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* Institut für Immunologie and
I Medizinische Klinik, Christian-Albrechts-Universität Kiel, Kiel, Germany;
Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Universität Köln, Köln, Germany; and
Laboratorium für Molekulare Biologie, Genzentrum der Universität München, München, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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80 amino acids, is responsible for the
initiation of programmed cell death and activation of NF-
B,
stress-activated protein kinases, and p38 mitogen-activated protein
kinase (2, 3). In contrast to the extensively studied
death domain, signaling mediated by other cytoplasmic domains of
TNF-R55 is less well defined. In previous studies, we have identified a
small region at the very N terminus of the death domain, designated the
neutral sphingomyelinase
(N-SMase)4 activating
domain (NSD) (4), that is responsible for the TNF-induced
activation of N-SMase. We have demonstrated that the adapter protein
factor associated with N-SMase activation (FAN) is required for the
stimulation of N-SMase. FAN represents a member of the WD40 (or
WD-repeat) family of proteins that includes mainly regulatory proteins.
The WD-repeat motif consists of a conserved core of 23–41 amino acids,
usually bounded by Gly-His (GH) and Trp-Asp (WD), and is implicated in
mediating protein-protein interactions (5). Accordingly,
we have provided evidence that the C-terminal WD repeats of FAN
interact with the NSD of human TNF-R55 and that this interaction is
required for N-SMase activation (6). N-terminally
truncated FAN, containing only the WD repeats, inhibits N-SMase
activation in a dominant negative manner (6), suggesting
that it lacks a putative effector domain in the N terminus. Notably,
the putative effector domain of FAN contains a region of significant
homology to the BEACH (Beige and CHS) domain present in the mouse
WD-repeat protein Beige and its human homolog, the CHS protein
(7). The BEACH domain has also been found in several other
WD-repeat proteins—Vps15, LvsA, LvsB, neurobeachin, and LBA
(8, 9, 10, 11, 12)—all of which have been implicated in the
regulation of vesicular trafficking. A second region of homology within
the N terminus of FAN shows similarity to the GRAM domain, a
motif found in glucosyltransferases, myotubularins, and other putative
membrane-associated proteins (13). Recently, a
WD-repeat-like domain has been defined for the LBA protein that
is also found in the putative effector domain of FAN (12).
However, so far, the significance of neither homology region for the
function of FAN has been examined. Aside from its role in TNF-induced
activation of N-SMase, and aside from a putative involvement of FAN in
apoptosis (14, 15), actin reorganization in macrophages
(16), as well as cutaneous barrier repair
(17), possible further signaling functions of FAN have
largely remained elusive.
Receptors for activated C-kinase (RACKs) comprise a group of cytosolic
proteins that can bind to activated isoforms of protein kinase C (PKC).
It has been suggested that RACKs facilitate the translocation of PKC
isoforms to cellular membranes and simultaneously stabilize PKC from
premature degradation (18). RACK1 is a 36-kDa protein with
homology to the 
subunit of heterotrimeric G proteins, that, like
FAN, is characterized by the presence of (seven) WD repeats
(19), structures which have the potential to fold into
multibladed-propeller-like structures (5). The WD repeats
in RACK1 are conserved from Chlamydomonas to human
(20), indicating that the function of RACK1 was presumably
established before the evolutionary divergence of plants and animals.
Recent results indicate that RACK1 is a scaffolding protein that is
involved in the recruitment, assembly, and regulation of a number of
different signaling molecules into membrane-associated complexes.
Proteins recruited by RACK1 comprise PKCII, PKC
, the cytoplasmic
tyrosine kinase c-src, the subunit of integrins, (18, 21, 22, 23), as well as a number of other cytoplasmic and viral
proteins (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). Functionally, RACK1 has been implicated
in the development of cardiac hypertrophy and failure, the regulation
of cell adhesion, protection of tumor cells from E1A-induced apoptosis,
age-associated decline in alveolar macrophage functions, assembly of
signaling complexes at the platelet-derived growth factor receptor,
integrin-dependent cell migration, negative signaling for superoxide
anion generation, and the recruitment of STAT1 to the human type I IFNR
(21, 27, 30, 38, 39, 40, 41, 42). Moreover, RACK1 interacts with the
pleckstrin homology (PH) domains of several proteins in vitro,
indicating that it may aid in colocalizing PKC with its PH
domain-containing substrates (43). However, only a subset
of these interactions depends upon PKC stimulation (23).
In addition, some of the interactions between the proteins listed above
and RACK1 appear to be mutually exclusive (22, 27),
suggesting that RACK1 forms distinct cellular signaling complexes in
response to unique cellular stimuli.
We have identified RACK1 as an interaction partner of FAN by means of the yeast interaction trap system. RACK1 specifically binds to FAN, as demonstrated by coimmunoprecipitation and GST coprecipitation experiments. Deletion studies in yeast map the interaction domains of both proteins to WD repeats IV to VII of RACK1 (minimally encompassing parts of WD repeat V to WD repeat VII), and to the C-terminal WD-repeat region of FAN. The interaction of FAN and RACK1 appears to be dependent on the folding of the WD repeats into a secondary structure–conceivably the proposed multibladed propeller—because no prominent linear binding motifs were identified in the WD-repeat region of FAN. Confocal laser-scanning microscopy studies suggest that overexpressed FAN and RACK colocalize at the plasma membrane together with TNF-R55. At the functional level, concurrent overexpression of FAN, RACK1, and a noncytotoxic deletion mutant of TNF-R55 strongly enhanced TNF-induced stimulation of N-SMase. Our findings suggest a novel linkage between TNF-R55 and RACK1/PKC-mediated signaling pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The plex.FAN constructs were generated by cloning restriction
fragments of FAN containing amino acids 3–917
(FAN3–917) and 3–547
(FAN3–547) into plex202. Sequencing verified
that the inserts were in the correct reading frame. The generation of
the Jurkat cDNA library fused to a synthetic activation domain has been
described elsewhere (44). The HeLa cDNA expression library
was purchased from Clontech Laboratories (Palo Alto, CA).
Transformation of the yeast strain EG48/JK103 (45) with
bait constructs and, subsequently, with the library DNA or selected
activation domain fusion constructs was performed as described
(44). Transformants were grown on
Ura-His-Trp-
glucose plates, before selection for leucine prototrophy on
Ura-His-Trp-Leu-
galactose plates was used to test for positive interaction. Expression
of the bait constructs was verified by immunoblots using a monoclonal
anti-lex Ab (BD Biosciences, Heidelberg, Germany). Testing
for -galactosidase expression was performed on either
Ura-His-Trp-
galactose X-Gal plates or, for quantification, in a liquid assay. Yeast
cells were grown overnight at 30°C in
Ura-His-Trp-
galactose medium to an OD600 of 1.8, diluted
1/5 in the same medium and grown at 30°C to an
OD600 of 0.5–0.8. Cells were washed, resuspended
in Z-buffer (60 mM Na2HPO4,
40 mM NaH2PO4, 10 mM KCl, 1
mM MgSO4, pH 7.0), and lysed by freezing/thawing.
The cells were diluted 1/8 in Z buffer containing 0.27% (v/v) 2-ME.
The reaction was started by adding 160 µl Z buffer containing 4 mg/ml
o-nitrophenyl--D-galactoside,
incubated at 37°C, and stopped with 0.4 ml of 1 M
Na2CO3. After precipitating
the cells, the OD420 of the supernatants was
determined. -Galactosidase units were calculated by the following
equation: units = 1000 x
OD420/(elapsed time of incubation (min) x
volume of cells (ml) x OD600).
In vitro interaction assay
The insert of pJG.RACK144–317 coding for
amino acids 144 to 317 was expressed as a GST fusion protein using the
bacterial expression vector pGEX4T1 (Amersham Biosciences, Freiburg,
Germany). Cultures of Escherichia coli XL1-Blue containing
pGEX-RACK1144–317 were induced with 2 mM
isopropyl--D-thiogalactoside for 6 h at
37°C. Bacteria were harvested, resuspended in PBS containing 1%
(v/v) Triton X-100, and lysed by three freeze/thaw cycles. After
centrifugation at 12,000 x g for 10 min to remove
debris, the supernatant was passed through a glutathione Sepharose 4B
column (Amersham Biosciences). The column was washed three times with
PBS, and GST fusion protein was eluted by the addition of 50 mM Tris
(pH 8.0)-10 mM glutathione according to the manufacturer’s
instructions. GST-RACK1144–317 fusion protein
(15 µg) or GST alone were preincubated with glutathione Sepharose 4B
beads for 1 h at 4°C in PBS containing 1% (v/v) Triton X-100
and collected by centrifugation. The precipitates were resuspended in
binding buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% (v/v)
Nonidet P-40 (NP40), 1 mM EDTA, and 5% (v/v) glycerol) and the entire
coding regions of FAN or cytosolic phospholipase
A2 (cPLA2) that had been
synthesized in vitro using the SP6-coupled TNT Reticulocyte Lysate
System (Promega, Madison, WI) and
[35S]methionine (Amersham Biosciences) were
added. After incubation for 2 h at 4°C, the beads were washed
three times in binding buffer containing 500 mM NaCl. Subsequently, the
bound proteins were eluted by boiling in SDS sample buffer, resolved by
8% (w/v) SDS-PAGE, and analyzed after autoradiography on a
phosphoimager.
Mapping of the interaction sites of RACK1 and FAN
For the isolation of a full-length cDNA for RACK1, the insert of pJG.RACK144–317, representing the largest of all clones isolated in the yeast interaction trap system, served as a probe to screen a human skeletal muscle cDNA library (lambda ZAP Express; Stratagene, Amsterdam, The Netherlands) by standard methods (46). The cDNA inserts of the isolated phage plaques were excised in vivo, following the instructions of the manufacturer, and sequenced. All clones isolated proved identical, containing the entire coding region of RACK1. Subsequently, the entire coding region of RACK1 (amino acids 1–317) as well as C-terminal fragments coding for amino acids 198–317 and 204–317 were amplified by PCR adding restrictions sites for EcoRI/SalI (RACK1–317) or EcoRI/XhoI (RACK1198–317 and RACK1204–317). The above fragments were then subcloned into both bait (plex202, EcoRI/SalI) and prey vector (pJG4–5, EcoRI/XhoI; Ref. 45) of the yeast interaction trap system. The insert of pJG.RACK144–317 was isolated as an EcoRI/XhoI fragment and subcloned into the EcoRI/SalI sites of plex202. As far as not yet available from the initial yeast interaction trap screens, bait and prey constructs of FAN3–917, FAN3–547, FAN703–917, as well as Apaf-1577–1182 (original construct kindly provided by Dr. X. Wang, Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX), were generated by the same procedure as above. All constructs were verified by sequencing.
Staggered peptide scanning assay
Cellulose filters containing FAN-derived peptide scanning libraries were purchased from Jerini Bio Tools (Berlin, Germany). Radiolabeled whole cell extracts from Jurkat cells were prepared by incubation of 3 x 107 cells with 2.5 mCi in vitro cell-labeling mix (>1000 Ci/mmol of L-[35S]methionine and L-[35S]cysteine; Amersham Biosciences) in methionine/cysteine-free medium for 4 h, followed by two washes in PBS and lysis in HDP (30 mM HEPES pH 7.9, 10% (v/v) glycerol, 7 mM MgCl2, 10 mM KCl, 1 mM DTT, 0.1% (v/v) NP40, 10 µg/ml aprotinin and leupeptin) by freezing/thawing. The cell lysate was incubated on peptide filters that had been prewashed twice in methanol and twice in PBS, in 1x SuperBlock blocking buffer (Pierce, Rockford, IL) in HDP overnight at 4°C with continuous shaking. After five washes at room temperature in NET (150 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 0.05% (v/v) NP40), filters were air-dried and autoradiographed. Binding of the cell lysates to individual peptides was quantified using a phosphoimager.
Cell culture
293 (human embryonic kidney) and COS-1 cells were originally obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in high glucose DMEM supplemented with 10% (v/v) fetal calf serum, 10 mM glutamine, 50 µg/ml penicillin, and 50 µg/ml streptomycin (Invitrogen, Groningen, The Netherlands) in a humidified incubator at 5% (w/v) CO2.
In vivo interaction assay
The coding region of RACK1 was amplified by PCR and ligated into
pcDNA3.1-/Myc-His A (Invitrogen) as an
N-terminal fusion to the myc tag of the vector, yielding
plasmid pMYC.RACK1–317. For
coimmunoprecipitation experiments, 1.8 x
106 293 cells were transiently transfected with 5
µg of pMYC.RACK1–317 or
pcDNA3.1-/Myc-His A in combination with 5 µg
of pFLAG.FAN3–917 or pFLAG.CMV2 (6)
by the calcium phosphate precipitation method. After 48 h, cells
were detached using 2 mM EDTA, lysed in TNE buffer (50 mM Tris, pH 8.0,
150 mM NaCl, 1% (v/v) NP40, and 2 mM EDTA) containing 10 µg/ml
aprotinin/leupeptin, and 3 mg of cellular lysates were precleared with
-bind-Sepharose (Amersham Biosciences). Immunoprecipitation was
performed overnight on ice using 3 µg of anti-FLAG M2 IgG1 mAb
(Sigma-Aldrich, Taufkirchen, Germany) or 1.4 µg of
anti-myc IgG1 mAb (Invitrogen) followed by collection of
the immunocomplexes by a 1-h incubation with -bind-Sepharose and
subsequent washing of the immunocomplexes for three times in TNE, once
in TNE containing 1 M NaCl, and once more in TNE. The
immunoprecipitated proteins were separated on 4–20% (w/v) gradient
gels by SDS-PAGE. For detection of expressed tagged RACK1 and FAN
protein in total cell lysates, 20 µg of cell protein per lane were
resolved by SDS-PAGE. After electrophoretic transfer to nitrocellulose
(Whatman-Biometra, Göttingen, Germany), reactive proteins were
detected using anti-myc and/or anti-FLAG Ab M2 by the
ECL detection kit (Amersham Biosciences). Coimmunoprecipitations of
endogenous RACK1 and FAN with TNF-R55 were performed essentially as
above with the following modifications: untransfected 293 cells were
lysed in TNB (20 mM Tris pH 8.0, 140 mM NaCl, and 0.5% (v/v) Brij58)
supplemented with the Protease Inhibitor Mix Complete (Roche
Diagnostics, Mannheim, Germany), 2.7 mg of cellular lysate were
immunoprecipitated without preclearing using 3 µg of anti-TNF-R55
htr-9 IgG1 mAb (Bachem, Heidelberg, Germany) or 1 µg of anti-FAN
N-19 goat polyclonal Ab (Santa Cruz Biotechnology, Heidelberg, Germany)
followed by SDS-PAGE, and endogenous RACK1 and FAN proteins present in
the immunoprecipitates were detected using either anti-FAN Ab N19
or anti-RACK1 IgM mAb (BD Biosciences).
Intracellular colocalization studies
A SalI-EcoRI fragment from the human
TNF-R55 encoding amino acids 1–345 was isolated from pADB-TR55
(47) and cloned into the expression vector pEF-BOS
(48) yielding pEF.TNF-R55
345. COS-1 cells were
transiently transfected by electroporation at 280 V/960 µF
using 5 µg of pMYC.RACK1–317,
pFLAG.FAN3–917, and/or pEF.TNF-R55345, and
grown to subconfluency on coverslips for 24–48 h before fixation in
2.5% (w/v) paraformaldehyde in PBS for 30 min at room temperature. The
coverslips were washed two times in PBS and once for 10 min in TBS to
remove the fixative followed by permeabilization and blocking of
nonspecific sites with 0.05% (w/v) Saponin and 0.1% (w/v) BSA in TBS
containing 0.1% (w/v) glycine for 1 h at room temperature and
three washes in TBS for 5 min each. Cells were incubated with primary
Abs (anti-myc mouse monoclonal IgG1 (1:100),
anti-FLAG mouse monoclonal IgG1 (1:200), affinity-purified
anti-FLAG rabbit polyclonal antiserum (1:50; Zymed Laboratories,
San Francisco, CA), anti-TNF-R55 goat polyclonal affinity-purified
IgG (1:50; R&D Systems, Minneapolis, MN) in TBS for 1 h at 37°C.
After washing three times in PBS (5, 10, and 15 min), cells were
incubated with secondary Abs (FITC-conjugated donkey anti-rabbit or
anti-goat IgG (1:100), rhodamine-conjugated donkey anti-mouse
IgG (1:100), CY5-conjugated donkey anti-rabbit IgG (1:350);
Dianova, Hamburg, Germany) for 1 h in TBS at 37°C. Cells were
again washed three times in PBS (5, 10, and 15 min) before stained
proteins were visualized using a Zeiss LSM 510 confocal laser-scanning
microscope (Zeiss, Jena, Germany). Final digital images were processed
by increasing contrast of all images by 50% using Adobe Photoshop 4.0
(Adobe Systems, Mountainview, CA).
Assays for N-SMase
293 cells (1.5 x 106) were
transiently transfected with 5 µg of
pcDNA3.1-/Myc-His A or with 5 µg of
pFLAG.FAN3–917,
pMYC.RACK1–317, or pEF.TNF-R55345, either
individually or as a combination of all three constructs, by the
calcium phosphate precipitation method. After 48 h, cells were
treated in triplicate in 0.5 ml medium with 100 ng/ml human recombinant
TNF (a gift from Dr. G. Adolf, Bender Research Institute, Vienna,
Austria) for 0–5 min. Cells were homogenized as described
(49), except that 0.5% (w/v)
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate was
substituted for 0.2% (v/v) Triton X-100 in the lysis buffer.
Radioactive phosphocholine produced from
[N-methyl-14C]-sphingomyelin (labeled in
the choline moiety, CFA566; Amersham Biosciences) was identified by TLC
and routinely determined in the aqueous phase by scintillation
counting.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify proteins that directly interact with FAN, we used the
yeast interaction trap system. A bait construct containing the
full-length FAN cDNA (plex.FAN3–917; Fig. 1
A) was used to screen a
Jurkat cDNA expression library. In 41 cases, the isolated colonies
contained inserts that were identical to various proportions of the C
terminus of RACK1, with the shortest insert spanning most of WD repeat
V to WD repeat VII (Fig. 1B), suggesting that this is the
minimal region through which RACK1 interacts with FAN. In quantitative
assays for relative -galactosidase expression, the interaction of
the largest RACK1 clone (pJG.RACK144–317) with
plex.FAN3–917 was about 13-fold stronger than
with the bait vector control. When the interaction of
RACK144–317 with a FAN bait construct containing
the putative N-SMase effector domain but lacking the WD repeats
(plex.FAN3–547; Fig. 1A) was tested,
only a weak -galactosidase expression of 2.8-fold above bait vector
control was measured. Because RACK1 was not isolated in additional
screens using FAN3–547 as bait (data not shown),
we conclude that binding of FAN to RACK1 is mediated through the
C-terminal WD repeats of FAN rather than through its N-terminal region.
In an independent screen using a cDNA expression library from HeLa
cells, 10 randomly chosen clones interacting with the full-length FAN
protein coded for C-terminal portions of RACK1 (data not shown),
strengthening the notion that RACK1 is as a potential interactor
for FAN.
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The association of FAN and RACK1 was further investigated by in
vitro binding experiments in a cell-free system. For this purpose, the
insert of pJG.RACK144–317 was expressed as a GST
fusion protein and tested for its ability to bind to
[35S]methionine-labeled, in-vitro-translated,
full-length FAN or the unrelated cPLA2. The
cPLA2 negative control showed the same pattern of
bands with GST alone and with GST-RACK1144–317,
indicating that no specific binding of RACK1 to
cPLA2 occurred (Fig. 2, lanes 2 and
4). In contrast, FAN bound to
GST-RACK1144–317 (Fig. 2, lane 3,
asterisk) but not to GST alone (lane 1).
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Bait and prey constructs for RACK11–317
(the entire coding region), RACK1144–317 (the
largest clone isolated in the above screen),
RACK1198–317 (the smallest clone isolated), and
RACK1204–317 (Fig. 3), as well as for
FAN3–917, FAN3–547,
FAN703–917 (containing WD repeats II to V at the
C terminus of FAN, Fig. 1
A), and
Apaf-1577–1182 (containing the C-terminal WD
repeats I to XII of Apaf-1) were generated as described under
Materials and Methods. Combinations of bait and prey
constructs were cotransformed into yeast, as outlined in Table I, and tested for -galactosidase
expression. In controls for nonspecific interaction with prey vector
pJG4-5 without insert, all bait constructs were negative (data not
shown). In line with the results from the previous screens,
RACK1144–317 strongly interacted with
full-length FAN regardless of which construct was used as bait or prey.
This RACK1 construct also interacted as both bait and prey with
FAN703–917 containing only the C-terminal WD
repeats, confirming that both proteins interact through their
C-terminal WD repeats. The RACK1144–317 bait
construct interacted with a RACK1144–317 prey
construct (self-association); however, it did not show any binding to
the WD-repeat-containing C terminus of Apaf-1. Therefore, the
interaction of RACK1144–317 and FAN is specific
and not due to a general stickiness of the WD-repeat regions of both
proteins.
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In line with the assumption that RACK1198–317 contained the minimal FAN-binding site, the even smaller construct RACK1204–317 did not bind to full-length FAN anymore in any bait/prey combination. Also, compared with RACK1198–317, the interaction with the WD-repeat coding construct FAN703–917 was strongly reduced. However, RACK1204–317 was still able to strongly associate with itself, indicating that this core fragment retained general binding activity but that specific binding to FAN required additional determinants.
When the entire RACK1 protein was used as bait or prey, we found
interaction with the C-terminal WD repeats of
FAN703–917, but, surprisingly, we could not
detect an interaction with full-length FAN. However, full-length FAN
also did not show an association with TNF-R55 in these experiments (as
measured by -galactosidase expression; data not shown), although
this association has been clearly shown in vivo (6).
Moreover, it has been demonstrated that the interaction of RACK1 with
the subunit of integrins or the HIV-1 Nef protein is detectable in
yeast with C-terminal RACK1 constructs containing WD repeats V to VII,
but not with full-length RACK1 (23, 33). These results are
most likely due to limitations of the yeast interaction trap system
when the expressed interaction partners become too large to allow the
fused DNA-binding and activation domains to drive -galactosidase
expression. In line with this assumption, neither full-length RACK1 nor
FAN showed self-association in these assays.
All RACK1 constructs showed no or only very weak interaction with
FAN3–547 harboring the putative N-terminal
effector domain. In summary, the above experiments indicate that the
interaction of RACK1 with FAN is mediated by a core region of RACK1
that comprises WD repeats IV to VII and that minimally must encompass
parts of WD repeat V (C-terminal of amino acid 198) as well as the WD
repeats VI and VII (Fig. 3).
In an independent approach, we wanted to determine whether FAN
contained further potential protein-binding sites. Similar to a
previous experiment that had identified the binding motif for FAN
within TNF-R55 (6), we used a cellulose-bound peptide
scanning library staggered by three amino acids each, containing
overlapping 13-mer peptides of the entire FAN protein (Fig. 4A). Jurkat cells were
metabolically labeled, and total cellular extracts were assayed for
binding to the staggered peptides. Within the putative effector domain
of FAN, regions of significant binding spanned peptides 14–18, 57–60,
79–80, and 189–191. However, none of the peptides contained in these
regions is localized entirely within the GRAM or BEACH domain of FAN.
The only peptides from these domains that bound cellular proteins
significantly over background were peptides 121 and 153–154, both
located in the BEACH domain (Fig. 4B). Apparently, the GRAM
and BEACH domains of FAN do not contain extended linear protein-binding
motifs detectable by the staggered peptide scanning assay.
Nevertheless, the peptide stretches detected outside of both domains
may mark the binding sites of yet-unidentified proteins potentially
participating in the signal transduction pathways of FAN. Within the WD
repeats of FAN, only peptide 288 displayed significant binding to the
labeled extracts (Fig. 4B). This was somewhat surprising,
because it could be expected that RACK1 or other WD-repeat binding
proteins present in the labeled extracts should produce a
"footprint" of labeled peptides in this assay. Apparently, the WD
repeats of FAN must be folded into the suggested multibladed-propeller
secondary structure for interaction with other proteins such as RACK1.
Because this secondary structure is not represented adequately by
linear binding motifs, the interaction of FAN and RACK1 is apparently
not detected by the staggered peptide scanning assay.
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To elucidate whether full-length RACK1 and FAN were able to
interact in intact cells, 293 cells were transiently transfected with a
myc-tagged version of RACK1
(pMYC.RACK1–317) in combination with a
FLAG-tagged full-length FAN construct
(pFLAG.FAN3–917; Ref. 6). For
control, cells were additionally transfected with
pMYC.RACK1–317 or
pFLAG.FAN3–917, in combination with the parental
vectors pFLAG.CMV2 or pcDNA3.1-/Myc-His A, and
finally with both empty vectors alone. The expression of the
transfected fusion proteins was confirmed in Western blots with
anti-myc and anti-FLAG Abs (Fig. 5, A and B;
lower panels). In parallel, identical amounts of cellular
lysates were immunoprecipitated using anti-FLAG Ab and
coimmunoprecipitating RACK1 was detected in Western blots with
anti-myc Ab. As shown in Fig. 5A (upper
panel), RACK1 was present only in FAN immunoprecipitates
(lane 2) but not in control immunoprecipitates
(lanes 1, 3, and 4). In reverse
experiments, RACK1 was immunoprecipitated with anti-myc
Ab and coimmunoprecipitating FAN was detected in Western blots with
anti-FLAG Ab. A band corresponding to the exact size of FAN was
detected specifically in RACK1 immunoprecipitates (Fig. 5B,
upper panel, lane 2). Similar results were
obtained when RACK1 was immunoprecipitated with a mAb directed against
RACK1 itself (data not shown). Because the interaction of overexpressed
proteins has to be interpreted with caution, we additionally examined
whether the endogenous RACK1 and FAN proteins could be detected in a
complex with TNF-R55. For this purpose, TNF-R55 was immunoprecipitated
from nontransfected 293 cells, and the presence of endogenous FAN and
RACK1 in the immunocomplexes was analyzed with Abs against the
endogenous proteins. In the case of FAN, a very faint band was detected
(Fig. 5C, lane 2). This band exactly corresponds
to the predicted size of the FAN protein, and also exactly corresponds
to the more prominent band detected in a control immunoprecipitation of
the total endogenous FAN protein followed by Western blot with the same
Ab (Fig. 5C, lane 1). Similarly, a band of the
predicted size of RACK1 was detected in the TNF-R55 immunoprecipitates
by the Ab against endogenous RACK1 (Fig. 5D). Identical
results (with proportionally stronger bands) were obtained when 293
cells transfected with pMYC.RACK1–317 or
pFLAG.FAN3–917 were analyzed (data not
shown). In summary, these data suggest that not only overexpressed but
also endogenous FAN and RACK1 are present in a complex with TNF-R55 in
intact cells.
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The intracellular distribution of FAN and RACK1 was investigated
by indirect immunofluorescence staining in combination with confocal
laser-scanning microscopy. Because the FAN Ab used in the
coimmunoprecipitation experiments did not recognize endogenous FAN with
sufficient specificity for immunofluorescence analyses (data not
shown), we analyzed COS-1 cells that were transiently transfected with
FLAG-tagged FAN (pFLAG.FAN3–917) in
combination with myc-tagged RACK1
(pMYC.RACK1–317) and detected the tagged
proteins with anti-FLAG and anti-myc Abs. Both FAN
(Fig. 6A, green) and—in line
with previous reports (27, 39, 42, 50)—RACK1 (Fig. 6B, red) were detected in the cytoplasm, but not in the
nucleus of the cells. The overlay image shows some overlap in the
localizations of FAN and RACK1 (Fig. 6C, yellow). However,
the association of FAN andRACK1 became much more prominent in
experiments in which we additionally transfected a truncated form of
TNF-R55 (TNF-R55345). This mutant contains a C-terminal deletion
that destroys the death domain, thereby eliminating the cytotoxic
effects of an overexpressed TNF-R55, but still carries a functional NSD
that permits binding of FAN (4, 49). In the presence of
TNF-R55345 (Fig. 6D, green), FAN (Fig. 6E,
blue) and RACK1 (Fig. 6F, red) showed a translocation from
the cytoplasm to the plasma membrane, and a pronounced colocalization
of all three proteins at the plasma membrane was detected in the
overlay of the individual detection channels (Fig. 6G,
white). In experiments in which only
pFLAG.FAN3–917 and pEF.TNF-R55345 were
transfected into COS-1 cells, an identical colocalization of FAN and
TNF-R55 at the plasma membrane could be observed as yellow coloring
(Fig. 6, H–J). However, when COS-1 cells were transfected
with pEF.TNF-R55345 in combination with
pMYC.RACK1–317, RACK1 did not colocalize with
TNF-R55 but stayed distributed throughout the cytoplasm (Fig. 6, K–M). This lack of colocalization did not change after
treatment of cells with TNF (although TNF clearly induced
internalization of TNF-R55; Fig. 6, N–P), indicating that
the endogenous FAN present in these cells is most likely not abundant
enough to induce a detectable colocalization of the overexpressed
TNF-R55 and RACK1 molecules, regardless of the presence of TNF.
Although experiments with overexpressed proteins have to be interpreted
with care, these results suggest that the association of FAN and RACK1
is apparently enhanced by the membrane receptor TNF-R55. As shown
above, TNF-R55 most likely first forms a complex with FAN at the plasma
membrane (Fig. 6, H–J). This complex may then recruit RACK1
more efficiently (Fig. 6, D–G) than FAN alone recruits
(Fig. 6, A–C). As TNF-R55 is expressed only in very low
receptor numbers due to the intrinsic cytotoxicity of its cytoplasmic
domain (51), the TNF-R55-FAN-RACK1 complexes might not
become visible in cells expressing only the endogenous TNF-R55 (Fig. 6C).
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Because FAN is required for the activation of N-SMase by TNF, we
investigated whether RACK1 is also part of the signaling
pathway leading from TNF-R55 to N-SMase. 293 cells were
transiently transfected with control vector
pcDNA3.1-/Myc-His A or with
pFLAG.FAN3–917,
pMYC.RACK1–317 or pEF.TNF-R55345 either as
individual plasmids or as a combination of all three constructs. As
shown in Fig. 7 and in line with previous
results (6), transfection of
pFLAG.FAN3–917 enhanced the activity of N-SMase
after TNF treatment. In contrast, transfection of
pMYC.RACK1–317 consistently resulted in a
diminished stimulation of N-SMase by TNF. Overexpression of
TNF-R55345 did not significantly alter the response of N-SMase
toward TNF, confirming that TNF-dependent N-SMase activity is not
elevated by stable expression of high numbers of noncytotoxic TNF-R55
deletion mutants (4). Similar to the colocalization
experiments where combined expression of all three proteins showed the
most pronounced colocalization, the simultaneous transfection of
pFLAG.FAN3–917,
pMYC.RACK1–317, and pEF.TNF-R55345 into
293 cells resulted in the strongest increase in the sensitivity
of N-SMase toward TNF, supporting the notion that a complex of TNF-R55,
FAN, and RACK1 may be most efficient in stimulating N-SMase. Taken
together, these results suggest that RACK1 has a modulating function in
N-SMase activation, and implicate RACK1 as a novel component in the
signaling pathways of TNF-R55.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains of heterotrimeric G proteins (52), the
phospholipase A2-activator protein PLAP
(53), the regulatory subunit of phosphatase 2A
(54), as well as FAN and RACK1 themselves (6, 19).
Here, we report the isolation of RACK1 as a FAN-binding protein
and present evidence that RACK1 modulates activation of N-SMase by TNF.
The interaction of RACK1 and FAN was established by multiple
independent methods, including 1) the yeast interaction trap system, 2)
the binding of in-vitro-translated FAN to a bacterially expressed
GST-RACK1 fusion protein, 3) coimmunoprecipitation experiments of
overexpressed as well as endogenous proteins, and 4) colocalization
studies of FAN, RACK1, and TNF-R55 in whole cells. Our results indicate
that the interaction of FAN and RACK1 most likely requires folding of
the WD repeats into a secondary structure–possibly the proposed
multibladed-propeller structure—because no prominent linear
protein-binding motifs were identified within the WD-repeat region of
FAN. Deletion studies confirm that binding of FAN and RACK1 requires a
crucial region in the C-terminal WD-repeat region of FAN. RACK1, in
turn, interacts with FAN through a region comprising at least parts of
WD repeat V to WD repeat VI. Because this region is sufficient
for binding, it is conceivable that folding of RACK1 into a
complete seven-bladed-propeller structure is not essential for its
association with FAN. The very same region of RACK1 has also been
implicated in its binding to the integrin subunit and to the HIV-1
Nef protein as well as to the cAMP-specific phosphodiesterase isoform
PDE4D5 (23, 33, 55). Obviously, this region constitutes an
interface through which RACK1 can interact with various different
proteins.
We have shown that, upon overexpression as tagged proteins, full-length RACK1 coimmunoprecipitates with full-length FAN and vice versa, demonstrating that the two proteins form a complex in intact cells. However, because RACK1 can bind to many different proteins, the significance of its association with FAN in artificial systems, such as yeast, or upon overexpression had to be verified by additional experiments. By immunoprecipitating the endogenous TNF-R55 from nontransfected cells, we were able to demonstrate the presence of endogenous FAN and RACK1 in the immunocomplexes by Western blots. Despite the limited specificity of the currently available reagents against endogenous FAN and RACK1, these experiments provide unequivocal evidence that both endogenous FAN and RACK1 do form a complex with TNF-R55 in untransfected cells, further strengthening the notion of RACK1 as a mediator of TNF-R55-induced signals. However, the question remains whether RACK1 precipitates by direct interaction with TNF-R55 or indirectly via endogenous FAN that is bound to TNF-R55. Because we had also isolated a fragment of RACK1 during the yeast interaction trap experiments in which FAN was identified as an interactor of TNF-R55 (our unpublished observation), we currently cannot exclude the possibility that RACK1 and TNF-R55 are able to interact directly to some extent, at least in the yeast system. In support of these findings, our colocalization data suggest that FAN and TNF-R55 form a complex at the plasma membrane, to which RACK1 colocalizes more efficiently than to either FAN or TNF-R55 alone. Therefore, we propose that TNF-R55, FAN, and RACK1 may be able to form a trimolecular complex in which all three proteins contain binding sites for the other two partners, similar to a complex between PKC, RACK1, and PH domain-containing proteins described by Rodriguez et al. (43). However, the formal proof for the existence of a trimolecular complex not only of overexpressed but also of endogenous TNF-R55, FAN, and RACK1 will have to await the development of more specific reagents.
Definitive evidence for a role of RACK1 in the signaling machinery of
TNF-R55 is provided by our observation that RACK1 modulates the
sensitivity of N-SMase toward TNF. The inhibitory effect of
overexpressed RACK1 on the activation of N-SMase by TNF suggests that
an excess of RACK1 may interfere by binding to endogenous FAN, thereby
making it unavailable for the transduction of signals from TNF-R55.
Notably, overexpression of TNF-R55345 alone did not significantly
alter activation of N-SMase by TNF, while overexpression of FAN
enhanced the response of N-SMase to TNF. Thus, it appears that, in
normal cells, the response of N-SMase to TNF seems to be limited by
downstream components of the signaling pathway, such as FAN. RACK1
apparently also belongs to these components, because the simultaneous
overexpression of TNF-R55345, FAN, and RACK1 increased the
responsiveness of N-SMase to TNF in a substantial manner when compared
with any individual transfection. In summary, these results clearly
define RACK1 as a functional component of the signaling pathway from
TNF-R55 to N-SMase.
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Acknowledgments |
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Footnotes |
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2 A.E.T. and S.A.-K. contributed equally to this paper.
3 Address correspondence and reprint requests to Dr. Dieter Adam, Institut für Immunologie, Christian-Albrechts-Universität Kiel, Michaelisstrasse 5, D-24105 Kiel, Germany. E-mail address: dadam{at}email.uni-kiel.de
4 Abbreviations used in this paper: N-SMase, neutral sphingomyelinase; NSD, N-SMase-activating domain; BEACH, Beige and CHS; FAN, factor associated with N-SMase activation; NP40, Nonidet P-40; PH, pleckstrin homology; PKC, protein kinase C; cPLA2, cytosolic phospholipase A2; RACK, receptor for activated C-kinase.
Received for publication March 1, 2002. Accepted for publication August 26, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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