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*
Intramural Research Support Program, Science Application International Corp.-Frederick, and
Basic Research Laboratory, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702;
Department of Medicine and Biochemistry/Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202; and
Department of Integrative Biology and Pharmacology, University of Texas, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain,
IL-2 receptor 
-chain, and erythropoietin receptor, can be
polyubiquitinated and degraded by proteasomes. The Ub-Pr pathway
regulates both the basal level turnover and the ligand-induced
degradation of the receptors. A previously identified putative
molecular chaperon, valosin-containing protein, undergoes tyrosine
phosphorylation in a cytokine-dependent manner and associates with the
receptor complexes following receptor engagement, suggesting that
valosin-containing protein may target the ubiquitinated receptors to
the proteasome for degradation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 26S proteasome is a large multisubunit complex (see Refs. 4, 5, 6 for a review) that is present in the nucleus and the cytoplasm and is frequently associated with the ER. Although many substrates of the Ub-Pr pathway have been identified as cytoplasmic or nuclear proteins, recent data indicate that this pathway also plays a role in the degradation of proteins involved in the membranous vacuolar system (see Refs. 7, 8, 9, 10 for a review). The vacuolar system consists of the secretory and endocytic/lysosomal pathways, including the plasma membrane, ER, Golgi apparatus, lysosomes, and various transport vesicles. The current model holds that unassembled proteins or abnormal proteins retained in the ER lumen can be retrotransported to the cytosolic face of the ER membrane, ubiquitinated, and ultimately degraded by the proteasomes. Prominent examples include the cystic fibrosis trans-regulator (11, 12), MHC class I heavy chains (13, 14), and TCR subunits (15, 16).
Ubiquitination of cell surface proteins is an evolutionarily conserved
process found in both yeast and mammalian cells. Recent evidence
suggests that ubiquitination can target cell surface receptors for
degradation by a lysosome- or proteasome-dependent pathway. In
Saccharomyces cerevisiae, ubiquitination of cell surface
receptors triggers the internalization and subsequent degradation via
the endocytic lysosomal pathway (8). Binding of mating
factor to Ste2p, a G protein-coupled plasma membrane receptor, induces
ubiquitination of the Ste2p cytoplasmic tail that stimulates the
internalization and degradation of the receptor-ligand complex by
lysosomes. In this example, polyubiquitination is not required, because
monoubiquitination is sufficient to trigger the degradative event
(17). In contrast to the role of ubiquitination in
endocytosis in yeast, its role in the regulation of mammalian plasma
membrane proteins is less clear. In animal cells, polyubiquitination
has been found to act on several cell surface receptor proteins,
including TCR -, 
-, and 
-chains, growth hormone, IgE,
epithelial growth factor (EGF), fibroblast growth factor (FGF),
platelet-derived growth factor (PDGF), CSF, stem cell factor (c-Kit),
and hepatocyte growth factor (c-Met) (see Refs. 7, 8, 9, 10 for
a review). Thus, ubiquitination serves to target these receptors for
internalization and degradation via the proteasome- or
lysosome-mediated pathway. Although the Ub-Pr pathway has been shown to
play an important role in regulating the degradation of c-Met
(18), PDGF receptor (19), TCR (15, 16), and TCR (16) chains, most of the
polyubiquitinated tyrosine kinase-type receptors (e.g., receptors for
EGF, PDGF, FGF, and c-Kit) are believed to be degraded via the
endocytic lysosomal pathways. In this report we demonstrate that the
non-tyrosine kinase-type cytokine receptors, IL-9 receptor -chain
(IL-9R), IL-2R -chain (IL-2R), and erythropoietin receptor
(EpoR) can be polyubiquitinated and degraded by the Ub-Pr pathway. In
addition, we report that these receptors coimmunoprecipitate with a
putative molecular chaperone, valosin-containing-protein (VCP),
previously shown to be a physical and functional link between the
ubiquitinated substrates and the proteasome (20). The
level of the coprecipitating VCP was found to directly correlate with
the level of receptor ubiquitination, further supporting a model of
Ub-Pr-mediated degradation of cytokine receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine T cell clone, TS1, was maintained in Click’s medium
(Irvine Scientific, Santa Ana, CA) containing 10% FCS, 2 mM
L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin,
and 1 ng/ml mIL-9 (R&D Systems, Minneapolis, MN). TS1 cells stably
transfected with human IL-9R variants (21) were
cultured in the same medium supplemented with 1 mg/ml G418 (Sigma, St.
Louis, MO). For cytokine stimulation, cells were cultured in medium
without IL-9 and serum for at least 15 h, then collected and
stimulated with 30 ng/ml recombinant human IL-9. YT cells (human
NK-like cell line) (22) were maintained in RPMI 1640
medium (BioWhittaker, Walkersville, MD) supplemented with 10% FCS, 2
mM L-glutamine, and antibiotics. Before stimulation with 10
nM human rIL-2 (Hoffmann-La Roche, Nutley, NJ), cells were starved in
medium without serum for at least 8 h. HCD-57 cells, an
Epo-dependent erythroleukemia cell line derived from an National
Institutes of Health Swiss mouse infected with Friend-murine leukemia
virus (23), were cultured in IMDM (Life Technologies,
Gaithersburg, MD) supplemented with 30% FCS, 50 µM 2-ME, 3 mg/ml
L-glutamine, antibiotics, and 0.3 U/ml Epo. Cells were
starved in medium containing 1.5% FCS and no Epo for 15 h before
Epo stimulation (100 U/ml). During the last 30 min of starvation, 0.05
nM sodium orthovanadate was added to the culture to inhibit phosphatase
activities.
Antisera and reagents
Polyclonal antiserum to Ub was purchased from Dako (Carpinteria,
CA). mAb against Flag (M2 clone), FITC-labeled anti-Flag, and
anti-phosphoserine antiserum were purchased from Sigma. Antisera
recognizing IL-2R (22) and VCP (anti-VCP-4 and -5
in Ref. 20) have been described previously. Anti-pY
(4G10), anti-EpoR, anti-STAT5a, and anti-Janus tyrosine
kinase-2 (anti-JAK2) sera were purchased from Upstate Biotechnology
(Lake Placid, NY; no. 05-321, 06-406, 06-553, and 06-255,
respectively). Anti-JAK3 and anti-STAT5b sera were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA; sc-513 and sc-835,
respectively). When anti-STAT5 immunoblotting was performed,
combined sera from equal volumes of anti-STAT5a and anti-STAT5b
were used. Proteasome inhibitors, lactacystin, LLnL (also known as
calpain inhibitor I,
N-acetyl-L-leucinyl-L-leucinyl-norleucinal),
and Z-Leu-Leu-Leu-H (aldehyde) (ZLLH) (also known as MG132) were
obtained from Affinity Research Products (Mamhead Castle, Mamhead,
U.K.), Sigma, and Peptide Institute (Osaka Japan), respectively. PMSF,
N-p-tosyl-L-phenylalanine
chloromethyl ketone,
N--p-tosyl-L-lysine
chloromethyl ketone, iodoacetamide (an isopeptidase inhibitor), and all
phosphatase inhibitors were purchased from Sigma.
Pulse-chase analysis
TS1 cells expressing the Flag-tagged wild-type IL-9R were
metabolically labeled with 0.1 mCi/ml (1000 Ci/mmol)
[35S]methionine/cysteine (ICN, Costa Mesa, CA)
at a density of 5 x 106/ml for 3 h. In
the final hour, 100 µM LLnL was added to one set of cultures to
inhibit the proteasome-mediated proteolysis, while control culture was
treated with DMSO alone. The cells were washed three times with PBS,
then cultured in fresh, nonradioactive medium either with or without
LLnL. Cells were harvested at various time points, washed, and
subjected to immunoprecipitation.
Plasma membrane isolation and flow cytometric analysis
For biochemical analysis, plasma membranes were isolated from
IL-9R-expressing TS1 cell as described by Thom et al.
(24), except that calcium was omitted. For flow cytometric
analysis, cells were starved for 15 h, then stimulated with IL-9
in the absence or the presence of proteasome inhibitor (LLnL or ZLLH)
for 20 min. Cells (1 x 106) from each
stimulated culture were washed with FACS buffer (1% BSA and 0.02%
sodium azide in PBS) and stained with FITC-conjugated anti-Flag Abs
(1 µg) or mouse isotype-matched control for 15 min at 4°C. After
washes, labeled cells were analyzed on a FACScan flow cytometer (Becton
Dickinson, Mountain View, CA).
Immunoprecipitations and immunoblot (Western) analyses
Both analyses were performed as described previously (20, 25) with minor modifications. Cells were lysed in 20 mM Tris-HCl
(pH 7.6), 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100 containing
protease inhibitors (1% aprotinin, 70 µg/ml PMSF, 40 µg/ml
N-p-tosyl-L-phenylalanine
chloromethyl ketone, 5 µg/ml
N--p-tosyl-L-lysine
chloromethyl ketone, 5 µg/ml leupeptin, and 50 µM LLnL),
isopeptidase inhibitor (10 mM iodoacetamide), and phosphatase
inhibitors (1 mM sodium orthovanadate, 30 mM sodium pyrophosphate, 0.4
mM -glycerophosphate, 50 mM sodium fluoride, and 10 µM sodium
molybdate). The lysates were clarified by centrifugation at 12,000
x g for 30 min and incubated with antisera. The immune
complexes were collected with protein A-conjugated Sepharose beads;
washed with 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, and 1%
Triton X-100; boiled in SDS-gel dissociation buffer; resolved by
SDS-PAGE; electrophoretically transferred onto polyvinylidene fluoride
(PVDF) membranes (Millipore, Bedford, MA), and further analyzed by
autoradiography or immunoblotting. For immunoblot analysis, equal
amounts of protein or immune complexes were resolved by SDS-PAGE and
transferred onto membranes. The membrane was blocked by milk buffer
(20, 25), washed, and incubated with antiserum (typically
at 1/1,000), followed by reaction with peroxidase-conjugated
anti-rabbit Ig serum (Roche, Indianapolis, IN) or anti-mouse
IgG (Southern Biotechnology Associates, Birmingham, AL), then developed
by the enhanced chemiluminescence detection system (ECL; Amersham,
Arlington Heights, IL). When serial blotting analyses were performed,
the previous Abs were removed by incubating the membrane in 60 mM
Tris-HCl (pH 6.8), 2% SDS, and 0.1 M 2-ME at 50°C for 30
min.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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turnover by the Ub-Pr pathway
IL-9 was initially identified as a growth-promoting factor for the
Ag-independent proliferation of murine Th cell clones
(26). Since then, its recognized biological functions have
expanded significantly to include direct effects on activated T cells,
B cells, mast cells, and hemopoietic progenitors (27, 28).
The functions of IL-9 are mediated by the IL-9R, which is a
non-tyrosine kinase member of the cytokine receptor superfamily. IL-9R
consists of a ligand-specific -chain and IL-2R 
-chain or common
-chain (c), which is shared by receptors
for IL-2, IL-4, IL-7, IL-9, and IL-15 (29, 30, 31, 32). To study
the turnover of IL-9R, murine TS1 cells stably transfected with
Flag-tagged human IL-9R (21) were used. Pulse-chase
experiments were performed in these cells in the absence or the
presence of the proteasome inhibitor, lactacystin or LLnL, over a
period of 180 min. As shown in Fig. 1
A, IL-9R was significantly
stabilized in the presence of LLnL (compare lanes 5 and
6 with lanes 11 and 12). In addition,
high Mr forms of receptors appear as a
broad band, typical of ubiquitinated proteins, accumulated at later
time points (marked by an asterisk). To identify the high
Mr proteins, the membrane was
immunoblotted with receptor-specific (Fig. 1B, top
panel) or Ub-specific (bottom panel) antiserum. Common
immunoreactivities were detected in both blots (lanes
11 and 12), confirming that they represent the
ubiquitinated receptors. Similar data were also obtained in experiments
conducted in the presence of lactacystin (data not shown). Because
these TS1 cells were cultured in the presence of a low concentration of
murine IL-9 (1 ng/ml), which does not bind to human IL-9R, we assume
that the measured turnover represents basal level degradation of human
IL-9R. Together, these results strongly suggest that the turnover of
IL-9R can be regulated by the Ub-Pr pathway. Because a number of
surface receptors have been shown to be ubiquitinated and targeted to
the lysosome for degradation (8), we examined whether the
lysosomal pathway plays a significant role in regulating the turnover
of IL-9R (Fig. 1C). IL-9R-expressing TS1 cells were
treated either with the lysosomal inhibitors, chloroquine and ammonium
chloride, or with the proteasomal inhibitor, LLnL. The cell lysates
were then analyzed by anti-receptor immunoprecipitation followed by
immunoblotting with anti-receptor or anti-Ub antiserum.
Although IL-9R molecules were detectably stabilized by the lysosomal
inhibitors, a higher level of receptor stabilization (Fig. 1C, upper panel) and ubiquitination (Fig. 1C, lower panel) was observed. These results
suggest that both lysosomes and proteasomes can regulate the turnover
of IL-9R, and the Ub-Pr pathway plays a more prominent role under
our experimental conditions.
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A); thus, the surface
receptors can be ubiquitinated. These receptors presumably can
internalize and be targeted for proteasomal degradation and/or
contribute to the intracellular receptor pool. These results were
further supported by flow cytometric studies, which demonstrated that
while the control cells lost surface receptors after ligand
stimulation, the same or increased numbers of surface receptors were
detected in proteasome inhibitor-treated cells (Fig. 2B).
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We next examined whether other non-tyrosine kinase-type cytokine
receptors, IL-2R and EpoR, are also regulated by the Ub-Pr pathways.
The IL-2 heterotrimeric receptor consists of an affinity-conferring
-chain, a ligand-specific -chain (IL-2R), and the
c (29, 32), whereas the EpoR
consists of homodimeric polypeptide chains (EpoR) (33).
The human YT cell line (22), which readily expresses
IL-2R and c, was used to study the
regulation of IL-2R degradation. Moreover, the murine HCD-57 cell line
(23), which expresses EpoR, was used to assess receptor
degradation. Both cell lines were treated with proteasome inhibitors
for various periods of time, and cell lysates were immunoprecipitated
with receptor-specific antisera. The precipitates were analyzed by
serial immunoblotting with receptor-specific and Ub-specific antisera.
Both Fig. 3A and Fig. 3B show an accumulation of high
Mr species, typical of ubiquitinated
receptors. Taken together, these data support the involvement of
Ub-Pr-mediated degradation of IL-2R and EpoR.
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Because polyubiquitination is capable of targeting the surface
receptors to both lysosomal and proteasomal degradations, we asked what
factors determine the final destination of the ubiquitinated receptor,
or more specifically, what directs certain ubiquitin-conjugated
substrates to the proteasome rather than lysosome (or vice versa). One
possible control point could be the association with specific molecular
chaperones that transfer the substrate to either the proteasome or the
lysosome. Previously, we demonstrated that VCP, a highly conserved
ATPase (34), is physically and functionally involved in
the Ub-Pr-mediated degradation of I
B (20). We
proposed a mechanism by which VCP acts as a molecular chaperone that
targets the ubiquitinated IB to the 26S proteasome for
degradation (20). Based on the hypothesis, we next tested
whether VCP may act as a general chaperone that also targets other
Ub-Pr substrates, including IL-2R, IL-9R, and EpoR, to the proteasome
for degradation.
It is well established that upon cytokine binding to the non-tyrosine
kinase receptors, the JAKs, physically associate and phosphorylate the
cytoplasmic domain of the receptors. The phosphorylated tyrosine
residues of the activated receptors serve as docking sites for the
recruitment of the transcription regulators, STATs, which normally
exist as inactive monomers in the cytoplasm. The receptor-associated
STATs become phosphorylated on tyrosines by JAKs, dissociate from the
respective receptors, dimerize, translocate to the nucleus, and
regulate specific cytokine-inducible genes (reviewed in Refs.
35, 36, 37). To study the involvement of VCP in the IL-2
signaling pathway, we stimulated YT cells with IL-2 and subjected the
cell lysates to anti-phosphotyrosine (pY) immunoprecipitation. The
precipitates were analyzed by immunoblotting with various antisera to
identify tyrosine-phosphorylated components captured within the immune
complex. As shown in Fig. 4A,
the receptor complex contained tyrosine-phosphorylated proteins with
sizes of 120, 97, and 70–75 kDa, similar to those of JAK3, STAT5/VCP,
and IL-2R, respectively (Fig. 4, top panel). Subsequent
immunoblotting confirmed that the 120- and 70/75-kDa proteins contain
JAK3 and IL-2R (data not shown), respectively. Phosphorylation of
p97 has been reported as a common event following stimulation of
responsive cells with a wide variety of cytokines/growth factors
(38, 39, 40). Western analysis showed the 97-kDa species to
consist of both STAT5 (STAT5a and STAT5b) and VCP, and thus is a
mixture. To demonstrate that VCP is itself tyrosine phosphorylated in
response to cytokine stimulation, cell lysates from IL-2-treated YT
cells were immunoprecipitated with anti-VCP antiserum followed by
immunoblotting with anti-pY Abs. As shown in Fig. 4B,
VCP was phosphorylated on tyrosine residues in an IL-2-dependent
fashion, while it was constitutively phosphorylated on serines in YT
cells (Fig. 3
B). Furthermore, when cell lysates were
immunoprecipitated with IL-2R-specific antiserum and subsequently
blotted with VCP antiserum, VCP was found to clearly associate with the
receptor complex (Fig. 4C). Because VCP and STAT5 are both
tyrosine phosphorylated following IL-2 stimulation and comigrate at
97 kDa, it is important to show that the STAT5 and VCP antisera do
not cross-react. The respective immunoprecipitation followed by
immunoblotting analyses clearly showed that the antisera are specific,
and no cross-reactivity was detected (Fig. 4D). Taken
together, these results indicate that upon IL-2 stimulation, VCP is
tyrosine phosphorylated and physically associates with the receptor
complex, which also contains JAK3 and STAT5.
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To determine whether VCP is also involved in Ub-Pr-mediated
degradation of non-c-containing cytokine
receptors, we examined the EpoR. HCD-57 cells were stimulated with Epo,
and cell lysates were subjected to immunoprecipitations with
anti-pY or anti-EpoR Abs followed by immunoblotting with
antisera against pY or VCP (Fig. 5). Fig. 5A shows that VCP is detected in anti-pY
immunoprecipitates (upper panels), and VCP
physically interacts with the EpoR complex during stimulation
(lower panels) in a manner similar to
IL-2-treated cells (Fig. 4). To further characterize the components of
the receptor complex, cell lysates were immunoprecipitated with
antisera reactive to specific components of the complex and analyzed by
immunoblotting (Fig. 5B). Consistent with the results
presented above, EpoR, JAK2, STAT5, and VCP all undergo tyrosine
phosphorylation in an Epo-dependent manner (Fig. 5, upper
panel). However, VCP was barely detected in JAK2 and STAT5 immune
complexes (lower panel, lanes 3–6),
whereas a significantly increased level of VCP was detected in the EpoR
complex after Epo stimulation (compare lanes 1 and
2). These results indicate that VCP physically associates
with the receptor complex, and the interaction between VCP and
JAK2/STAT5 is more likely indirect.
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To investigate the significance of tyrosine phosphorylation in
receptor turnover, we first examined whether tyrosine-phosphorylated
receptors can be ubiquitinated (Fig. 6).
IL-9R-expressing TS1 cells were treated with the proteasome
inhibitor, ZLLH, for various periods of time. The cell lysates were
either directly analyzed by immunoblotting (lanes
1–3 and 7–9) or subjected to anti-phosphotyrosine
immunoprecipitation and immunoblotting (lanes 4–6
and 10–12). Both anti-receptor and anti-Ub
immunoblotting detected increased levels of high
Mr species (>200 kDa in
lanes 4–6 and lanes 10–12) during
the treatment. These results strongly suggest that the high
Mr proteins are tyrosine
phosphorylated and ubiquitinated IL-9R molecules. Thus, tyrosine
phosphorylated receptors can be ubiquitinated.
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associationBased on the data obtained to date, at least two possibilities could be envisioned to explain the involvement of VCP in the cytokine signaling pathways activated by IL-9, IL-2, and Epo. First, VCP may be a signal transducer and serve as an adaptor for other molecules (e.g., JAKs and STATs) in the signaling cascade. Under this assumption, a certain degree of specificity is expected with a prediction that some surface receptors defective in transducing signals would lose the ability to coprecipitate VCP. Second, VCP may act as a molecular chaperone that simply interacts with the ubiquitinated substrates, thus associating with various conjugates. In this scenario, a low degree of substrate specificity would be expected.
To differentiate between these two possibilities, we used a panel of
TS1 cell lines stably transfected with IL-9R mutants
(21) (summarized in Fig. 7)
to examine whether any cytoplasmic domains of IL-9R are critical for
VCP interaction. Previous work using these deletion and site-specific
mutants demonstrated that the BOX1 domain (aa 298–315) and the STAT3
binding motif (YLPQ) are essential for IL-9-mediated cell proliferation
and signal transduction (21) (Fig. 7). As shown in Fig. 8, while all the IL-9R
variants were efficiently expressed in TS1 cells (upper
panel), VCP coprecipitated with all but the D4 mutant
(lower panel). This indicates that VCP is still
capable of interacting with the receptors defective in IL-9-induced
signaling and/or proliferation, presumably through the ubiquitinated
domains. Because the D4 mutant only contains a four-residue cytoplasmic
tail, which is too short to interact with the ubiquitination machinery,
it is probably not ubiquitinated and thus could not interact with VCP.
This low substrate specificity supports the hypothesis that VCP
interacts with ubiquitinated substrates and targets them for proteasome
degradation. In accordance with this hypothesis, when the parental TS1
cells and TS1 cells expressing the wild-type or the D4 mutant of
IL-9R were analyzed (Fig. 9), the
ubiquitinated IL-9R -chains were only detected in the wild-type
cells (lanes 3 and 6), and not in the
control (lanes 1 and 4) or D4-expressing
cells (lanes 2 and 5). The lack of
ubiquitinated D4 molecules is probably not due to a general deficiency
of ubiquitination, because abundant ubiquitinated proteins were
detected in all cell lines (lanes 7–9).
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ubiquitination and
associated VCP
Our model predicts that while ubiquitinated receptors accumulate
in the cell, the level of associating chaperone, VCP, should also
increase. To address this hypothesis, we stimulated IL-9R-expressing
cells with human IL-9 in the presence of proteasome inhibitors and
analyzed cell lysates by anti-IL-9R immunoprecipitation followed
by immunoblotting with receptor- and VCP-specific antisera (Fig. 10). The results clearly demonstrate
that while ubiquitinated receptors accumulated (upper
panels), the coprecipitating VCP also increased (bottom
panel).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2R, and EpoR, can be regulated by the Ub-Pr-mediated
degradation pathway in both cytokine-dependent and independent
fashions. Although our data suggest a Ub-Pr mechanism, they do not
preclude a role for other proteolytic systems. We tested whether these
receptors are degraded through the lysosomal pathway by treating cells
with lysosome-specific inhibitors, such as weak alkalines and
chloroquine. Whereas varying degrees of receptor stabilization were
detected in all cases, a major population of the receptors was
stabilized and ubiquitinated in proteasome inhibitor-treated cells
(IL-9R study shown in Fig. 1C). Based on these results,
we conclude that although the receptors could be degraded by the
lysosomes, a significant proportion of the receptors is regulated by
the Ub-Pr pathways.
Cytokine binding to their respective receptors stimulates not only
positive activation pathways, such as the JAK/STAT pathway, but also
negative regulatory pathways, which are involved in the termination of
the signal transduction. Negative regulation can be achieved by at
least three families of proteins: suppressors of cytokine signaling,
protein inhibitors of activated STATs, and the
SH2-containing phosphatase (41). Another effective
negative regulatory pathway involves the down-regulation of cell
surface receptors. The present studies suggest that upon cytokine
stimulation, these receptors are tyrosine phosphorylated and
polyubiquitinated, and the ubiquitinated receptors are targeted to
proteasomal degradation through association with VCP, a
proteasome-associated putative chaperone. The hypothesis of VCP being a
proteasome-associated molecular chaperone that targets a wide variety
of ubiquitinated substrates to the proteasome is supported by several
lines of evidence: 1) VCP preferentially binds the polyubiquitinated
substrates (e.g., IB) rather than the nonubiquitinated substrates
(20); 2) VCP association with IB is necessary for
final degradation of IB by the 26S proteasome (20);
3) VCP physically associates with the 26S proteasome, copurifies with
the proteasome, and coimmunoprecipitates with subunits of the
proteasome (20); 4) Cdc48p (yeast orthologue of VCP) is
required for Ub-dependent proteolytic system in yeast
(42), and mutation of CDC48 gene results in cell cycle
arrest (43), which is probably a result of defective Ub-Pr
degradation; and 5) Koegl et al. (44) recently reported
that Cdc48p acts downstream from a polyubiquitin chain assembly enzyme,
E4, and upstream from the 26S proteasome in the Ub-Pr pathway.
Furthermore, E4 has been linked to a stress tolerance pathway,
which probably requires the mediation of molecular chaperones. 6) We
recently found that VCP is present in the immune complexes of other
Ub-Pr substrates, e.g., cyclins, and is necessary for their degradation
via the Ub-Pr pathway (data not shown). Taken together, it is apparent
that VCP physically and functionally links the Ub-Pr substrates and the
26S proteasome, supporting our model that VCP binds to ubiquitinated
receptors and preferentially targets them to the proteasome for
degradation. Whether VCP indeed functions as a molecular chaperone that
assists in the unfolding and subsequent degradation of these substrates
awaits to be determined.
A number of tyrosine kinase-containing surface receptors (e.g., EGF receptor, PDGF receptor, FGF receptor, and c-Kit) have been demonstrated to be degraded by the lysosomes after polyubiquitination (see Refs. 7, 8, 9, 10 for a review). It was suggested that the ubiquitin system is required in the signaling steps leading to receptor internalization and/or localization to the lysosomes. Because the molecular mechanisms involved in this signaling pathway are not well elucidated, we propose that there may exist other lysosome-associated chaperones or adaptors, and binding of the ubiquitinated receptors to either prolysosome or proproteasome chaperones determines the final destination of these receptors. Because our hypothesis predicts that VCP will bind a large population of ubiquitinated proteins, it would be interesting to examine whether these lysosome-destined ubiquitinated receptors also bind to VCP and, furthermore, whether VCP physically interacts with lysosomes.
Phosphorylation of a 97-kDa protein (p97) has been reported as a common
event following stimulation of hemopoietic cells with a wide variety of
cytokines, including GM-CSF, IL-2, IL-3, and Epo (38, 39, 40).
In this study we also observed a 97-kDa tyrosine-phosphorylated protein
activated by IL-2 (Fig. 4), Epo (Fig. 5), and IL-9 (data not shown) and
associated with the receptor complex. Previous characterization of p97
has led to the cloning and identification of STATs, which generally
have a molecular size of 97 kDa. The present study identified a
distinct protein, VCP, as another component of the p97 complex.
Therefore, p97 is not a single protein and may represent a mixture of
STAT and VCP molecules. Lastly, a recent report identified another
97-kDa protein, Gab2, as a cytokine-induced SH2-containing
phosphatase-binding protein in hemopoietic cells (45).
Whether this protein exists in a IL-2, Epo, or IL-9 receptor complex
remains to be determined.
In addition to the present study, tyrosine phosphorylation of VCP has been observed in a variety of cellular activities. Interestingly, VCP was originally identified as one of the first tyrosine-phosphorylated proteins in response to T cell activation and a component of the TCR-mediated tyrosine kinase activation pathway (34). It was also shown that stimulation of B cells with hydrogen peroxide induces high levels of tyrosine phosphorylation of VCP (46). Furthermore, tyrosine phosphorylation of Cdc48p results in its nuclear translocation during late G1 phase; thus, it is believed to play a role in cell cycle control (47). These observations suggest that phosphorylation of VCP must play a role in a common mechanism that underlies these seemingly unrelated activities. We propose that VCP is tyrosine phosphorylated following activation of the Ub-Pr pathway and represents another mechanism for down-regulating the activation signal distinct from suppressors of cytokine signaling, protein inhibitors of activated STATs, and phosphatases. Further work is required to examine how tyrosine phosphorylation activates VCP in the context of the Ub-Pr pathway. Preliminary experiments suggest that tyrosine phosphorylation is not required for VCP association with the ubiquitinated substrates (data not shown), but probably triggers a later event in the degradation pathway.
Whether other components of the receptor complex, such as the
cytokines, JAKs, or STATs, are also degraded by the proteasome is not
clear. What is clearly evident from our cytokine signaling studies is
an increase in receptor ubiquitination over time (
Figs. 1–3, 6, and
10), as revealed by anti-Ub Western analyses. These findings
support the idea that the entire receptor complex may be targeted for
proteasomal degradation. Thus, even though JAK and STAT proteins may
not be directly ubiquitinated, they may be degraded with the receptors
gratuitously.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chou-Chi H. Li, Science Applications International Corp.-Frederick, Laboratory of Leukocyte Biology, National Cancer Institute-Frederick Cancer Research and Development Center, P.O. Box B, Building 567, Room 252, Frederick, MD 21702-1201.
3 Abbreviations used in this paper: Ub-Pr, ubiquitin-dependent proteasome-mediated degradation; ER, endoplasmic reticulum; EGF, epithelial growth factor; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; Epo, erythropoietin; EpoR, Epo receptor; VCP, valosin-containing-protein; LLnL, N-acetyl-L-leucinyl-L-leucinyl-norleucinal; PVDF, polyvinylidene fluoride; c, common -chain; JAK, Janus tyrosine kinase; ZLLH, Z-Leu-Leu-Leu-H (aldehyde).
Received for publication December 9, 1999. Accepted for publication September 5, 2000.
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chain of the T cell antigen receptor is degraded in the cytosol. Immunity 7:113.[Medline]
receptor involves ubiquitin-proteasome proteolytic pathway. J. Biol. Chem. 270:29447.B and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IB. J. Biol. Chem. 273:3562. chain in interleukin-9-mediated cell proliferation and signal transduction. J. Biol. Chem. 272:21334.-subunit. J. Biol. Chem. 268:22765. chain of the human IL-2 receptor. Science 257:379. and chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13:2822.[Medline]
chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu. Rev. Immunol. 14:179.[Medline]
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