The ubiquitin-dependent proteasome-mediated (Ub-Pr) degradation pathway has been shown to regulate a large variety of substrates, including nuclear, cytosolic, and membrane proteins. In mammalian systems, polyubiquitin modification has been identified in a number of cell surface receptors for more than a decade; however, its biological significance has remained unclear until recently. For growth factor receptors with intrinsic tyrosine kinase domains, polyubiquitination is believed to trigger the internalization and subsequent degradation via the lysosomal pathway. In this study we provide the first evidence that non-tyrosine kinase-type cytokine surface receptors, IL-9R α-chain, IL-2 receptor β-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.

The ubiquitin-dependent proteasome-mediated degradation (Ub-Pr)3 pathway regulates a wide variety of cellular activities, including cell growth, differentiation, immune and inflammatory responses, endoplasmic reticulum (ER) degradation, gene transcription, and intracellular signaling pathways that regulate apoptosis, senescence, and oncogenesis (see Refs. 1, 2, 3 for a review). The Ub-Pr pathway consists of two sequential steps: 1) short-lived and long-lived substrates, which include abnormal proteins, are first conjugated with polyubiquitin chains that tag the substrate for degradation; then 2) ubiquitinated substrates, presumably assisted by a molecular chaperone(s), are transferred to the proteasome where they are denatured and degraded.

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 α 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 α-, δ-, and ζ-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), TCRα (15, 16), and TCRδ (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 α-chain (IL-9Rα), IL-2R β-chain (IL-2Rβ), 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.

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-9Rα 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.

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-2Rβ (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-α-p-tosyl-l-lysine chloromethyl ketone, iodoacetamide (an isopeptidase inhibitor), and all phosphatase inhibitors were purchased from Sigma.

TS1 cells expressing the Flag-tagged wild-type IL-9Rα were metabolically labeled with 0.1 mCi/ml (1000 Ci/mmol) [35S]methionine/cysteine (ICN, Costa Mesa, CA) at a density of 5 × 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.

For biochemical analysis, plasma membranes were isolated from IL-9Rα-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 × 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).

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-α-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 β-glycerophosphate, 50 mM sodium fluoride, and 10 μM sodium molybdate). The lysates were clarified by centrifugation at 12,000 × 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.

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 α-chain and IL-2R γ-chain or common γ-chain (γc), 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-9Rα (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-9Rα 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. 1,B, 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-9Rα. Together, these results strongly suggest that the turnover of IL-9Rα 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-9Rα (Fig. 1,C). IL-9Rα-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-9Rα molecules were detectably stabilized by the lysosomal inhibitors, a higher level of receptor stabilization (Fig. 1,C, upper panel) and ubiquitination (Fig. 1 C, lower panel) was observed. These results suggest that both lysosomes and proteasomes can regulate the turnover of IL-9Rα, and the Ub-Pr pathway plays a more prominent role under our experimental conditions.

FIGURE 1.

Stabilization of IL-9Rα and accumulation of ubiquitinated IL-9Rα in proteasome inhibitor-treated cells. A, TS1 cells expressing Flag-tagged wild-type IL-9Rα were metabolically labeled with [35S]methionine and [35S]cysteine for 3 h, then chased with fresh medium without radioactivity from 0–180 min in the absence (lanes 1–6) or the presence (lanes 7–12) of the proteasome inhibitor, LLnL. At the indicated time points (minutes), cells were lysed, and the lysates were immunoprecipitated with anti-Flag Abs. Precipitates were resolved by SDS-PAGE, transferred to PVDF membrane, and visualized by autoradiography. B, The membrane was subsequently immunoblotted with anti-Flag Abs (upper panel) and anti-Ub antiserum (lower panel). IL-9Rα is indicated on the right, and the asterisk designates the high Mr ubiquitinated IL-9Rα conjugates. C, Cells were treated with chloroquine (0.15 mM), LLnL (100 mM), or ammonium chloride (20 mM) for the indicated period (hours). The cell lysates were immunoprecipitated with anti-Flag Abs, and the precipitates were analyzed by SDS-PAGE followed by immunoblotting with anti-Flag (upper panel) or anti-Ub (lower panel) antiserum. The bottom band in both panels represents the Ig reactivity. The heavy band around and above 68 kDa in the top panel is the nonubiquitinated IL-9Rα.

FIGURE 1.

Stabilization of IL-9Rα and accumulation of ubiquitinated IL-9Rα in proteasome inhibitor-treated cells. A, TS1 cells expressing Flag-tagged wild-type IL-9Rα were metabolically labeled with [35S]methionine and [35S]cysteine for 3 h, then chased with fresh medium without radioactivity from 0–180 min in the absence (lanes 1–6) or the presence (lanes 7–12) of the proteasome inhibitor, LLnL. At the indicated time points (minutes), cells were lysed, and the lysates were immunoprecipitated with anti-Flag Abs. Precipitates were resolved by SDS-PAGE, transferred to PVDF membrane, and visualized by autoradiography. B, The membrane was subsequently immunoblotted with anti-Flag Abs (upper panel) and anti-Ub antiserum (lower panel). IL-9Rα is indicated on the right, and the asterisk designates the high Mr ubiquitinated IL-9Rα conjugates. C, Cells were treated with chloroquine (0.15 mM), LLnL (100 mM), or ammonium chloride (20 mM) for the indicated period (hours). The cell lysates were immunoprecipitated with anti-Flag Abs, and the precipitates were analyzed by SDS-PAGE followed by immunoblotting with anti-Flag (upper panel) or anti-Ub (lower panel) antiserum. The bottom band in both panels represents the Ig reactivity. The heavy band around and above 68 kDa in the top panel is the nonubiquitinated IL-9Rα.

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To determine whether the intracellular or the surface membrane receptor was ubiquitinated, TS1 cells were treated with or without the proteasome inhibitor, ZLLH, and the two forms of receptors were isolated. Immunoprecipitation and Western blotting experiments showed that ubiquitinated receptors can be detected in both fractions (Fig. 2,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. 2 B).

FIGURE 2.

Ubiquitination and degradation of the cell surface receptors. A, IL-9Rα-expressing TS1 cells were left untreated or were treated with the proteasome inhibitor, ZLLH, for 3 h, and the cell lysates were separated into plasmic membrane (M) and intracellular (C) fractions. One tenth of the lysates were resolved by SDS-gel (lanes 1–4), and the rest were immunoprecipitated with anti-Flag Abs then resolved by the gel (lanes 5–8). After electrophoretic transfer, the membrane was immunoblotted with anti-Ub antiserum. The high Mr ubiquitinated receptors are marked with an asterisk. B, TS1 ells were stimulated with hIL-9 in the presence of nothing (control), LLnL, or ZLLH for 20 min. The surface expression of IL-9Rα was monitored by flow cytometry using FITC-conjugated anti-Flag Abs. The solid area and the open area represent staining before and after treatment, respectively.

FIGURE 2.

Ubiquitination and degradation of the cell surface receptors. A, IL-9Rα-expressing TS1 cells were left untreated or were treated with the proteasome inhibitor, ZLLH, for 3 h, and the cell lysates were separated into plasmic membrane (M) and intracellular (C) fractions. One tenth of the lysates were resolved by SDS-gel (lanes 1–4), and the rest were immunoprecipitated with anti-Flag Abs then resolved by the gel (lanes 5–8). After electrophoretic transfer, the membrane was immunoblotted with anti-Ub antiserum. The high Mr ubiquitinated receptors are marked with an asterisk. B, TS1 ells were stimulated with hIL-9 in the presence of nothing (control), LLnL, or ZLLH for 20 min. The surface expression of IL-9Rα was monitored by flow cytometry using FITC-conjugated anti-Flag Abs. The solid area and the open area represent staining before and after treatment, respectively.

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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 α-chain, a ligand-specific β-chain (IL-2Rβ), and the γc (29, 32), whereas the EpoR consists of homodimeric polypeptide chains (EpoR) (33). The human YT cell line (22), which readily expresses IL-2Rβ and γc, 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. 3,A and Fig. 3 B 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-2Rβ and EpoR.

FIGURE 3.

Ub-Pr regulation of IL-2Rβ and EpoR. A, YT cells were treated with LLnL (100 μM) for 0, 2, 4, or 6 h. The cell lysates were immunoprecipitated (IP) with IL-2Rβ-specific antiserum, and immunoprecipitates were resolved by SDS-PAGE, then transferred to a PVDF membrane. The membrane was immunoblotted first with IL-2Rβ (upper panel), then with anti-Ub antiserum (lowerpanel). B, HCD-57 cells were treated with lactacystin (10 μM) for 0, 1, or 3 h. The cell lysates were treated exactly as described in A, except that EpoR-specific antiserum instead of IL-2Rβ antiserum was used. The unmodified receptors are indicated, and the high Mr ubiquitinated proteins are marked with an asterisk.

FIGURE 3.

Ub-Pr regulation of IL-2Rβ and EpoR. A, YT cells were treated with LLnL (100 μM) for 0, 2, 4, or 6 h. The cell lysates were immunoprecipitated (IP) with IL-2Rβ-specific antiserum, and immunoprecipitates were resolved by SDS-PAGE, then transferred to a PVDF membrane. The membrane was immunoblotted first with IL-2Rβ (upper panel), then with anti-Ub antiserum (lowerpanel). B, HCD-57 cells were treated with lactacystin (10 μM) for 0, 1, or 3 h. The cell lysates were treated exactly as described in A, except that EpoR-specific antiserum instead of IL-2Rβ antiserum was used. The unmodified receptors are indicated, and the high Mr ubiquitinated proteins are marked with an asterisk.

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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 IκBα (20). We proposed a mechanism by which VCP acts as a molecular chaperone that targets the ubiquitinated IκBα 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. 4,A, 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-2Rβ, respectively (Fig. 4, top panel). Subsequent immunoblotting confirmed that the 120- and 70/75-kDa proteins contain JAK3 and IL-2Rβ (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. 4,B, 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-2Rβ-specific antiserum and subsequently blotted with VCP antiserum, VCP was found to clearly associate with the receptor complex (Fig. 4,C). 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. 4 D). 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.

FIGURE 4.

Tyrosine phosphorylation and coprecipitation of VCP in an IL-2Rβ complex following IL-2 stimulation. A, YT cells were stimulated with rhIL-2 (10 nM) for the indicated periods of time (shown on the top). The cell lysates were immunoprecipitated (IP) with anti-pY Abs, and the precipitates were resolved by SDS-PAGE and transferred to a PVDF membrane. The membrane was then immunoblotted, in series, with antisera directed to pY, VCP, JAK3, and STAT5 (indicated on the left). The control lane, marked M, represents a mock precipitation performed without cell lysates. The positions of the respective proteins are indicated on the right. The 97-kDa species (p97) contains the comigrating VCP and STAT5. B, YT cells were treated as described in A. Immunoprecipitation (IP) was conducted using anti-VCP antiserum, and immunoblot analyses were performed with antisera reactive to VCP, pY, and phosphoserine (pS). C, The experiment was conducted exactly as described in A, except that different antisera (as indicated) were used. D, Crude YT cell lysates (40 μg), recombinant VCP-His fusion protein (10 μg), STAT5, and VCP immunoprecipitates isolated from the YT lysates were resolved by SDS-PAGE, transferred to membrane, and immunoblotted with anti-STAT5 (left panel) or anti-VCP (right panel) antiserum. The lines between the panels represent the positions of the molecular size markers corresponding to 200, 97, 68, and 43 kDa.

FIGURE 4.

Tyrosine phosphorylation and coprecipitation of VCP in an IL-2Rβ complex following IL-2 stimulation. A, YT cells were stimulated with rhIL-2 (10 nM) for the indicated periods of time (shown on the top). The cell lysates were immunoprecipitated (IP) with anti-pY Abs, and the precipitates were resolved by SDS-PAGE and transferred to a PVDF membrane. The membrane was then immunoblotted, in series, with antisera directed to pY, VCP, JAK3, and STAT5 (indicated on the left). The control lane, marked M, represents a mock precipitation performed without cell lysates. The positions of the respective proteins are indicated on the right. The 97-kDa species (p97) contains the comigrating VCP and STAT5. B, YT cells were treated as described in A. Immunoprecipitation (IP) was conducted using anti-VCP antiserum, and immunoblot analyses were performed with antisera reactive to VCP, pY, and phosphoserine (pS). C, The experiment was conducted exactly as described in A, except that different antisera (as indicated) were used. D, Crude YT cell lysates (40 μg), recombinant VCP-His fusion protein (10 μg), STAT5, and VCP immunoprecipitates isolated from the YT lysates were resolved by SDS-PAGE, transferred to membrane, and immunoblotted with anti-STAT5 (left panel) or anti-VCP (right panel) antiserum. The lines between the panels represent the positions of the molecular size markers corresponding to 200, 97, 68, and 43 kDa.

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To determine whether VCP is also involved in Ub-Pr-mediated degradation of non-γc-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. 5,A shows that VCP is detected in anti-pY immunoprecipitates (upperpanels), and VCP physically interacts with the EpoR complex during stimulation (lowerpanels) 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. 5,B). 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.

FIGURE 5.

Involvement of VCP in Epo signaling. A, Starved HCD-57 cells were stimulated with Epo (100 U/ml) for 0–30 min. The cell lysates were subjected to serial immunoprecipitation (IP) and immunoblotting (Blot) with the indicated antisera. B, HCD-57 cells were either unstimulated (−) or stimulated with Epo (+) for 30 min. The cell lysates were analyzed by immunoprecipitation (IP) followed by immunoblotting (Blot) with the indicated antisera.

FIGURE 5.

Involvement of VCP in Epo signaling. A, Starved HCD-57 cells were stimulated with Epo (100 U/ml) for 0–30 min. The cell lysates were subjected to serial immunoprecipitation (IP) and immunoblotting (Blot) with the indicated antisera. B, HCD-57 cells were either unstimulated (−) or stimulated with Epo (+) for 30 min. The cell lysates were analyzed by immunoprecipitation (IP) followed by immunoblotting (Blot) with the indicated antisera.

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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-9Rα-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 lanes4–6 and lanes 10–12) during the treatment. These results strongly suggest that the high Mr proteins are tyrosine phosphorylated and ubiquitinated IL-9Rα molecules. Thus, tyrosine phosphorylated receptors can be ubiquitinated.

FIGURE 6.

Ubiquitination of the tyrosine-phosphorylated receptors. IL-9Rα-expressing TS1 cells were treated with ZLLH for 0, 1.5, or 3 h. Cell lysates were either separated by SDS-PAGE (lanes 1–3 and 7–9) or subjected to anti-pY immunoprecipitation (IP) and SDS-PAGE (lanes 4–6 and 10–12). After electrophoretic transfer, the membrane was immunoblotted (Blot) with Flag- or Ub-specific antiserum. The molecular size standards are shown on the left. The smeary-patterned reactivities detected above the 200-kDa standard presumably represent the ubiquitinated pY-containing receptors.

FIGURE 6.

Ubiquitination of the tyrosine-phosphorylated receptors. IL-9Rα-expressing TS1 cells were treated with ZLLH for 0, 1.5, or 3 h. Cell lysates were either separated by SDS-PAGE (lanes 1–3 and 7–9) or subjected to anti-pY immunoprecipitation (IP) and SDS-PAGE (lanes 4–6 and 10–12). After electrophoretic transfer, the membrane was immunoblotted (Blot) with Flag- or Ub-specific antiserum. The molecular size standards are shown on the left. The smeary-patterned reactivities detected above the 200-kDa standard presumably represent the ubiquitinated pY-containing receptors.

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Based 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-9Rα mutants (21) (summarized in Fig. 7) to examine whether any cytoplasmic domains of IL-9Rα 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-9Rα 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-9Rα were analyzed (Fig. 9), the ubiquitinated IL-9R α-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).

FIGURE 7.

Structure-function analyses of the IL-9Rα variants. A schematic diagram of the Flag-tagged wild-type, deletion mutants, and site-specific mutants of IL-9Rα is shown. The resulting effects on IL-9-induced cell proliferation (21 ), signaling (21 ), polyubiquitination, and VCP coprecipitation (VCP-Co-IP) are summarized.

FIGURE 7.

Structure-function analyses of the IL-9Rα variants. A schematic diagram of the Flag-tagged wild-type, deletion mutants, and site-specific mutants of IL-9Rα is shown. The resulting effects on IL-9-induced cell proliferation (21 ), signaling (21 ), polyubiquitination, and VCP coprecipitation (VCP-Co-IP) are summarized.

Close modal
FIGURE 8.

Detection of VCP in receptor complexes of all IL-9Rα variants except D4. Parental TS1 or TS1 cells expressing the various IL-9Rα variants (indicated on the top) were stimulated with rhIL-9 in the presence of LLnL for 8 h. The cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting with Flag-specific (upper panel) and VCP-specific (lower panel) antisera.

FIGURE 8.

Detection of VCP in receptor complexes of all IL-9Rα variants except D4. Parental TS1 or TS1 cells expressing the various IL-9Rα variants (indicated on the top) were stimulated with rhIL-9 in the presence of LLnL for 8 h. The cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting with Flag-specific (upper panel) and VCP-specific (lower panel) antisera.

Close modal
FIGURE 9.

Lack of ubiquitination on IL-9Rα D4 mutant. Parental TS1 (−; lanes 1 and 4) and TS1 cells expressing the wild-type (WT; lanes 3 and 6) or D4 mutant (lanes 2 and 5) of IL-9Rα were treated with proteasome inhibitors for 12 h. The cell lysates were immunoprecipitated (IP) with anti-Flag Abs to isolate the receptor complexes, which were further assessed by immunoblotting (Blot) with the anti-Flag (lanes 1–3) or anti-Ub (lanes 4–6) serum. An aliquot of 50 μg of the total cell lysates from each cell line was resolved by SDS-PAGE and immunoblotted with Ub-specific antiserum (lanes 7–9).

FIGURE 9.

Lack of ubiquitination on IL-9Rα D4 mutant. Parental TS1 (−; lanes 1 and 4) and TS1 cells expressing the wild-type (WT; lanes 3 and 6) or D4 mutant (lanes 2 and 5) of IL-9Rα were treated with proteasome inhibitors for 12 h. The cell lysates were immunoprecipitated (IP) with anti-Flag Abs to isolate the receptor complexes, which were further assessed by immunoblotting (Blot) with the anti-Flag (lanes 1–3) or anti-Ub (lanes 4–6) serum. An aliquot of 50 μg of the total cell lysates from each cell line was resolved by SDS-PAGE and immunoblotted with Ub-specific antiserum (lanes 7–9).

Close modal

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-9Rα-expressing cells with human IL-9 in the presence of proteasome inhibitors and analyzed cell lysates by anti-IL-9Rα 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).

FIGURE 10.

Coprecipitation of increasing amounts of VCP with IL-9Rα in proteasome inhibitor-treated cells. TS1 cells expressing the wild-type IL-9Rα were treated with human IL-9 and 10 μM LLnL for 0–10 h. Cell lysates were then immunoprecipitated (IP) with anti-Flag Abs, resolved by SDS-PAGE, transferred to PVDF membrane, and immunoblotted (Blot) with anti-Flag or anti-VCP antiserum. IL-9Rα is denoted on the right along with the high relative molecular mass species indicated by asterisks.

FIGURE 10.

Coprecipitation of increasing amounts of VCP with IL-9Rα in proteasome inhibitor-treated cells. TS1 cells expressing the wild-type IL-9Rα were treated with human IL-9 and 10 μM LLnL for 0–10 h. Cell lysates were then immunoprecipitated (IP) with anti-Flag Abs, resolved by SDS-PAGE, transferred to PVDF membrane, and immunoblotted (Blot) with anti-Flag or anti-VCP antiserum. IL-9Rα is denoted on the right along with the high relative molecular mass species indicated by asterisks.

Close modal

In the present study we provide the first evidence that non-tyrosine kinase-type members of the cytokine receptor superfamily, IL-9Rα, IL-2Rβ, 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-9Rα study shown in Fig. 1 C). 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., IκBα) rather than the nonubiquitinated substrates (20); 2) VCP association with IκBα is necessary for final degradation of IκBα 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.

We thank Eying Chen, Charlotte Hanson, and Rebecca Erwin-Cohen for technical assistance. We also thank C. Sadowski and L. Finch for the FACS analysis, and Dr. Q. Wang for comments on the manuscript.

1

This work was supported by the National Cancer Institute under Contract NO1CO56000 (to Science Applications International Corp.). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

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; γc, common γ-chain; JAK, Janus tyrosine kinase; ZLLH, Z-Leu-Leu-Leu-H (aldehyde).

1
Hershko, A., A. Ciechanover.
1998
. The ubiquitin system.
Annu. Rev. Biochem.
67
:
425
2
Ciechanover, A..
1998
. The ubiquitin-proteasome pathway: on protein death and cell life.
EMBO J.
17
:
7151
3
Baumeister, W., J. Walz., F. Zuhl, E. Seemuller.
1998
. The proteasome: paradigm of a self-compartmentalizing protease.
Cell
92
:
367
4
Bochtler, M., L. Ditzel, M. Grol, C. Hartmann, R. Huber.
1999
. The proteasome.
Annu. Rev. Biophys. Biomol. Struct.
28
:
295
5
Rechsteiner, M. 1998. The 26 S proteasome. In Ubiquitin and the Biology of the Cell. J.-M. Peters, J. R. Harris, and D. Finley, eds. Plenum Press, New York and London, p. 147.
6
Coux, O., K. Tanaka, A. L. Goldberg.
1996
. Structure and functions of the 20S and 26S proteasomes.
Annu. Rev. Biochem.
65
:
801
7
Kopito, R. R. 1998. Ubiquitination of integral membrane proteins and proteins in the secretory pathway. In Ubiquitin and the Biology of the Cell. J.-M. Peters, J. R. Harris, and D. Finley, eds. Plenum Press, New York and London, p. 389.
8
Hicke, L..
1999
. Gettin’ down with ubiquitin: turning off cell-surface receptors, transporters and channels.
Trends Cell Biol.
9
:
107
9
Bonifacino, J. S., A. M. Weissman.
1998
. Ubiquitin and the control of protein fate in the secretory and endocytic pathways.
Annu. Rev. Cell Dev. Biol.
14
:
19
10
Strous, G. J., R. Govers.
1999
. The ubiquitin-proteasome system and endocytosis.
J. Cell Sci.
112
:
1417
11
Ward, C. L., S. Omura, R. R. Kopito.
1995
. Degradation of CFTR by the ubiquitin proteasome pathway.
Cell
83
:
121
12
Jensen, T. J., M. A. Loo, S. Pind, D. B. Williams, A. L. Goldberg, J. R. Riordan.
1995
. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing.
Cell
83
:
129
13
Wiertz, E. J ., T. R. Jones, L. Sun, M. Bogyo, H. J. Geuze, H. L. Ploegh.
1996
. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol.
Cell
84
:
769
14
Wiertz, E. J., D. Tortorella, M. Bogyo, J. Yu, W. Mothes, T. R. Jones, T. A. Rapoport, H. L. Ploegh.
1996
. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction.
Nature
384
:
432
15
Huppa, J. B., H. L. Ploegh.
1997
. The α chain of the T cell antigen receptor is degraded in the cytosol.
Immunity
7
:
113
16
Yang, M., S. Omura, J. S. Bonifacino, A. M. Weissman.
1998
. Novel aspects of degradation of T cell receptor subunits from the endoplasmic reticulum (ER) in T cells: importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from ER membranes.
J. Exp. Med.
187
:
835
17
Terrell, J., S. Shih, R. Dunn, L. Hicke.
1998
. A function for monoubiquitination in the internalization of a G protein-coupled receptor.
Mol. Cell
1
:
193
18
Jaffers, M., G. A. Taylor, K. M. Weidner, S. Omura, G. F. Vande Woude.
1997
. Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.
Mol. Cell. Biol.
17
:
799
19
Mori, S., K. Tanaka, S. Omural, Y. Saito.
1995
. Degradation process of ligand-stimulated platelet-derived growth factor β receptor involves ubiquitin-proteasome proteolytic pathway.
J. Biol. Chem.
270
:
29447
20
Dai, R.-M., E. Chen, D. L. Longo, C. M. Gorbea, C.-C. H. Li.
1998
. Involvement of valosin-containing protein, an ATPase co-purified with IκBα and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IκBα.
J. Biol. Chem.
273
:
3562
21
Zhu, Y. X., H. B. Sun, M. L.-S. Tsang, J. McMahel, S. Grigsby, T. Yin, Y.-C. Yang.
1997
. Critical cytoplasmic domains of human interleukin-9 receptor α chain in interleukin-9-mediated cell proliferation and signal transduction.
J. Biol. Chem.
272
:
21334
22
Kirken, R. A., H. Rui, G. A. Evans, W. L. Farrar.
1993
. Characterization of an interleukin-2 (IL-2) induced tyrosine phosphorylated 116-kDa protein associated with the IL-2 receptor β-subunit.
J. Biol. Chem.
268
:
22765
23
Ruscetti, S. K., N. J. Janesch, A. Chakvaborti, S. T. Sawyer, W. D. Hankins.
1990
. Friend spleen focus-forming virus induces factor independence in an erythropoietin-dependent erythroleukemia cell line.
J. Virol.
63
:
1057
24
Thom, D., A. J. Powell, C. W. Lloyd, D. A. Reed.
1977
. Rapid isolation of plasma membranes in high yield from cultured fibroblasts.
Biochem. J.
168
:
187
25
Chen, E., R. Hrdlickova, J. Nehyba, D. L. Longo, H. R. Bose, C.-C. H. Li.
1998
. Degradation of proto-oncoprotein c-Rel by the ubiquitin-proteasome pathway.
J. Biol. Chem.
273
:
35201
26
Uyttenhove, C., R. J. Simpson, J. Van Snick.
1988
. Functional and structural characterization of P40, a mouse glycoprotein with T-cell growth factor activity.
Proc. Natl. Acad. Sci. USA
85
:
6934
27
Renauld, J. C., F. Houssiau, J. Louahed, A. Vink, J. Van Snick, C. Uyttenhove.
1993
. Interleukin-9.
Adv. Immunol.
54
:
79
28
Yang, Y.-C., S. Ricciardi, A. Ciarletta, J. Calvetti, K. Kelleher, S. C. Clark.
1989
. Expression cloning of cDNA encoding a novel human hematopoietic growth factor: human homologue of murine T-cell growth factor P40.
Blood
74
:
1880
29
Takeshita, T., H. Asao, K. Ohtani, N. Ishii, D. Kumaki, N. Tanaka, H. Munataka, M. Nakamura, K. Sugamura.
1992
. Cloning of the γ chain of the human IL-2 receptor.
Science
257
:
379
30
Noguchi, M., Y. Nakamura, S. M. Russell, S. F. Ziegler, M. Tsang, X. Cao, W. J. Leonard.
1993
. Interleukin-2 receptor gamma chain: a functional component of the interleukin-7 receptor.
Science
262
:
1877
31
Giri, J. G., M. Andieh, J. Eisenman, K. Shanebeck, K. Grabstein, S. Kumaki, A. Namen, L. S. Park, D. Cosman, D. Anderson.
1994
. Utilization of the β and γ chains of the IL-2 receptor by the novel cytokine IL-15.
EMBO J.
13
:
2822
32
Sugamura, K., H. Asao, M. Kondo, N. Tanaka, N. Ishii, K. Ohbo, M. Nakamur, T. Takeshita.
1996
. The interleukin-2 receptor γ chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID.
Annu. Rev. Immunol.
14
:
179
33
D’Andrea, A., G. Fasman, G. Wong, H. Lodish.
1990
. Erythropoietin receptor: cloning strategy and structural features.
Int. J. Cell Cloning
1
: (Suppl.):
173
34
Egerton, M., O. R. Ashe, D. Chen, B. J. Druker, W. H. Burgess, L. E. Samelson.
1992
. VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation.
EMBO J.
11
:
3533
35
Darnell, J. E., Jr.
1997
. STATs and gene regulation.
Science
277
:
1630
36
O’Shea, J. J..
1997
. Jaks, Stats, cytokine signal transduction, and immunoregulation: are we there yet?.
Immunity
7
:
1
37
Ihle, J. N..
1995
. Cytokine receptor signalling.
Nature
377
:
591
38
Linnekin, D., G. Evans, D. Michiel, W. L. Farrar.
1992
. Characterization of 97 kDa phosphotyrosylprotein regulated by multiple cytokines.
J. Biol. Chem.
267
:
23993
39
Miura, O., A. D’Andrea, D. Kabat, J. N. Ihle.
1991
. Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis.
Mol. Cell. Biol.
11
:
4895
40
Showers, M. O., J. F. Moreau, D. Linnekin, B. Druker, A. D. D’Andrea.
1992
. Activation of the erythropoietin receptor by the Friend spleen focus-forming virus gp55 glycoprotein induces constitutive protein tyrosine phosphorylation.
Blood
80
:
3070
41
Hilton, D. J..
1999
. Negative regulators of cytokine signal transduction.
Cell. Mol. Life Sci.
55
:
1568
42
Ghislain, M., R. J. Dohmen, F. Levy, A. Varshavsky.
1996
. Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae.
EMBO J.
15
:
4884
43
Moir, D., S. E. Steward, B. C. Osmond, D. Botstein.
1982
. Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies.
Genetics
100
:
547
44
Koegl, M., T. Hoppe, S. Schlenker, H. D. Ulrich, T. U. Mayer, S. Jentsch.
1999
. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly.
Cell
96
:
635
45
Gu, H., J. D. Griffin, B. G. Neel.
1997
. Characterization of two SHP-2-associated binding proteins and potential substrates in hematopoietic cells.
J. Biol. Chem.
272
:
16421
46
Schulte, R. J., M.-A. Campbell, W. H. Fischer, B. M. Sefton.
1994
. Tyrosine phosphorylation of VCP, the mammalian homologue of the Saccharomyces cerevisiae CDC48 protein, is unusually sensitive to stimulation by sodium vanadate and hydrogen peroxide.
J. Immunol.
153
:
5465
47
Madeo, F., J. Schlauer, H. Zischka, D. Mecke, K.-U. Frohlich.
1998
. Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p.
Mol. Biol. Cell
9
:
131