Investigation of the role of ß-TrCP in growth hormone transduction defect (GHTD).

Background Growth hormone(GH) and epidermal growth factor (EGF) stimulate cell growth and differentiation, and crosstalking between their signaling pathways is important for normal cellular development. Growth hormone transduction defect (GHTD) is characterized by excessive GH receptor (GHR) degradation, due to over-expression of the E3 ubiquitin ligase, cytokine inducible SH2-containing protein (CIS). GH induction of GHTD fibroblasts after silencing of messenger RNA (mRNA) CIS (siCIS) or with higher doses of GH restores normal GH signaling. ß-Transducing-repeat-containing protein (ß-TrCP), another E3 ubiquitin ligase, also plays a role in GHR endocytosis. We studied the role of ß-TrCP in the regulation of the GH/GHR and EGF/EGF receptor (EGFR) pathways in normal and GHTD fibroblasts. Materials and methods Fibroblast cultures were developed from gingival biopsies of a GHTD (P) and a control child (C). Protein expression and cellular localization of ß-TrCP were studied by Western immunoblotting and immunofluorescence, respectively, after: (1) GH 200 µg/L human GH (hGH) induction, either with or without silence CIS (siCIS), and (2) inductions with 200 µg/L GH or 1000 µg/L GH or 50 ng/mL EGF. Results After induction with: (1) GH200/siCIS, the protein expression and cytoplasmic-membrane localization of ß-TrCP were increased in the patient, (2) GH200 in the control and GH1000 in the patient, the protein and cytoplasmic-membrane localization of ß-TrCP were increased and (3) EGF, the protein expression and cytoplasmic-membrane localization of ß-TrCP were increased in both the control and the patient. Conclusions (1) ß-TrCP appears to be part of the negative regulatory mechanism of the GH/GHR and EGF/EGFR pathways. (2) There appears to be a negative correlation between ß-TrCP and CIS. (3) In the control and GHTD patient, ß-TrCP increases when CIS is suppressed, possibly as a compensatory inhibitor of the GH/GHR pathway.

NF-κB activation is a turn on for vaccinia virus phosphoprotein A49 to turn off NF-κB activation.

Vaccinia virus protein A49 inhibits NF-κB activation by molecular mimicry and has a motif near the N terminus that is conserved in IκBα, ß-catenin, HIV Vpu, and some other proteins. This motif contains two serines, and for IκBα and ß-catenin, phosphorylation of these serines enables recognition by the E3 ubiquitin ligase ß-TrCP. Binding of IκBα and ß-catenin by ß-TrCP causes their ubiquitylation and thereafter proteasome-mediated degradation. In contrast, HIV Vpu and VACV A49 are not degraded. This paper shows that A49 is phosphorylated at serine 7 but not serine 12 and that this is necessary and sufficient for binding ß-TrCP and antagonism of NF-κB. Phosphorylation of A49 S7 occurs when NF-κB signaling is activated by addition of IL-1ß or overexpression of TRAF6 or IKKß, the kinase needed for IκBα phosphorylation. Thus, A49 shows beautiful biological regulation, for it becomes an NF-κB antagonist upon activation of NF-κB signaling. The virulence of viruses expressing mutant A49 proteins or lacking A49 (vΔA49) was tested. vΔA49 was attenuated compared with WT, but viruses expressing A49 that cannot bind ß-TrCP or bind ß-TrCP constitutively had intermediate virulence. So A49 promotes virulence by inhibiting NF-κB activation and by another mechanism independent of S7 phosphorylation and NF-κB antagonism. Last, a virus lacking A49 was more immunogenic than the WT virus.

ß-TrCP upregulates HIF-1 in prostate cancer cells.

The substantial availability of hypoxia-inducible factor 1 (HIF-1) for pathophysiological states, such as malignancies and ischemia, is primarily regulated post-translationally through the ubiquitin proteolytic system. The balance between degradation and stabilization of HIF-1α protein is determined by specific E3 ligases. In our search for new E3 ligases that might affect HIF-1α protein expression, we studied the effects of beta-transducin repeat-containing protein (ß-TrCP) on the hypoxic pathway in cancer cells. ß-TrCP is overexpressed in many tumors and regulates various cellular processes through mediating the degradation of important targets. Unexpectedly, we found that ß-TrCP overexpression increases HIF-1α protein expression level as well as HIF-1 transcriptional activity by stabilizing HIF-1α protein and preventing its ubiquitination and proteasomal degradation in prostate cancer cells. By using a proteomic approach, we succeeded in demonstrating that ß-TrCP interferes with the association between HIF-1α and HSP70/CHIP, a HIF-1α established E3 ligase complex. Whereas the E3 ligase activity of ß-TrCP is well known, antagonizing another E3 ligase is a new mechanism of action of this important E3. We suggest that destroying or suppressing ß-TrCP and thereby interrupting the HIF-1 pathway, could be valuable antitumor therapy.

MicroRNA-182 promotes pancreatic cancer cell proliferation and migration by targeting ß-TrCP2.

Pancreatic cancer is an aggressive malignancy. The median survival rate remains low, indicating that the identification of novel biomarkers and therapeutic targets is critical. Here, we examined the role of microRNA-182 (miR-182) in pancreatic cancer development. Analysis of human pancreatic cancer specimens and cell lines showed that miR-182 is overexpressed in pancreatic cancer and promotes tumor proliferation and invasion. ß-TrCP2 was confirmed as a direct target of miR-182. Silencing of ß-TrCP2 increased the levels of ß-catenin, which is similar to miR-182 overexpression. Ectopic expression of ß-TrCP2 inhibited the miR-182-induced activation of ß-catenin signaling. The oncogenic effect of miR-182 and its reversal by ß-TrCP2 were confirmed in vivo This study suggests that ß-TrCP and miR-182 may be possible biomarkers and targets for early detection and treatment of pancreatic cancer.

S6 Kinase- and ß-TrCP2-Dependent Degradation of p19Arf Is Required for Cell Proliferation.

The kinase mTOR (mammalian target of rapamycin) promotes translation as well as cell survival and proliferation under nutrient-rich conditions. Whereas mTOR activates translation through ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP), how it facilitates cell proliferation has remained unclear. We have now identified p19(Arf), an inhibitor of cell cycle progression, as a novel substrate of S6K that is targeted to promote cell proliferation. Serum stimulation induced activation of the mTOR-S6K axis and consequent phosphorylation of p19(Arf) at Ser(75). Phosphorylated p19(Arf) was then recognized by the F-box protein ß-TrCP2 and degraded by the proteasome. Ablation of ß-TrCP2 thus led to the arrest of cell proliferation as a result of the stabilization and accumulation of p19(Arf). The ß-TrCP2 paralog ß-TrCP1 had no effect on p19(Arf) stability, suggesting that phosphorylated p19(Arf) is a specific substrate of ß-TrCP2. Mice deficient in ß-TrCP2 manifested accumulation of p19(Arf) in the yolk sac and died in utero. Our results suggest that the mTOR pathway promotes cell proliferation via ß-TrCP2-dependent p19(Arf) degradation under nutrient-rich conditions.

Regulation of Nrf2 signaling and longevity in naturally long-lived rodents.

The preternaturally long-lived naked mole-rat, like other long-lived species and experimental models of extended longevity, is resistant to both endogenous (e.g., reactive oxygen species) and environmental stressors and also resists age-related diseases such as cancer, cardiovascular disease, and neurodegeneration. The mechanisms behind the universal resilience of longer-lived organisms to stress, however, remain elusive. We hypothesize that this resilience is linked to the activity of a highly conserved transcription factor, nuclear factor erythroid 2-related factor (Nrf2). Nrf2 regulates the transcription of several hundred cytoprotective molecules, including antioxidants, detoxicants, and molecular chaperones (heat shock proteins). Nrf2 itself is tightly regulated by mechanisms that either promote its activity or increase its degradation. We used a comparative approach and examined Nrf2-signaling activity in naked mole-rats and nine other rodent species with varying maximum lifespan potential (MLSP). We found that constitutive Nrf2-signaling activity was positively correlated (P = 0.0285) with MLSP and that this activity was also manifested in high levels of downstream gene expression and activity. Surprisingly, we found that species longevity was not linked to the protein levels of Nrf2 itself, but rather showed a significant (P < 0.01) negative relationship with the regulators Kelch-like ECH-Associated Protein 1 (Keap1) and ß-transducin repeat-containing protein (ßTrCP), which target Nrf2 for degradation. These findings highlight the use of a comparative biology approach for the identification of evolved mechanisms that contribute to health span, aging, and longevity.

Substrate trapping proteomics reveals targets of the ßTrCP2/FBXW11 ubiquitin ligase.

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study ßTrCP2/FBXW11, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to "trap" ubiquitylated substrates on the SCF(FBXW11) E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCF(FBXW11) bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.

Oncogenic Y641 mutations in EZH2 prevent Jak2/ß-TrCP-mediated degradation.

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase ß-TrCP (FBXW1). ß-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs ß-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of ß-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2(Y641) mutants recurrently implicated in lymphoma pathogenesis are unable to bind ß-TrCP. Further, endogenous EZH2(Y641) mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that ß-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

KRAS protein stability is regulated through SMURF2: UBCH5 complex-mediated ß-TrCP1 degradation.

Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, ß-transducing repeat containing protein 1 (ß-TrCP1). Conversely, ß-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, ß-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutation of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.

FOXO3a-Dependent Mechanism of E1A-Induced Chemosensitization.

Gene therapy trials in human breast, ovarian, and head and neck tumors indicate that adenovirus E1A can sensitize cancer cells to the cytotoxic effects of paclitaxel in vitro and in vivo. Resistance to paclitaxel has been reported to occur in cells expressing low levels of the Forkhead transcription factor FOXO3a. In this article, we report that FOXO3a is critical for E1A-mediated chemosensitization to paclitaxel. RNA interference-mediated knockdown of FOXO3a abolished E1A-induced sensitivity to paclitaxel. Mechanistic investigations indicated that E1A indirectly stabilized FOXO3a by acting at an intermediate step to inhibit a ubiquitin-dependent proteolysis pathway involving the E3 ligase ßTrCP and the FOXO3a inhibitory kinase IKKß. E1A derepressed this inhibitory pathway by stimulating expression of the protein phosphatase 2A (PP2A)/C protein phosphatases, which by binding to the TGF-ß-activated kinase TAK1, inhibited its ability to activate IKKß and, thereby, to suppress ßTrCP-mediated degradation of FOXO3a. Thus, by stimulating PP2A/C expression, E1A triggers a signaling cascade that stabilizes FOXO3a and mediates chemosensitization. Our findings provide a leap forward in understanding paclitaxel chemosensitization by E1A, and offer a mechanistic rational to apply E1A gene therapy as an adjuvant for improving therapeutic outcomes in patients receiving paclitaxel treatment.

Inhibition of human melanoma cell growth by the dietary flavonoid fisetin is associated with disruption of Wnt/ß-catenin signaling and decreased Mitf levels.

The prognosis of advanced melanoma remains poor in spite of treatment advances, emphasizing the importance of additional preventive measures. Flavonoids, natural components of our diet, are being investigated for their chemopreventive/therapeutic properties. Microphthalmia-associated transcription factor (Mitf), downstream of the Wnt/ß-catenin pathway, has become an important prognostic marker of melanoma. In this study, we show that treatment of 451Lu melanoma cells with the dietary flavonoid fisetin (3,7,3',4'-tetrahydroxyflavone) resulted in decreased cell viability with G1-phase arrest and disruption of Wnt/ß-catenin signaling. This was accompanied by a decrease in the expression of Wnt protein and its co-receptors, as well as by a parallel increase in the expression of endogenous Wnt inhibitors. Fisetin-treated cells showed increased cytosolic levels of Axin and ß-TrCP and decreased phosphorylation of glycogen synthase kinase 3ß associated with decreased ß-catenin stabilization. Fisetin-mediated interference with the functional cooperation between ß-catenin and T-cell factor (TCF)-2 resulted in the downregulation of positively regulated TCF targets, such as c-myc, Brn-2, and Mitf. Flow-cytometric analysis of Mitf-overexpressing cells showed that fisetin repressed Mitf-induced cell proliferation. Finally, administration of fisetin to 451Lu-xenografted nude mice resulted in the inhibition of tumor development and decreased Mitf expression. Our data suggest that fisetin can be developed as an effective agent against melanoma because of its potential inhibitory effect on ß-catenin/Mitf signaling.

Blockade of JAK2 activity suppressed accumulation of ß-catenin in leukemic cells.

The Wnt/ß-catenin pathway has been implicated in leukemogenesis. We found ß-catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. ß-Catenin can be significantly down-regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/ß-catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of ß-catenin, APC, Axin, ß-Trcp, GSK3α, and GSK3ß were up-regulated within 12-16 h. However, only the protein levels of GSK3ß and ß-Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490-induced inhibition of ß-catenin can be attenuated by shRNA targeting ß-TrCP. Taken together; these results suggest that ß-Trcp plays a key role in the cross-talk between JAK/STAT and Wnt/ß-catenin signaling in leukemia cells.

[Transducin beta-TrCP expressed in developmental hair follicle of mice].

OBJECTIVE: To identify the expression of antagonist beta-TrCP protein in Sonic hedgehog signal transduction pathway and Wnt signal transduction pathway in hair follicle tissues. METHODS: The heads of day 18 embryo, and one day and six-days-old postnatal mice were acquired and treated with 40 g/L paraformaldehyde fixation for 48 h and paraffin embedding. The expression of beta-TrCP proteins was examined using LsAB (labelled streptavidin-biotin) method. RESULTS: beta-TrCP proteins were expressed in the cytoplasm of the hair stems of hair follicle, hair cuticle, cuticle of root sheath, Huxley's layer of internal root sheath cells, external root sheath and mesenchymal tissues, but not in connective tissue sheath and Henle's layer of internal root sheath. CONCLUSION: TrCP express in the developmental hair follicle tissues, which implicates that beta-TrCP regulate the developmental hair follicle by mediating the signal transduction pathways.

Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer.

The maintenance and preservation of distinct phases during the cell cycle is a highly complex and coordinated process. It is regulated by phosphorylation--through the activity of cyclin-dependent kinases (CDKs)--and protein degradation, which occurs through ubiquitin ligases such as SCF (SKP1-CUL1-F-box protein) complexes and APC/C (anaphase-promoting complex/cyclosome). Here, we explore the functionality and biology of the F-box proteins, SKP2 (S-phase kinase-associated protein 2) and beta-TrCP (beta-transducin repeat-containing protein), which are emerging as important players in cancer biogenesis owing to the deregulated proteolysis of their substrates.

Epigenetic silencing of AXIN2/betaTrCP and deregulation of p53-mediated control lead to wild-type beta-catenin nuclear accumulation in lung tumorigenesis.

Beta-catenin accumulation is often found in lung tumors, but only a few patients have mutations in beta-catenin gene. In addition, activated p53 downregulates beta-catenin. Therefore, we postulated that alteration of the degradation complex AXIN2 (axis inhibition protein 2) and betaTrCP (beta-transducin repeat-containing protein) and p53 regulation could result in beta-catenin protein accumulation in lung cancer. Using the immunohistochemical and sequencing analyses, we found that patients with beta-catenin accumulation without mutation were associated with patients with p53 overexpression and low AXIN2 expression (P=0.023 approximately 0.041). Alteration of AXIN2 was associated with poor survival in early stage patients (P=0.016). Low expression of AXIN2 and betaTrCP was significantly associated with promoter hypermethylation and histone deacetylation. Ectopic expression and knockdown of p53, AXIN2 and betaTrCP genes in A549 (p53 wild-type) and H1299 (p53 null) lung cancer cell lines showed cooperation between p53 and AXIN2/betaTrCP in the reduction of beta-catenin expression. Our clinical and cell model findings provide new evidence that epigenetic silencing of AXIN2/betaTrCP in the degradation complex and deregulation of p53-mediated control lead to wild-type beta-catenin nuclear accumulation in non-small cell lung cancer tumorigenesis. In addition, a high level of p53 downregulates the beta-catenin expression, but this effect is attenuated by non-functional AXIN2 or betaTrCP in lung cancer.

Beta-TrCP1-mediated degradation of PERIOD2 is essential for circadian dynamics.

Regulated degradation of circadian clock proteins is a crucial step for rhythm generation per se but also for establishing a normal circadian period. Here, the authors show that the F-box protein beta-transducin repeat containing protein 1 (beta-TrCP1) as part of the E3 ubiquitin ligase complex is an essential component of the mammalian circadian oscillator. Down-regulation of endogenous beta-TrCP1 as well as expression of a dominant-negative form both result in lengthening of the circadian period in oscillating fibroblasts. These phenotypes are due to an impaired degradation of PERIOD (PER) proteins, since expression of beta-TrCP interaction-deficient PER2 variants--but not wild-type PER2--results in a dramatic stabilization of PER2 protein as well as in the disruption of circadian rhythmicity. Mathematical modeling conceptualizes the authors' findings and suggests that loss of sustained rhythmicity in cells with eliminated beta-TrCP-mediated PER2 degradation is due to excessive nuclear repression, a prediction they verified experimentally.

Mechanisms of CD4 downregulation by the Nef and Vpu proteins of primate immunodeficiency viruses.

Human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), and simian immunodeficiency virus (SIV) are the etiological agents of acquired immunodeficiency syndrome (AIDS) in humans and a related disease in non-human primates. These viruses infect T cells and macrophages that express the surface glycoprotein, CD4, because this glycoprotein acts as a co-receptor for incoming virus particles. Once infection has occurred, however, the presence of CD4 poses problems for the virus life cycle, including the possibility of superinfection, premature binding of CD4 to nascent virus particles, and inhibition of virus release. Accordingly, primate immunodeficiency viruses have evolved at least two distinct mechanisms, mediated by the Nef and Vpu viral proteins, to "downregulate" CD4 in the host cells. Nef and Vpu are mainly expressed early and late, respectively, in the viral life cycle, ensuring continuous removal of CD4. Nef links mature CD4 to components of clathrin-dependent trafficking pathways at the plasma membrane, and perhaps in intracellular compartments, leading to internalization and delivery of CD4 to lysosomes for degradation. Vpu, on the other hand, interacts with newly-synthesized CD4 in the endoplasmic reticulum, linking CD4 to the SCF ubiquitin ligase and facilitating the entry of CD4 into the endoplasmic-reticulum-associated degradation pathway. These two mechanisms lead to a dramatic reduction of CD4 expression in infected cells and are essential for efficient virus replication and disease progression.

beta-Trcp mediates ubiquitination and degradation of the erythropoietin receptor and controls cell proliferation.

Control of intensity and duration of erythropoietin (Epo) signaling is necessary to tightly regulate red blood cell production. We have recently shown that the ubiquitin/proteasome system plays a major role in the control of Epo-R signaling. Indeed, after Epo stimulation, Epo-R is ubiquitinated and its intracellular part is degraded by the proteasome, preventing further signal transduction. The remaining part of the receptor and associated Epo are internalized and degraded by the lysosomes. We show that beta-Trcp is responsible for Epo-R ubiquitination and degradation. After Epo stimulation, beta-Trcp binds to the Epo-R. This binding, like Epo-R ubiquitination, requires Jak2 activation. The Epo-R contains a typical DSG binding sequence for beta-Trcp that is highly conserved among species. Interestingly, this sequence is located in a region of the Epo-R that is deleted in patients with familial polycythemia. Mutation of the serine residue of this motif to alanine (Epo-RS462A) abolished beta-Trcp binding, Epo-R ubiquitination, and degradation. Epo-RS462A activation was prolonged and BaF3 cells expressing this receptor are hypersensitive to Epo, suggesting that part of the hypersensitivity to Epo in familial polycythemia could be the result of the lack of beta-Trcp recruitment to the Epo-R.

Structural studies on 24P-IkappaBalpha peptide derived from a human IkappaB-alpha protein related to the inhibition of the activity of the transcription factor NF-kappaB.

The IkappaB-alpha protein, inhibitor of the transcription factor nuclear factor-kappaB (NF-kappaB), is a cellular substrate of beta-transducin repeat containing protein (beta-TrCP). beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. beta-TrCP targets the protein IkappaB-alpha for ubiquitination, followed by proteasome degradation. The SCF-beta-TrCP complex specifically recognizes an IkappaB-alpha peptide containing the DpSGXXpS motif in a phosphorylation-dependent manner. A fragment comprising 24 amino acids residues for the phosphorylated peptide at the two sites Ser32 and Ser36 and thus termed 24P-IkappaBalpha (P-IkappaBalpha21-44) was characterized conformationally by NMR spectroscopy and molecular dynamics simulation. In the free states, 24P-IkappaBalpha exhibits mainly a random coil conformation, although the presence of a nascent bend was detected between residues 30 and 36, flanked by two N- and C-terminal disordered regions. The bound conformation of the phosphorylated IkappaB-alpha peptide was obtained using transfer nuclear Overhauser effect spectroscopy (TRNOESY) experiments. To further elucidate the basis of the beta-TrCP interaction, a complex between 24P-IkappaBalpha peptide and beta-TrCP protein was studied using saturation transfer difference (STD) NMR experiments. The conformation of 24P-IkappaBalpha bound to beta-TrCP presents a bend corresponding to the 31DpSGLDpS36 motif and on both sides N- and C-terminal turn regions (Lys22-Asp31 and Met37-Glu43). The bound structure of the phosphorylated peptide suggests that these domains are crucial for the interaction of the peptide with its receptor showing the protons identified by STD NMR as exposed in close proximity to the beta-TrCP surface.

Stabilization of prolactin receptor in breast cancer cells.

The role of the hormone prolactin (PRL) in the pathogenesis of breast cancer is mediated by its cognate receptor (PRLr). Ubiquitin-dependent degradation of the PRLr that negatively regulates PRL signaling is triggered by PRL-mediated phosphorylation of PRLr on Ser349 followed by the recruitment of the beta-transducin repeats-containing protein (beta-TrCP) ubiquitin-protein isopeptide ligase. We report here for the first time that interaction between PRLr and beta-TrCP is less efficient in human breast cancer cells than in non-tumorigenic human mammary epithelial cells. Furthermore, we demonstrate that both PRLr degradation and PRLr phosphorylation on Ser349 are impaired in breast tumor cells and tissues, an observation that directly correlates with enhanced expression of the PRLr in malignant breast epithelium. These findings represent a novel mechanism through which altered PRLr stability may directly influence the pathogenesis of breast cancer.