Atypical protein kinase C regulates dual pathways for degradation of the oncogenic coactivator SRC-3/AIB1

SRC-3/AIB1 is a steroid receptor coactivator with potent growth-promoting activity, and its overexpression is sufficient to induce tumorigenesis. Previous studies indicate that the cellular level of SRC-3 is tightly regulated by both ubiquitin-dependent and ubiquitin-independent proteasomal degradation pathways. Atypical protein kinase C (aPKC) is frequently overexpressed in cancers. In the present study, we show that aPKC phosphorylates and specifically stabilizes SRC-3 in a selective ER-dependent manner. We further demonstrate that an acidic residue-rich region in SRC-3 is an important determinant for aPKC-mediated phosphorylation and stabilization. The mechanism of the aPKC-mediated stabilization appears due to a decreased interaction between SRC-3 and the C8 subunit of the 20S core proteasome, thus preventing SRC-3 degradation. Our results demonstrate a potent signaling mechanism for regulating SRC-3 levels in cells by coordinate enzymatic inhibition of both ubiquitin-dependent and ubiquitin-independent proteolytic pathways.     

Feeding the machine: mechanisms of proteasome-catalyzed degradation of ubiquitinated proteins

The proteasome plays a role in a myriad of intracellular processes from cell-cycle control to antigen presentation. Central to these processes is the targeting of selected proteins for proteasomal degradation via their conjugation to ubiquitin. The mechanisms by which the ubiquitin-dependent proteasomal proteolysis occurs can be divided into four steps: first, substrate protein recognition by its cognate E3 ubiquitin ligase; second, polyubiquitinated protein substrate recruitment to the proteasome; third, protein substrate deubiquitination; and four, proteolytic chamber pore opening/substrate translocation followed by proteolysis. Recent advances include the identification of novel E3 ubiquitin ligase recognition determinants, a new isopeptidase activity, and a better understanding of how the proteasome's axial channels are gated.     

Downregulation of the upstream binding factor1 by glycogen synthase kinase3beta in myeloid cells induced to differentiate

The upstream binding factor 1 (UBF1), one of the proteins that regulate the activity of RNA polymerase I, is downregulated in 32D myeloid cells induced to differentiate into granulocytes, either by the type 1 insulin-like growth factor (IGF-1) or the granulocytic colony stimulating factor (G-CSF). Downregulation of UBF1 is largely due to protein degradation, while mRNA levels are not affected. Inhibition of UBF1 degradation by lithium chloride (LiCl)and lactacystin suggest a role of glycogen synthase kinase beta (GSK3beta) in a proteasome-dependent degradation of UBF. GSK3beta phosphorylates in vitro and in vivo the UBF protein, which has five putative motifs for phosphorylation by GSK3beta. Elimination and/or mutations of these motifs stabilize the UBF1 protein even in cells induced to differentiate. Conversely, a stably transfected, constitutively active GSK3beta accelerates the downregulation of UBF1. We show further that activation of the differentiating protein C/EPBalpha in 32D cells transformed by the oncogenic BCR/ABL protein causes downregulation of UBF1. Finally, inhibition of differentiation of myeloid cells by a dominant negative mutant of Stat3 stabilizes the UBF1 protein, while rapamycin-induced differentiation of myeloid cells downregulates UBF1 levels. Taken together, our results indicate that the induction of granulocytic differentiation in 32D murine myeloid cells causes the degradation of UBF1, via GSK3beta and the proteasome pathway.     

Degradation of antizyme inhibitor, an ornithine decarboxylase homologous protein, is ubiquitin-dependent and is inhibited by antizyme

Ornithine decarboxylase (ODC) is the most notable example of a protein degraded by the 26 S proteasome without ubiquitination. Instead, ODC is targeted to degradation by direct binding to a polyamine-induced protein termed antizyme (Az). Antizyme inhibitor (AzI) is an ODC-related protein that does not retain enzymatic activity yet binds Az with higher affinity than ODC. We show here that like ODC, AzI is also a short-lived protein that undergoes proteasomal degradation. However, in contrast to ODC degradation, the degradation of AzI is ubiquitin-dependent and does not require interaction with Az. Moreover, Az binding actually stabilizes AzI by inhibiting its ubiquitination. Substituting the C terminus of AzI with that of ODC, which together with Az constitutes the complete degradation signal of ODC, does not subvert AzI degradation from the ubiquitin-dependent mode to the Az-dependent mode, suggesting dominance of the ubiquitination signal. Our results suggest opposing roles of Az in regulating the degradation of AzI and ODC.     

C/EBPalpha triggers proteasome-dependent degradation of cdk4 during growth arrest

CCAAT/enhancer binding protein alpha (C/EBPalpha) causes growth arrest via direct interaction with the cyclin-dependent kinases cdk2 and cdk4. In this paper, we present evidence showing that C/EBPalpha enhances a proteasome-dependent degradation of cdk4 during growth arrest in liver of newborn mice and in cultured cells. Overexpression of C/EBPalpha in several biological systems leads to a reduction of cdk4 protein levels, but not mRNA levels. Experiments with several tissue culture models reveal that C/EBPalpha enhances the formation of cdk4-ubiquitin conjugates and induces degradation of cdk4 through a proteasome-dependent pathway. As a result, the half-life of cdk4 is shorter and protein levels of cdk4 are reduced in cells expressing C/EBPalpha. Gel filtration analysis of cdk4 complexes shows that a chaperone complex cdk4-cdc37-Hsp90, which protects cdk4 from degradation, is abundant in proliferating livers that lack C/EBPalpha, but this complex is weak or undetectable in livers expressing C/EBPalpha. Our studies show that C/EBPalpha disrupts the cdk4-cdc37-Hsp90 complex via direct interaction with cdk4 and reduces protein levels of cdk4 by increasing proteasome-dependent degradation of cdk4.     

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation.     

Leishmania express a functional Cdc20 homologue

Our knowledge concerning the mechanisms of cell cycle regulation in organisms belonging to the Trypanosometidae family is limited. Leishmania donovani are parasitic protozoa that cause kala azar, a fatal form of visceral leishmaniasis in humans. Here we provide evidence that the L. donovani genome contains a Cdc20 homologue. Cdc20 is a regulator of the Anaphase Promoting Complex/Cyclosome (APC/C) that mediates ubiquitin-dependent proteasomal degradation of key cell cycle regulators in eukaryotes. We show that L. donovani Cdc20 protein (LdCdc20p) can complement a lack of yeast Cdc20 protein in Saccharomyces cerevisiae cells, validating the functionality of LdCdc20p. Furthermore, we demonstrate cyclic expression of LdCdc20p and that it contains an active RXXL destruction motif, a distinctive feature of proteins targeted for proteasomal degradation by APC/C. Finally, in line with the proteasome mediating LdCdc20p degradation, promastigotes exposed to proteasome inhibitor display elevated LdCdc20p levels. Taken together our data indicate that Leishmania regulate their cell cycle by ubiquitin-dependent proteasomal degradation mediated by the APC/C.     

The Proteasome-associated Protein Ecm29 Inhibits Proteasomal ATPase Activity and in Vivo Protein Degradation by the Proteasome

Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.     

Defining the role of ubiquitin-interacting motifs in the polyglutamine disease protein, ataxin-3

Polyglutamine (polyQ) expansions cause neurodegeneration that is associated with protein misfolding and influenced by functional properties of the host protein. The polyQ disease protein, ataxin-3, has predicted ubiquitin-specific protease and ubiquitin-binding domains, which suggest that ataxin-3 functions in ubiquitin-dependent protein surveillance. Here we investigate direct links between the ubiquitin-proteasome pathway and ataxin-3. In neural cells we show that, through its ubiquitin interaction motifs (UIMs), normal or expanded ataxin-3 binds a broad range of ubiquitinated proteins that accumulate when the proteasome is inhibited. The expression of a catalytically inactive ataxin-3 (normal or expanded) causes ubiquitinated proteins to accumulate in cells, even in the absence of proteasome inhibitor. This accumulation of ubiquitinated proteins occurs primarily in the cell nucleus in transfected cells and requires intact UIMs in ataxin-3. We further show that both normal and expanded ataxin-3 can undergo oligoubiquitination. Although this post-translational modification occurs in a UIM-dependent manner, it becomes independent of UIMs when the catalytic cysteine residue of ataxin-3 is mutated, suggesting that ataxin-3 ubiquitination is itself regulated in trans by its own de-ubiquitinating activity. Finally, pulse-chase labeling reveals that ataxin-3 is degraded by the proteasome, with expanded ataxin-3 being as efficiently degraded as normal ataxin-3. Mutating the UIMs does not alter degradation, suggesting that UIM-mediated oligoubiquitination of ataxin-3 modulates ataxin-3 function rather than stability. The function of ataxin-3 as a de-ubiquitinating enzyme, its post-translational modification by ubiquitin, and its degradation via the proteasome link this polyQ protein to ubiquitin-dependent pathways already implicated in disease pathogenesis.     

Role of ubiquitin in proteasomal degradation of mutant alpha(1)-antitrypsin Z in the endoplasmic reticulum

A delay in intracellular degradation of the mutant alpha(1)-antitrypsin (alpha(1)AT)Z molecule is associated with greater retention within the endoplasmic reticulum (ER) and susceptibility to liver disease in a subgroup of patients with alpha(1)AT deficiency. Recent studies have shown that alpha(1)ATZ is ordinarily degraded in the ER by a mechanism that involves the proteasome, as demonstrated in intact cells using human fibroblast cell lines engineered for expression of alpha(1)ATZ and in a cell-free microsomal translocation assay system programmed with purified alpha(1)ATZ mRNA. To determine whether the ubiquitin system is required for proteasomal degradation of alpha(1)ATZ and whether specific components of the ubiquitin system can be implicated, we have now used two approaches. First, we overexpressed a dominant-negative ubiquitin mutant (UbK48R-G76A) by transient transfection in the human fibroblast cell lines expressing alpha(1)ATZ. The results showed that there was marked, specific, and selective inhibition of alpha(1)ATZ degradation mediated by UbK48R-G76A, indicating that the ubiquitin system is at least in part involved in ER degradation of alpha(1)ATZ. Second, we subjected reticulocyte lysate to DE52 chromatography and tested the resulting well-characterized fractions in the cell-free system. The results showed that there were both ubiquitin-dependent and -independent proteasomal mechanisms for degradation of alpha(1)ATZ and that the ubiquitin-conjugating enzyme E2-F1 may play a role in the ubiquitin-dependent proteasomal mechanism.     

Combined treatment of human multiple myeloma cells with bortezomib and doxorubicin alters the interactome of 20S proteasomes

The proteasome is the key player in targeted degradation of cellular proteins and serves as a therapeutic target for treating several blood malignancies. Although in general, degradation of proteins via the proteasome requires their ubiquitination, a subset of proteins can be degraded independently of their ubiquitination by direct interaction with subunits of the 20S proteasome core. Thus, investigation of the proteasome-associated proteins may help identify novel targets of proteasome degradation and provide important insights into the mechanisms of malignant cell proteostasis. Here, using biochemical purification of proteasomes from multiple myeloma (MM) cells followed by mass-spectrometry we have uncovered 77 proteins in total that specifically interacted with the 20S proteasome via its PSMA3 subunit. Our GST pull-down assays followed by western blots validated the interactions identified by mass-spectrometry. Eleven proteins were confirmed to bind PSMA3 only upon apoptotic conditions induced by a combined treatment with the proteasome inhibitor, bortezomib, and genotoxic drug, doxorubicin. Nine of these eleven proteins contained bioinformatically predicted intrinsically disordered regions thus making them susceptible to ubiquitin-independent degradation. Importantly, among those proteins five interacted with the ubiquitin binding affinity matrix suggesting that these proteins may also be ubiquitinylated and hence degraded via the ubiquitin-dependent pathway. Collectively, these PSMA3-interacting proteins represent novel potential substrates for 20S proteasomes upon apoptosis. Furthermore, these data may shed light on the molecular mechanisms of cellular response to chemotherapy.

Abbreviations: BD: bortezomib/doxorubicin treatment; CDK: cyclin-dependent kinases; CHCA: α-cyanohydroxycinnamic acid; IDP: intrinsically disordered proteins; IDR: intrinsically disordered regions; IPG: immobilized pI gradient; MALDI TOF/TOF: matrix-assisted laser desorption/ionization time-of-flight tandem mass-spectrometry; MM: multiple myeloma; ODC: ornithine decarboxylase; PI: proteasomal inhibitors; PSMA: alpha-type 20S proteasome subunits; PTMs: post-translational modifications; SDS-PAGE: sodium dodecylsulphate polyacrylamide gel electrophoresis; UIP: ubiquitin-independent proteasomal proteolysis.     

Insulin-like growth factor (IGF)-II regulates CCAAT/enhancer binding protein alpha expression via phosphatidyl-inositol 3 kinase in human hepatoblastoma cell lines

To reveal growth factor and its signal pathway to CCAAT/enhancer binding protein alpha (C/EBPalpha) in hepatocyte differentiation, we used Huh-6 and HepG2, human hepatoblastoma (HBL) cell lines that maintain the expression of genes in hepatoblasts and remain at that stage of differentiation. Insulin-like growth factor (IGF)-II, hepatocyte growth factor (HGF), and dexamethasone (Dex) stimulated HBL cells for Northern blot analysis. Bromodeoxyuridine (BrdU) up-take assay and Western blot analysis on albumin was performed to unveil proliferation and differentiation activity of IGF-II. C/EBPalpha and phosphorylation of Akt were analyzed by Western blot analysis. LY294002 and wortmannin, specific inhibitors of PI3 kinase, and PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase, were used to examine the signaling pathway of C/EBPalpha upregulated by IGF-II. Luciferase assay was performed to study the promoter activity of C/EBPalpha. Actinomycin D was used to analyze half-life of C/EBPalpha mRNA. IGF-II up-regualted C/EBPalpha by Northern blot and Western blot while HGF and Dex did not by Northern blot. IGF-II promoted proliferation and differentiation by BrdU up-take assay and Western blot analysis on albumin. Akt phosphorylated by IGF-II, suggested that phosphatidyl-inositol (PI) 3 kinase mediated the signaling pathway of IGF-II. LY294002 and wortmannin suppressed expression of C/EBPalpha. IGF-II activated the promoter activity and prolonged half-life of mRNA, suggesting that IGF-II activated promoter and stabilized mRNA. LY294002 and wortmannin suppressed the promoter activity of C/EBPalpha while PD98059 did not, suggesting that activation of the promoter was mediated by PI3 kinase.     

Rictor Undergoes Glycogen Synthase Kinase 3 (GSK3)-dependent,FBXW7-mediated Ubiquitination and Proteasomal Degradation

Rictor, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of rictor (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased rictor ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor. Hence, rictor is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with rictor and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination, and increased rictor stability. Finally, enforced activation of Akt enhanced rictor levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation.     

Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism

The 26S proteasome is a large multi-subunit protein complex that exerts specific degradation of proteins in the cell. The 26S proteasome consists of the 20S proteolytic particle and the 19S regulator. In order to be targeted for proteasomal degradation most of the proteins must undergo the post-translational modification of poly-ubiquitination. However, a number of proteins can also be degraded by the proteasome via a ubiquitin-independent pathway. Such degradation is exercised largely through the binding of substrate proteins to the PSMA3 (alpha 7) subunit of the 20S complex. However, a systematic analysis of proteins interacting with PSMA3 has not yet been carried out. In this report, we describe the identification of proteins associated with PSMA3 both in the cytoplasm and nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and tandem mass-spectrometry revealed a large number of PSMA3-bound proteins that are involved in various aspects of mRNA metabolism, including splicing. In vitro biochemical studies confirmed the interactions between PSMA3 and splicing factors. Moreover, we show that 20S proteasome is involved in the regulation of splicing in vitro of SMN2 (survival motor neuron 2) gene, whose product controls apoptosis of neurons.