cell Adhes. Commun. 2, 455-464[Medline] [Order article via Infotrieve] |
34. |
Weitzman, J. B.,
Pasqualini, R.,
Takada, Y.,
and Hemler, M. E.
(1993)
J. Biol. Chem.
268,
8651-8657 |
35. |
Hemler, M. E.,
Huang, C.,
Takada, Y.,
Schwarz, L.,
Strominger, J. L.,
and Clabby, M. L.
(1987)
J. Biol. Chem.
262,
11478-11485 |
36. | Pujades, C., Teixidó, J., Bazzoni, G., and Hemler, M. E. (1996) Biochem. J. 313, 899-908[Medline] [Order article via Infotrieve] |
37. |
Lee, R. T.,
Berditchevski, F.,
Cheng, G. C.,
and Hemler, M. E.
(1995)
Circ. Res.
76,
209-214 |
38. |
Hemler, M. E.,
Ware, C. F.,
and Strominger, J. L.
(1983)
J. Immunol.
131,
334-340 |
39. |
Hemler, M. E.,
Sánchez-Madrid, F.,
Flotte, T. J.,
Krensky, A. M.,
Burakoff, S. J.,
Bhan, A. K.,
Springer, T. A.,
and Strominger, J. L.
(1984)
J. Immunol.
132,
3011-3018 |
40. | Amiot, M. (1995) in Leukocyte Typing V-White Cell Differentiation Antigens (Schlossman, S. F. , Boumsell, L. , Gilks, W. , Harlan, J. M. , Kishimoto, T. , Morimoto, C. , Ritz, J. , Shaw, S. , Silverstein, R. , Springer, T. , Tedder, T. F. , and Todd, R. F., eds) , pp. 556-558, Oxford University Press, Oxford |
41. | Fukudome, K., Furuse, M., Imai, T., Nishimura, M., Takagi, S., Hinuma, Y., and Yoshie, O. (1992) J. Virol. 66, 1394-1401[Abstract] |
42. |
Yauch, R. L.,
Kazarov, A. R.,
Desai, B.,
Lee, R. T.,
and Hemler, M. E.
(2000)
J. Biol. Chem.
275,
9230-9238 |
43. | Takagi, S., Fujikawa, K., Imai, T., Fukuhara, N., Fukudome, K., Minegishi, M., Tsuchiya, S., Konno, T., Hinuma, Y., and Yoshie, O. (1995) Int. J. Cancer 61, 706-715[Medline] [Order article via Infotrieve] |
44. | Mannion, B. A., Berditchevski, F., Kraeft, S.-K., Chen, L. B., and Hemler, M. E. (1996) J. Immunol. 157, 2039-2047[Abstract] |
45. | Chan, B. M. C., and Hemler, M. E. (1993) J. Cell Biol. 120, 537-543[Abstract] |
46. |
Bazzoni, G.,
Ma, L.,
Blue, M.-L.,
and Hemler, M. E.
(1998)
J. Biol. Chem.
273,
6670-6678 |
47. |
Manger, B.,
Weiss, A.,
Imboden, J.,
Laing, T.,
and Stobo, J. D.
(1987)
J. Immunol.
139,
2755-2760 |
48. | Hocevar, B. A., Morrow, D. M., Tykocinski, M. L., and Fields, A. P. (1992) J. Cell Sci. 101, 671-679[Abstract] |
49. |
Pontremoli, S.,
Melloni, E.,
Sparatore, B.,
Michetti, M.,
Salamino, F.,
and Horecker, B. L.
(1990)
J. Biol. Chem.
265,
706-712 |
50. |
Nakamura, K.,
Mitamura, T.,
Takahashi, T.,
Kobayashi, T.,
and Mekada, E.
(2000)
J. Biol. Chem.
275,
18284-18290 |
51. | Matsumoto, A. K., Martin, D. R., Carter, R. H., Klickstein, L. B., Ahearn, J. M., and Fearon, D. T. (1993) J. Exp. Med. 178, 1407-1417[Abstract] |
52. |
Pileri, P.,
Uematsu, Y.,
Campagnoli, S.,
Galli, G.,
Falugi, F.,
Petracca, R.,
Weiner, A. J.,
Houghton, M.,
Rosa, D.,
Grandi, G.,
and Abrignani, S.
(1998)
Science
282,
938-941 |
53. | Emi, N., Kitaori, K., Seto, M., Ueda, R., Saito, H., and Takahashi, T. (1993) Immunogenetics 37, 193-198[Medline] [Order article via Infotrieve] |
54. | Hogervorst, F., Admiraal, L. G., Niessen, C., Kuikman, I., Janssen, H., Daams, H., and Sonnenberg, A. (1993) J. Cell Biol. 121, 179-191[Abstract] |
55. |
Kitadokoro, K.,
Bordo, D.,
Galli, G.,
Petracca, R.,
Falugi, F.,
Abrignani, S.,
Grandi, G.,
and Bolognesi, M.
(2001)
EMBO J.
20,
12-18 |
56. | Carmo, A. M., and Wright, M. D. (1995) Eur. J. Immunol. 25, 2090-2095[Medline] [Order article via Infotrieve] |
57. | Seehafer, J. G., and Shaw, A. R. (1991) Biochem. Biophys. Res. Commun. 179, 401-406[Medline] [Order article via Infotrieve] |
58. |
Mineo, C.,
Ying, Y. S.,
Chapline, C.,
Jaken, S.,
and Anderson, R. G.
(1998)
J. Cell Biol.
141,
601-610 |
59. |
Yashiro-Ohtani, Y.,
Zhou, X. Y.,
Toyo-Oka, K.,
Tai, X. G.,
Park, C. S.,
Hamaoka, T.,
Abe, R.,
Miyake, K.,
and Fujiwara, H.
(2000)
J. Immunol.
164,
1251-1259 |
60. |
Claas, C.,
Stipp, C. S.,
and Hemler, M. E.
(2001)
J. Biol. Chem.
276,
7974-7984 |
61. |
Mochly-Rosen, D.,
and Gordon, A. S.
(1998)
FASEB J.
12,
35-42 |
62. |
Liliental, J.,
and Chang, D. D.
(1998)
J. Biol. Chem.
273,
2379-2383 |
63. |
Berditchevski, F.,
and Odintsova, E.
(1999)
J. Cell Biol.
146,
477-492 |
64. | Aderem, A. (1995) Biochem. Soc. Trans. 23, 587-591[Medline] [Order article via Infotrieve] |
65. |
Yánez-Mó, M.,
Alfranca, A.,
Cabañas, C.,
Marazuela, M.,
Tejedor, R.,
Ursa, M. A.,
Ashman, L. K.,
De Landázuri, M. O.,
and Sánchez-Madrid, F.
(1998)
J. Cell Biol.
141,
791-804 |
66. | Blume-Jensen, P., Siegbahn, A., Stabel, S., Heldin, C. H., and Ronnstrand, L. (1993) EMBO J. 12, 4199-4209[Abstract] |
67. | Woods, A., and Couchman, J. R. (1992) J. Cell Sci. 101, 277-290[Abstract] |
68. | Shimizu, Y., Van Seventer, G. A., Ennis, E., Newman, W., Horgan, K. J., and Shaw, S. (1992) J. Exp. Med. 175, 577-582[Abstract] |