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Previous Article | Next Article 
Molecular and Cellular Biology, May 2004, p. 4038-4048, Vol. 24, No. 9
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.9.4038-4048.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Animal Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104,1 AMC Cancer Center, Lakewood, Colorado 802142
Received 24 November 2003/ Returned for modification 22 December 2003/ Accepted 10 February 2004
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ABSTRACT |
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INTRODUCTION |
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Limitation of the extent and duration of hormonal signaling in cells is required for the physiological regulation of cellular responses. Negative regulation of PRL signaling appears to play an important role in cellular physiology, as abnormal activation of PRL signaling results in uncontrollable cell proliferation in vitro (35) and abnormal development of mammary glands and deficient lactation in vivo (19). Recently described mechanisms mediating negative regulation of the PRL-induced Jak-Stat pathway include the inactivation of Jak2 and Stat5 by specific phosphatases, suppressors of cytokine signaling, and protein inhibitors of activated STAT (reviewed in reference 6). In addition, modulation of this and other PRL-dependent signal transduction pathways is thought to rely on down-regulation of PRLR in response to the ligand (11, 13), which was shown to occur via PRLR endocytosis and lysosomal degradation (12, 14, 17). However, the determination of the role of PRLR down-regulation in restricting the extent of PRL signaling, as well as the delineation of the mechanisms that mediate PRLR down-regulation, remains a work in progress.
Ubiquitin-mediated proteolysis plays a universal role in the irreversible negative regulation of various signaling pathways (52), including those induced by pituitary hormones (46). Whereas the polyubiquitination of proteins leads to their degradation by 26S proteasome complexes, the oligoubiquitination of plasma membrane proteins targets them for endocytosis and degradation in lysosomes (25, 26). In both instances, the fate of protein substrates depends on the activity of specific E3 ubiquitin protein ligases that recognize these substrates and mediate their ubiquitination. The ubiquitin pathway is essential for the endocytosis and degradation of the receptor for growth hormone (GHR), although the ubiquitination of GHR per se seems not to be required (reviewed in references 46 and 47). The role of the ubiquitination of PRLR in its down-regulation and degradation has not previously been determined, and putative E3 ubiquitin protein ligases that catalyze such ubiquitination have not previously been identified.
By analogy with other receptors whose down-regulation and degradation is mediated by ubiquitination of their intracellular domains (25, 26), signals for the degradation of PRLR are expected to be present within its cytoplasmic tail. The sequences of the cytoplasmic tails of the long form of PRLR are poorly conserved among various species, but we noticed that the DSGRGS sequence, which is similar to a recognition motif for the SCFß-TrCP E3 ligase, is present in all species whose PRLR sequences have been determined (Table 1). The ß-TrCP1 (39) and HOS/ß-TrCP2 (16, 48) members of the ß-TrCP/Fbw1 subfamily of F-box proteins (4, 53) are known to recognize the phosphorylated DSG(X)2+nS motif within their substrates, which include the I
B (16, 23, 44, 48, 54, 55), ß-catenin (16, 22, 29, 34, 54), ATF4 (33), Emi1 (21, 40), Cdc25A (3, 28), Dlg (38), and IFNAR1 (32) proteins. ß-TrCP proteins recognize phosphorylated substrate and recruit the core Skp1-Cul1-Roc1/Rbx1 ubiquitin ligase complex to ubiquitinate the substrates (reviewed in reference 10). Unlike the predominantly nuclear ß-TrCP1 protein, the ß-TrCP2 member of the ß-TrCP subfamily exhibits mostly cytoplasmic localization (9, 33).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(New England Biolabs) were purchased. Recombinant E2 (UbcH5C) was a gift of Z.-Q. Pan. E1 and ubiquitin (Affiniti Inc.) were purchased.
DNA constructs.
Human pCDNA3-PRLR-Flag (35) was kindly provided by S. M. Anderson. The replacement of serine 349 with alanine was carried out with a QuikChange site-directed mutagenesis kit (Stratagene). Glutathione S-transferase (GST)-tagged bacterial expression constructs were prepared by subcloning sequences encoding C-terminal amino acids 259 to 622 (the cytoplasmic tail) of PRLR into the pGEX-2T vector (Amersham). Hemagglutinin (HA)-tagged ubiquitin (50) and Stat5-driven luciferase reporter (pLHRE-luc) (43) were generous gifts from D. Bohmann and P. A. Kelly. HA-tagged ß-TrCP1 (kindly provided by R. Benarous) (33), ß-TrCP2- and ß-TrCP2
N-encoding plasmids (16), and the constructs for the expression of Flag-Cul1, Skp1, and Roc1 (49) were previously described. Plasmids for the expression of GFP (Clontech), Renilla luciferase, and ß-galactosidase (Promega) were purchased. The plasmid for the expression of short hairpin RNA (shRNA) directed against both ß-TrCP1 and ß-TrCP2 (shBTR1,2) was kindly provided by J. W. Harper (28). Plasmids for the specific knock-down of ß-TrCP2 (shBTR2) and the control shRNA construct (shCON) were constructed on a backbone of pSilencer 1.0-U6 vector (Ambion) by using the sequence of chemically synthesized duplex small interfering RNA described elsewhere (32). Sequences that formed the hairpin duplexes were 5'-GAGGCCATCAGAAGGAAACTTCAAGAGAGTTTCCTTCTGATGGCCTC-3' for shBTR2 and 5'-GAGGCCATCAGTGGGAAACTTCAAGAGAGTTTCCCACTGATGGCCTC-3' for shCON. The effects of shRNA constructs were verified by immunoblotting analysis.
Tissue culture and transfections. 293T human embryo kidney cells were kindly provided by Z. Ronai. Hamster CHO-K1 cells and mouse embryo fibroblasts from ß-TrCP1 knockout mice (21) were generous gifts from R. J. Eisenberg and M. Pagano. Cells were grown in Dulbecco modified Eagle medium (DMEM) in the presence of 10% fetal bovine serum (FBS) and antibiotics at 37°C and at 5% CO2. Transfections were performed by the calcium phosphate procedure or lipofection (with Lipofectamine Plus or Lipofectamine 2000 [Invitrogen]) 24 to 48 h before harvesting. Stable mass cultures of CHO-K1 cells expressing PRLR proteins were obtained by cotransfecting PRLR constructs with pBABE-puro vector, followed by selection in medium containing puromycin (1 µg/ml).
Antibodies and immunotechniques. Antibodies against HA (Roche), Flag (M2; Sigma), PRLR (Affinity BioReagents, Inc., or Neomarkers, Inc.), Skp2 (Zymed), GST (Santa Cruz), and phospho-Stat5 and Stat5 (Cell Signaling Technology) were purchased. HOS-N antibody, which specifically reacts with ß-TrCP2, and HOS-C antibody, which recognizes both ß-TrCP1 and ß-TrCP2, were previously described (45). Secondary antibodies conjugated with horseradish peroxidase (Chemicon) were purchased. Immunoprecipitation and immunoblotting procedures were described elsewhere (16). Digital images were prepared with Adobe Photoshop 7.0 software.
In vitro binding assay.
Recombinant PRLR proteins expressed in 293T cells (grown in the presence of 10% FBS) were immunopurified with Flag antibody and protein A beads, stringently washed with stripping buffer containing 50 mM Tris HCl (pH 7.5), 1 M NaCl, 50 mM NaF, 10 nM okadaic acid, and 0.1% Nonidet P-40, and equilibrated with binding buffer (50 mM Tris HCl [pH 7.5], 100 mM NaCl, 50 mM NaF, 10 nM okadaic acid, 0.1% Nonidet P-40). For treatment with phosphatase , the beads were washed with the binding buffer without phosphatase inhibitors and incubated with the phosphatase for 1 h at 37°C, followed by washes in stripping buffer and reequilibration with binding buffer. Flag-PRLR proteins immobilized on the beads were incubated with in vitro-translated and 35S-labeled ß-TrCP2 for 60 min at 4°C. The beads were extensively washed with binding buffer, and associated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.
In a separate experiment, 293T cells were starved overnight in medium containing 0.5% FBS and treated with PRL (200 ng/ml) for 0 to 120 min (as indicated in Fig. 2B) and lysates were prepared from harvested cells. GST-PRLR proteins were expressed and purified from bacterial cells by using glutathione-Sepharose. Purified bacterial proteins (1 µg) immobilized on glutathione beads were phosphorylated with 293T cell lysates (25 µg) of 293T cells in the presence or absence of ATP for 30 min at 30°C, followed by stringent washing with stripping buffer and reequilibration with binding buffer. GST-PRLR proteins immobilized on the beads were incubated with in vitro-translated and 35S-labeled ß-TrCP2 for 60 min at 4°C. The beads were extensively washed with binding buffer, and associated proteins were analyzed by SDS-PAGE, autoradiography, and immunoblotting with GST antibody.
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]ATP for 30 min at 30°C. Proteins were captured on SCFß-TrCP isolated from transfected 293T cells with HA antibody (Roche) and protein A beads (Invitrogen) as previously described (49) and washed with buffer containing 300 mM NaCl, 0.5% Nonidet P-40, and phosphatase inhibitors. Ubiquitination reactions were carried out with a total volume of 20 µl containing 2 µg of ubiquitin, 20 pmol of E1, and 100 pmol of E2 as well as 2 mM ATP at 37°C for 60 min. Boiling in SDS-sample buffer terminated the reactions, and the products were analyzed by SDS-PAGE and autoradiography. Pulse-chase analysis was carried out with 293T cells as described elsewhere (16). Briefly, cells were grown in 100-mm-diameter dishes and transfected with the indicated plasmids. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35S]methionine-[35S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM). Harvested cells were lysed, and PRLR proteins were immunoprecipitated with Flag or PRLR antibody, separated on SDS-PAGE gel, and analyzed by autoradiography. Luciferase reporter assays. 293T or CHO cells were transfected with pLHRE-luc and Renilla luciferase vectors (Promega) as well as with small interfering RNA plasmids where indicated, starved in medium with 0.5% serum overnight, and treated with PRL (200 ng/ml) for 24 h before harvesting. Luciferase activities were determined with the aid of a Dual luciferase kit (Promega).
Cell proliferation. CHO stable mass cultures were seeded into 96-well plates (5 x 103 of the trypan blue-negative live cells per well) in medium containing 0.5% FBS, and cell proliferation was assessed after 3 days of incubation with or without PRL (50 ng/ml) by using a colorimetric WST-1 cell proliferation kit (Roche) according to the manufacturer's recommendations. Acceleration of growth by PRL was calculated as [1 – (absorbance with PRL/absorbance without PRL)] x 100%.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S1 splicing form of human PRLR (31) but in neither the short nor the intermediate form of the receptor. Although the overall sequence of the intracellular domain of PRLR is poorly conserved among different species (6), the DSGRGS sequence is present in all species whose PRLR sequence has been identified to date (Table 1). We tested whether ß-TrCP2 interacts with PRLR by means of coimmunoprecipitation with the lysates of 293T cells grown under low-serum conditions (0.5% FBS). Analysis of these reactions revealed that ß-TrCP2 was indeed found in immunoprecipitates with PRLR antibody but not with an irrelevant monoclonal (Flag) antibody (Fig. 1A, lanes 2 and 3). We could not detect an interaction between endogenous ß-TrCP2 and PRLR in a reciprocal coimmunoprecipitation experiment (data not shown) either because of possible interfering effects of anti-ß-TrCP antibody or because of low levels of PRLR in 293T cells and/or the transience of the interaction. Treatment of 293T cells with PRL noticeably increased the extent of the binding of ß-TrCP2 to PRLR (Fig. 1A, lane 3 versus lane 6). These results suggest that ß-TrCP2 interacts with PRLR and that treatment with the hormone ligand promotes this interaction.
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Phosphorylation of PRLR, which is promoted by the ligand, is required for the ability of PRLR to interact with ß-TrCP2. We further tested the role of PRLR phosphorylation in binding to ß-TrCP2. Recombinant Flag-PRLRwt purified from 293T cells interacted with ß-TrCP2 in vitro, but no interaction was observed with the Flag-PRLRS349A mutant (Fig. 2A). Phosphatase treatment of Flag-PRLRwt decreased its ability to bind ß-TrCP2 in vitro (Fig. 2A), indicating that specific phosphorylation of PRLR was essential for the ability of PRLR to interact with ß-TrCP2. This result also indicates that recombinant Flag-PRLR overexpressed in 293T cells which were grown in medium supplemented with 10% FBS was already phosphorylated and capable of binding to ß-TrCP2 in the absence of added ligand. This finding does not rule out the possibility of a role for PRL in ß-TrCP2-PRLR interaction, since PRL is contained in FBS (41) and the overexpression of receptors alone (e.g., tumor necrosis factor alpha receptor [24] or IFNAR1 [32]) is known to mediate signaling events.
To test the role of PRL in the PRLR phosphorylation that enables ß-TrCP2-PRLR interaction, we prepared extracts from serum-starved cells at various time points after treatment with PRL. We further used these extracts as a source of kinase activity to phosphorylate immobilized bacterially produced GST-PRLR proteins, followed by repeated stringent washing and an in vitro ß-TrCP2 binding assay. Binding of ß-TrCP2 was detected with GST-PRLRwt protein phosphorylated with cell extracts from PRL-treated cells, with peak of activity being observed at 30 and 90 min after the addition of PRL (Fig. 2B, lanes 4 to 8). Importantly, the omission of ATP from the kinase reaction prevented interaction with ß-TrCP2, indicating that GST-PRLR needs to be phosphorylated to exhibit affinity for ß-TrCP2 (lane 2). Binding to ß-TrCP2 was also not detected with the GST-PRLRS349A mutant (Fig. 2B, lanes 9 to 14). These data suggest that the phosphorylation of PRLR at Ser349 by a kinase, whose activity is increased by the hormone ligand treatment, is required for the interaction of PRLR with ß-TrCP2.
Ubiquitination of PRLR depends on ß-TrCP2.
The binding of ß-TrCP2 to specifically phosphorylated substrates such as IB, ß-catenin, and IFNAR1 often results in the ubiquitination of such proteins. To determine a role for ß-TrCP in the ubiquitination of PRLR, we assessed the effects of decreasing ß-TrCP expression by the shRNA-mediated knock-down approach. Transfection of 293T cells with control shRNA (shCON) that differs from shBTR2 by a 2-bp substitution (see Materials and Methods) did not decrease ß-TrCP levels (Fig. 3A). shRNA directed against ß-TrCP2 (shBTR2) (see Materials and Methods) or that directed against both ß-TrCP forms (shBTR1,2) (28) suppressed the expression of ß-TrCP proteins in 293T cells as measured by immunoblotting with the antibody, which recognizes ß-TrCP1 and ß-TrCP2 (45). These shRNA constructs did not affect the steady-state levels of another F-box protein, Skp2 (Fig. 3A). In the absence of good-quality antibody specifically recognizing ß-TrCP1, we used the expression of HA-tagged ß-TrCP proteins to further characterize the effects of shRNA constructs. As seen in Fig. 3B, shBTR1,2 inhibited the expression of both ß-TrCP1 and ß-TrCP2 (lanes 7 and 4). These data are consistent with results recently reported by the group of J. W. Harper (28). shBTR2 decreased the levels of ß-TrCP2 without substantially affecting the expression of ß-TrCP1 (Fig. 3B, lanes 3 and 6).
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To further confirm the role that ß-TrCP might play in the ubiquitination of PRLR, we compared the extent of the ubiquitination of Flag-tagged PRLRwt with that of the PRLRS349A mutant, whose interaction with ß-TrCP2 is impaired (Fig. 1B and 2A). The ubiquitination of this mutant was substantially decreased compared to the ubiquitination of wild-type PRLR (Fig. 3D, lane 6 versus lane 3), and the residual ubiquitination of the PRLRS349A mutant was not affected by shBTR2 (lane 6 versus lane 7). These results suggest that binding to ß-TrCP is important for PRLR ubiquitination, although the possibility of a contribution of ß-TrCP-independent mechanisms cannot be ruled out.
In addition to its effects on recombinant PRLR protein, the transfection of shBTR2 inhibited the extent of the ubiquitination of endogenous PRLR in 293T cells (Fig. 3E, lane 2 versus lane 3). Taken together, the data presented in Fig. 3 support the conclusion that ß-TrCPs play a major role in the ubiquitination of PRLR in cells.
We next sought to investigate whether SCFß-TrCP E3 ligase is capable of direct ubiquitination of PRLR. The extracts from 293T cells pretreated with PRL were used to phosphorylate GST-PRLR proteins in the presence of [32P-]ATP. These proteins were then captured on immobilized recombinant SCFß-TrCP2 ubiquitin ligase. Whereas the GST-PRLRS349A mutant protein was phosphorylated to an extent similar to that of its wild-type counterpart (data not shown), no binding between this mutant and SCFß-TrCP2 was detected (Fig. 4, lane 1). This result is in line with the data showing that the interaction of the PRLRS349A mutant with ß-TrCP2 is impaired (Fig. 1B and 2). Similarly, omitting ß-TrCP2 from immobilized complexes prevented the capture of phospho-GST-PRLR as well as the recruitment of Cul1 (Fig. 4, lane 4) and precluded testing of the ubiquitination of GST-PRLR under these experimental conditions.
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ß-TrCPs regulate proteolysis of PRLR. Ubiquitination of plasma membrane receptors is known to result in their internalization and degradation via the lysosomal pathway. We measured the rate of PRLR degradation by pulse-chase analysis and found that treatment of cells with inhibitors of the lysosomal pathway (methylamine HCl, MA) led to stabilization of PRLR (Fig. 5A). This result implicates the lysosomal pathway in the proteolysis of PRLR.
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3 h. Coexpression of the dominant-negative ß-TrCP2N mutant, which is known to compete with endogenous ß-TrCP2 and ß-TrCP1 for substrates and to inhibit the activities SCFß-TrCP ubiquitin ligase (16, 48), noticeably delayed the degradation of Flag-PRLRwt (Fig. 5B). This result indicates that ß-TrCPs are required for the efficient degradation of PRLR. We next sought genetic evidence for the role of ß-TrCP in regulating PRLR proteolysis. We observed that the rate of Flag-PRLRwt degradation was modestly decreased in mouse embryo fibroblasts derived from ß-TrCP1 knockout mice (Fig. 5C). Since the stability of ß-TrCP substrates in these cells is thought to be regulated by ß-TrCP2 (21), we used the shRNA approach to confirm the role of ß-TrCP in PRLR degradation. Flag-PRLRwt was noticeably stabilized by the coexpression of shRNA either directed against ß-TrCP2 alone or directed against both ß-TrCP1 and ß-TrCP2 (Fig. 5D). These data suggest that SCFß-TrCP E3 ligase plays a key role in regulating PRLR stability.
To further confirm the role of ß-TrCP in the proteolysis of PRLR, we compared the rate of degradation of Flag-tagged PRLRwt with that of the PRLRS349A mutant, which poorly interacts with ß-TrCP2 (Fig. 1A and 2B). This mutant exhibited substantially higher stability (half-life, >10 h) than the wild-type PRLR (half-life, 3 h) (Fig. 5E). In addition, proteolysis of endogenous PRLR was impaired in cells transfected with shBTR2 (Fig. 5F). These observations support other data presented here and are consistent with the idea that ß-TrCP plays an important role in regulating the degradation of PRLR.
Role of ß-TrCP in regulating cellular responses to PRL. Considering the role of ß-TrCP in regulating the stability of PRLR, as well as the fact that this receptor is required for most of the cellular effects elicited by PRL, we sought to determine whether ß-TrCP is involved in the control of the extent of PRL signaling. For these experiments, we used the shBTR2 construct, which inhibited the ubiquitination and degradation of PRLR to an extent similar to that of the effects of shRNA against both ß-TrCP forms (Fig. 3C and 5D). Transfection of 293T cells with shBTR2 noticeably augmented PRL-induced phosphorylation of Stat5 at Tyr694 (Fig. 6A). The measurement of the PRL-induced transcriptional activity of Stat5 as evaluated by the Stat5-driven luciferase reporter assay revealed activity levels (normalized per the activity of Renilla luciferase and calculated in arbitrary units) of 110 ± 13 units in cells transfected with shCON. The expression of the shBTR2 construct increased the activity of the Stat5-dependent reporter to 260 ± 58 units (P = 0.029, Student's t test). These findings indicate that ß-TrCP contributes to negative regulation of the extent of PRL signaling.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our findings characterize PRLR as a novel substrate for SCFß-TrCP E3 protein ligase. Substrate recognition of this ligase is mediated by the closely related F-box proteins ß-TrCP1 and ß-TrCP2. These proteins are interchangeable in biochemical assays and experiments with ß-TrCP overexpression (10). Knock-down experiments with shRNA showed that, in 293T cells, the inhibition of ß-TrCP2 levels attenuates the ubiquitination and degradation of PRLR protein to an extent similar to that of the effects of shRNA directed against both ß-TrCP1 and ß-TrCP2 (Fig. 3 and 5). This result indicates that ß-TrCP2 may represent a major regulator of PRLR ubiquitination and of PRLR stability in these cells (Fig. 3 and 5), although the possibility of a more prominent role for ß-TrCP1 in other cell types and/or under various physiological or pathological conditions should not be ruled out. The predominantly cytoplasmic localization of ß-TrCP2 compared to the nuclear localization of ß-TrCP1 (9, 33) may provide a plausible explanation of why ß-TrCP2 might be preferred by cytoplasmic substrates of SCFß-TrCP such as IFNAR1 (32) and PRLR (this study). Modest stabilization of PRLR was observed in embryo fibroblasts from mice with genetic elimination of ß-TrCP1 (Fig. 5C). Since knock-down experiments rarely (if ever) allow complete inhibition of protein expression, the future development of ß-TrCP2 knockout animal models will enable the resolution of the issues of redundancy and relative substrate specificity for these two forms of ß-TrCP.
Ubiquitination of PRLR by SCFß-TrCP E3 ligase depends on interaction between PRLR and an F-box protein subunit of the ligase (i.e., ß-TrCP). This interaction is promoted by PRL, suggesting that ß-TrCP2-mediated ubiquitination may play a role in the down-regulation of PRLR in response to the ligand. As is the case for other SCFß-TrCP substrates, PRLR ubiquitination and proteolysis appear to depend on specific phosphorylation of PRLR within the ß-TrCP2 recognition motif. Our data indicate that PRL treatment induces activity of a yet-to-be-identified kinase(s) that phosphorylates PRLR with the DSGRGS motif and enables the recruitment of E3 ligase by PRLR as well as PRLR ubiquitination and degradation. Future studies will identify the nature of such a kinase and determine its role in regulating the extent of PRL signaling.
In human cells, PRL is known to interact with various splicing forms of PRLR (2, 6, 7). Given the presence of the DSGRGS sequence, it is expected that the SCFß-TrCP-dependent pathway regulates proteolysis of the long form of PRLR as well as its S1 variant (31). At the same time, the down-regulation of the intermediate and short forms of PRLR, which lack the ß-TrCP2 recognition motif, may be regulated by other E3 ligases, or these forms may undergo endocytosis and degradation in a ubiquitin-independent manner. For example, multiple endocytosis motifs (including phenylalanine and dileucine motifs) were found to bring about internalization of short forms of PRLR as well as of truncation mutants of the long form (37, 51). Since intermediate or short forms of human PRLR are either partially or entirely deficient in mediating the signal transduction pathways induced by PRL (30, 42), it is expected that ß-TrCP2-dependent control of stability of the long-form PRLR is important for regulating a complete repertoire of PRL signaling.
Future studies are required to determine how the ubiquitination of PRLR by SCFß-TrCP E3 ligase leads to the proteolysis of PRLR. Whereas ß-TrCP-dependent ubiquitination of IFNAR1 targets this protein for endocytosis and degradation by a lysosomal pathway (32), other known substrates of SCFß-TrCP are degraded by 26S proteasome complexes (10). The treatment of cells with inhibitors of the lysosomal pathway substantially increased the half-life of Flag-PRLRwt in our assays (Fig. 5A). This result is in line with evidence that lysosomotropic agents prevent down-regulation of PRLR by its ligand (14). Thus, it is likely that ß-TrCP-mediated ubiquitination of PRLR targets this protein for degradation via the lysosomal pathway.
Conjugation of ubiquitin to the plasma membrane-localized receptors often provides them with a potent internalization signal (reviewed in reference 27). Interestingly, it has been shown that bovine PRLR mutants, which lack a substantial portion of the cytoplasmic tail encompassing the ß-TrCP recognition motif, undergo efficient endocytosis (37). In addition, the removal of a large portion of the cytoplasmic tail of GHR, which is closely related to PRLR and also contains a similar ß-TrCP recognition motif (DSGRTS), does not impair the internalization of these GHR mutants (20). However, a putative role for ß-TrCP-mediated ubiquitination in the internalization of endogenous PRLR (as well as that of GHR) cannot be entirely ruled out, since it remains possible that the endocytosis of native receptor protein is antagonized by a factor(s) whose effects rely on the integrity of sequences that are absent in such mutants. In addition, ß-TrCP-dependent ubiquitination may play a role in sorting already-internalized PRLR and targeting it to lysosomes for degradation. A similar function in the regulation of growth factor receptor proteolysis is attributed to c-Cbl E3 ubiquitin ligase (36).
Putative roles of ß-TrCP in PRLR endocytosis and postinternalization sorting remain to be determined. The development of antibodies specifically recognizing long and S1 forms of PRLR will improve the feasibility of this analysis in future studies. However, it is important to note that, regardless of the mode of PRLR proteolysis, the ß-TrCP-dependent pathway is stimulated by PRL (Fig. 1A and 2B). These data suggest an important physiological role of the ß-TrCP-mediated mechanism in the down-regulation of PRLR levels and the restriction of the extent of PRL signaling.
Future studies will also determine whether ß-TrCP-dependent regulation of PRLR stability and PRL signaling is important in restricting the effects of PRL on breast tissue. It is conceivable that these mechanisms may be utilized in limiting the extent of mammary gland development during pregnancy or in the gland's postlactation involution. In addition, considering that breast cancer cells secrete PRL and are subjected to its autocrine regulation (1, 8), putative aberrations in the ß-TrCP-mediated pathway in these cells would be expected to lead to sustained stimulation of their growth.
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ACKNOWLEDGMENTS |
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This work was supported in part by The University of Pennsylvania Cancer Center Pilot Grant and NCI grant CA 92900 (to S.Y.F.) and American Cancer Society award RSG-02-140-01-CNE (to V.S.S.).
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Bole-Feysot, C., V. Goffin, M. Edery, N. Binart, and P. A. Kelly. 1998. Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr. Rev. 19:225-268.
3. Busino, L., M. Donzelli, M. Chiesa, D. Guardavaccaro, D. Ganoth, N. V. Dorrello, A. Hershko, M. Pagano, and G. F. Draetta. 2003. Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage. Nature 426:87-91.[CrossRef][Medline]
4. Cenciarelli, C., D. S. Chiaur, D. Guardavaccaro, W. Parks, M. Vidal, and M. Pagano. 1999. Identification of a family of human F-box proteins. Curr. Biol. 9:1177-1179.[CrossRef][Medline]
5. Clevenger, C. V., D. O. Freier, and J. B. Kline. 1998. Prolactin receptor signal transduction in cells of the immune system. J. Endocrinol. 157:187-197.[CrossRef][Medline]
6. Clevenger, C. V., P. A. Furth, S. E. Hankinson, and L. A. Schuler. 2003. The role of prolactin in mammary carcinoma. Endocr. Rev. 24:1-27.
7. Clevenger, C. V., and J. B. Kline. 2001. Prolactin receptor signal transduction. Lupus 10:706-718.
8. Clevenger, C. V., and T. L. Plank. 1997. Prolactin as an autocrine/paracrine factor in breast tissue. J. Mammary Gland Biol. Neoplasia 2:59-68.[CrossRef][Medline]
9. Davis, M., A. Hatzubai, J. S. Andersen, E. Ben-Shushan, G. Z. Fisher, A. Yaron, A. Bauskin, F. Mercurio, M. Mann, and Y. Ben-Neriah. 2002. Pseudosubstrate regulation of the SCFß-TrCP ubiquitin ligase by hnRNP-U. Genes Dev. 16:439-451.
10. Deshaies, R. J. 1999. SCF and Cullin/Ring H2-based ubiquitin ligases. Annu. Rev. Cell Dev. Biol. 15:435-467.[CrossRef][Medline]
11. Djiane, J., H. Clauser, and P. A. Kelly. 1979. Rapid down-regulation of prolactin receptors in mammary gland and liver. Biochem. Biophys. Res. Commun. 90:1371-1378.[CrossRef][Medline]
12. Djiane, J., C. Delouis, and P. A. Kelly. 1982. Prolactin receptor turnover in explants of pseudopregnant rabbit mammary gland. Mol. Cell. Endocrinol. 25:163-170.[CrossRef][Medline]
13. Djiane, J., L. M. Houdebine, and P. A. Kelly. 1981. Down-regulation of prolactin receptors in rabbit mammary gland: differential subcellular localization. Proc. Soc. Exp. Biol. Med. 168:378-381.[Medline]
14. Djiane, J., P. A. Kelly, and L. M. Houdebine. 1980. Effects of lysosomotropic agents, cytochalasin B and colchicine on the "down-regulation" of prolactin receptors in mammary gland explants. Mol. Cell. Endocrinol. 18:87-98.[CrossRef][Medline]
15. Freeman, M. E., B. Kanyicska, A. Lerant, and G. Nagy. 2000. Prolactin: structure, function, and regulation of secretion. Physiol. Rev. 80:1523-1631.
16. Fuchs, S. Y., A. Chen, Y. Xiong, Z. Q. Pan, and Z. Ronai. 1999. HOS, a human homolog of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IB and beta-catenin. Oncogene 18:2039-2046.[CrossRef][Medline]
17. Genty, N., J. Paly, M. Edery, P. A. Kelly, J. Djiane, and R. Salesse. 1994. Endocytosis and degradation of prolactin and its receptor in Chinese hamster ovary cells stably transfected with prolactin receptor cDNA. Mol. Cell. Endocrinol. 99:221-228.[CrossRef][Medline]
18. Goffin, V., N. Binart, P. Touraine, and P. A. Kelly. 2002. Prolactin: the new biology of an old hormone. Annu. Rev. Physiol. 64:47-67.[CrossRef][Medline]
19. Gourdou, I., J. Paly, C. Hue-Beauvais, L. Pessemesse, J. Clark, and J. Djiane. 2004. Expression by transgenesis of a constitutively active mutant form of the prolactin receptor induces premature abnormal development of the mouse mammary gland and lactation failure. Biol. Reprod. 70:718-728.
20. Govers, R., P. van Kerkhof, A. L. Schwartz, and G. J. Strous. 1998. Di-leucine-mediated internalization of ligand by a truncated growth hormone receptor is independent of the ubiquitin conjugation system. J. Biol. Chem. 273:16426-16433.
21. Guardavaccaro, D., Y. Kudo, J. Boulaire, M. Barchi, L. Busino, M. Donzelli, F. Margottin-Goguet, P. K. Jackson, L. Yamasaki, and M. Pagano. 2003. Control of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo. Dev. Cell 4:799-812.[CrossRef][Medline]
22. Hart, M., J. P. Concordet, I. Lassot, I. Albert, R. del los Santos, H. Durand, C. Perret, B. Rubinfeld, F. Margottin, R. Benarous, and P. Polakis. 1999. The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell. Curr. Biol. 9:207-210.[CrossRef][Medline]
23. Hatakeyama, S., M. Kitagawa, K. Nakayama, M. Shirane, M. Matsumoto, K. Hattori, H. Higashi, H. Nakano, K. Okumura, K. Onoe, and R. A. Good. 1999. Ubiquitin-dependent degradation of IB
is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1. Proc. Natl. Acad. Sci. USA 96:3859-3863.
24. Heller, R. A., K. Song, N. Fan, and D. J. Chang. 1992. The p70 tumor necrosis factor receptor mediates cytotoxicity. Cell 70:47-56.[CrossRef][Medline]
25. Hicke, L. 1999. Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels. Trends Cell Biol. 9:107-112.[CrossRef][Medline]
26. Hicke, L. 2001. Protein regulation by monoubiquitin. Nat. Rev. Mol. Cell Biol. 2:195-201.[CrossRef][Medline]
27. Hicke, L., and R. Dunn. 2003. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol. 19:141-172.[CrossRef][Medline]
28. Jin, J., T. Shirogane, L. Xu, G. Nalepa, J. Qin, S. J. Elledge, and J. W. Harper. 2003. SCFß-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase. Genes Dev. 17:3062-3074.
29. Kitagawa, M., S. Hatakeyama, M. Shirane, M. Matsumoto, N. Ishida, K. Hattori, I. Nakamichi, A. Kikuchi, and K. Nakayama. 1999. An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin. EMBO J. 18:2401-2410.[CrossRef][Medline]
30. Kline, J. B., H. Roehrs, and C. V. Clevenger. 1999. Functional characterization of the intermediate isoform of the human prolactin receptor. J. Biol. Chem. 274:35461-35468.
31. Kline, J. B., M. A. Rycyzyn, and C. V. Clevenger. 2002. Characterization of a novel and functional human prolactin receptor isoform (deltaS1PRLr) containing only one extracellular fibronectin-like domain. Mol. Endocrinol. 16:2310-2322.
32. Kumar, K. G., W. Tang, A. K. Ravindranath, W. A. Clark, E. Croze, and S. Y. Fuchs. 2003. SCF(HOS) ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-alpha receptor. EMBO J. 22:5480-5490.[CrossRef][Medline]
33. Lassot, I., E. Segeral, C. Berlioz-Torrent, H. Durand, L. Groussin, T. Hai, R. Benarous, and F. Margottin-Goguet. 2001. ATF4 degradation relies on a phosphorylation-dependent interaction with the SCFßTrCP ubiquitin ligase. Mol. Cell. Biol. 21:2192-2202.
34. Latres, E., D. S. Chiaur, and M. Pagano. 1999. The human F box protein beta-Trcp associates with the Cul1/Skp1 complex and regulates the stability of beta-catenin. Oncogene 18:849-854.[CrossRef][Medline]
35. Lee, R. C., J. A. Walters, M. E. Reyland, and S. M. Anderson. 1999. Constitutive activation of the prolactin receptor results in the induction of growth factor-independent proliferation and constitutive activation of signaling molecules. J. Biol. Chem. 274:10024-10034.
36. Levkowitz, G., H. Waterman, E. Zamir, Z. Kam, S. Oved, W. Y. Langdon, L. Beguinot, B. Geiger, and Y. Yarden. 1998. c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12:3663-3674.
37. Lu, J. C., P. Scott, G. J. Strous, and L. A. Schuler. 2002. Multiple internalization motifs differentially used by prolactin receptor isoforms mediate similar endocytic pathways. Mol. Endocrinol. 16:2515-2527.
38. Mantovani, F., and L. Banks. 2003. Regulation of the discs large tumor suppressor by a phosphorylation-dependent interaction with the ß-TrCP ubiquitin ligase receptor. J. Biol. Chem. 278:42477-42486.
39. Margottin, F., S. P. Bour, H. Durand, L. Selig, S. Benichou, V. Richard, D. Thomas, K. Strebel, and R. Benarous. 1998. A novel human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif. Mol. Cell 1:565-574.[CrossRef][Medline]
40. Margottin-Goguet, F., J. Y. Hsu, A. Loktev, H. M. Hsieh, J. D. Reimann, and P. K. Jackson. 2003. Prophase destruction of Emi1 by the SCFßTrCP/Slimb ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase. Dev. Cell 4:813-826.[CrossRef][Medline]
41. Root, A. W. 1976. Growth hormone and prolactin in the fetus. Prog. Clin. Biol. Res. 10:107-126.[Medline]
42. Ross, R. J., N. Esposito, X. Y. Shen, S. Von Laue, S. L. Chew, P. R. Dobson, M. C. Postel-Vinay, and J. Finidori. 1997. A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein. Mol. Endocrinol. 11:265-273.
43. Sotiropoulos, A., S. Moutoussamy, F. Renaudie, M. Clauss, C. Kayser, F. Gouilleux, P. A. Kelly, and J. Finidori. 1996. Differential activation of Stat3 and Stat5 by distinct regions of the growth hormone receptor. Mol. Endocrinol. 10:998-1009.[Abstract]
44. Spencer, E., J. Jiang, and Z. J. Chen. 1999. Signal-induced ubiquitination of IB by the F-box protein Slimb/ß-TrCP. Genes Dev. 13:284-294.
45. Spiegelman, V. S., W. Tang, M. Katoh, T. J. Slaga, and S. Y. Fuchs. 2002. Inhibition of HOS expression and activities by Wnt pathway. Oncogene 21:856-860.[CrossRef][Medline]
46. Strous, G. J., and J. Gent. 2002. Dimerization, ubiquitylation and endocytosis go together in growth hormone receptor function. FEBS Lett. 529:102-109.[CrossRef][Medline]
47. Strous, G. J., and P. van Kerkhof. 2002. The ubiquitin-proteasome pathway and the regulation of growth hormone receptor availability. Mol. Cell. Endocrinol. 197:143-151.[CrossRef][Medline]
48. Suzuki, H., T. Chiba, M. Kobayashi, M. Takeuchi, T. Suzuki, A. Ichiyama, T. Ikenoue, M. Omata, K. Furuichi, and K. Tanaka. 1999. IB ubiquitination is catalyzed by an SCF-like complex containing Skp1, cullin-1, and two F-box/WD40-repeat proteins, ßTrCP1 and ßTrCP2. Biochem. Biophys. Res. Commun. 256:127-132.[CrossRef][Medline]
49. Tan, P., S. Y. Fuchs, A. Chen, K. Wu, C. Gomez, Z. Ronai, and Z. Q. Pan. 1999. Recruitment of a ROC1-CUL1 ubiquitin ligase by Skp1 and HOS to catalyze the ubiquitination of IB. Mol. Cell 3:527-533.[CrossRef][Medline]
50. Treier, M., L. M. Staszewski, and D. Bohmann. 1994. Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78:787-798.[CrossRef][Medline]
51. Vincent, V., V. Goffin, M. Rozakis-Adcock, J. P. Mornon, and P. A. Kelly. 1997. Identification of cytoplasmic motifs required for short prolactin receptor internalization. J. Biol. Chem. 272:7062-7068.
52. Weissman, A. M. 2001. Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell Biol. 2:169-178.[CrossRef][Medline]
53. Winston, J. T., D. M. Koepp, C. Zhu, S. J. Elledge, and J. W. Harper. 1999. A family of mammalian F-box proteins. Curr. Biol. 9:1180-1182.[CrossRef][Medline]
54. Winston, J. T., P. Strack, P. Beer-Romero, C. Y. Chu, S. J. Elledge, and J. W. Harper. 1999. The SCFß-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IB and ß-catenin and stimulates IB ubiquitination in vitro. Genes Dev. 13:270-283.
55. Yaron, A., A. Hatzubai, M. Davis, I. Lavon, S. Amit, A. M. Manning, J. S. Andersen, M. Mann, F. Mercurio, and Y. Ben-Neriah. 1998. Identification of the receptor component of the IB-ubiquitin ligase. Nature 396:590-594.[CrossRef][Medline]
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