DNA polymerase eta is targeted by Mdm2 for polyubiquitination and proteasomal degradation in response to ultraviolet irradiation

DNA polymerase eta (PolH), the product of the xeroderma pigmentosum variant (XPV) gene and a Y-family DNA polymerase, plays a pivotal role in translesion DNA synthesis. Loss of PolH leads to early onset of malignant skin cancer in XPV patients and increases UV-induced carcinogenesis. Thus, the pathways by which PolH expression and activity are controlled may be explored as a strategy to prevent UV-induced cancer. In this study, we found that Mdm2, a RING finger E3 ligase, promotes PolH degradation. Specifically, we showed that knockdown of Mdm2 increases PolH expression in both p53-proficient and -deficient cells. In addition, we showed that UV-induced PolH degradation is attenuated by Mdm2 knockdown. In contrast, ectopically expression of Mdm2 decreases PolH expression, which can be abrogated by the proteasome inhibitor MG132. Moreover, we showed that Mdm2 physically associates with PolH and promotes PolH polyubiquitination in vivo and in vitro. Finally, we showed that knockdown of Mdm2 increases the formation of PolH replication foci and decreases the sensitivity of cells to UV-induced lesions in a PolH-dependent manner. Taken together, we uncovered that Mdm2 serves as an E3 ligase for PolH polyubiquitination and proteasomal degradation in cells under the basal condition and in response to UV irradiation.     

DBC2 resistance is achieved by enhancing 26S proteasome-mediated protein degradation

Tumor suppressor gene DBC2 stops growth of tumor cells through regulation of CCND1. Interference of CCND1 down-regulation prevented growth arrest caused by DBC2 [T. Yoshihara, D. Collado, M. Hamaguchi, Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function, Biochemical and biophysical research communications 358 (2007) 1076-1079]. It was also noted that DBC2 resistant cells eventually arose after repeated induction of DBC2 with muristerone A treatment [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. In order to elucidate the mechanism of resistance acquisition, we analyzed DBC2 sensitive and resistant cells derived from the same progenitor cells (T-47D). We discovered that DBC2 protein was abundantly expressed in the sensitive cells when DBC2 was induced. In contrast, it was undetectable by western blot analysis in the resistant cells. We confirmed that the inducible gene expression system was responsive in both cells by detecting induced GFP. Additionally, inhibition of 26S proteasome by MG132 revealed production of DBC2 protein in the resistant cells. These findings indicate that the resistant T-47D cells survive DBC2 induction by rapid destruction of DBC2 through 26S proteasome-mediated protein degradation.     

Tip60 acetyltransferase activity is controlled by phosphorylation

Here we show that the phosphorylation of histone acetyltransferase Tip60, a target of human immunodeficiency virus, type 1-encoded transactivator Tat, plays a crucial role in the control of its catalytic activity. Baculovirus-based expression and purification of Tip60 combined with mass spectrometry allowed the identification of serines 86 and 90 as two major sites of phosphorylation in vivo. The phosphorylation of Tip60 was found to modulate its histone acetyltransferase activity. One of the identified phosphorylated serines, Ser-90, was within a consensus cyclin B/Cdc2 site. Ser-90 was specifically phosphorylated in vitro by the cyclin B/Cdc2 complex. Accordingly, the phosphorylation of Tip60 was enhanced after drug-induced arrest of cells in G(2)/M. This G(2)/M-dependent phosphorylation of Tip60 was abolished by treating cells with a specific inhibitor of the cyclin-dependent kinase, roscovitin. All together, these results strongly suggest a G(2)/M-dependent control of Tip60 activity.     

Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53

The product of the Mdm2 oncogene directly interacts with p53 and promotes its ubiquitination and proteasomal degradation. Initial biological studies identified nuclear export sequences (NES), similar to that of the Rev protein from the human immunodeficiency virus, both in Mdm2 and p53. The reported phenotypes resulting from mutation of these NESs, together with results obtained using the nuclear export inhibitor leptomycin B (LMB), have led to a model according to which nuclear export of p53 (via either the NES of Mdm2 or its own NES) is required for efficient p53 degradation. In this study we demonstrate that Mdm2 can promote degradation of p53 in the nucleus or in the cytoplasm, provided both proteins are colocalized. We also investigated if nuclear export is an obligate step on the p53 degradation pathway. We find that (1) when proteasome activity is inhibited, ubiquitinated p53 accumulates in the nucleus and not in the cytoplasm; (2) Mdm2 with a mutated NES can efficiently mediate degradation of wild type p53 or p53 with a mutated NES; (3) the nuclear export inhibitor LMB can increase the steady-state level of p53 by inhibiting Mdm2-mediated ubiquitination of p53; and (4) LMB fails to inhibit Mdm2-mediated degradation of the p53NES mutant, demonstrating that Mdm2-dependent proteolysis of p53 is feasible in the nucleus in the absence of any nuclear export. Therefore, given cocompartmentalization, Mdm2 can promote ubiquitination and proteasomal degradation of p53 with no absolute requirement for nuclear to cytoplasmic transport.     

Mdm2 is involved in the ubiquitination and degradation of G-protein-coupled receptor kinase 2

G-protein-coupled receptor kinase 2 (GRK2) is a central regulator of G-protein-coupled receptor signaling. We report that Mdm2, an E3-ubiquitin ligase involved in the control of cell growth and apoptosis, plays a key role in GRK2 degradation. Mdm2 and GRK2 association is enhanced by beta(2)-adrenergic receptor stimulation and beta-arrestin. Increased Mdm2 expression accelerates GRK2 proteolysis and promotes kinase ubiquitination at defined residues, whereas GRK2 turnover is markedly impaired in Mdm2-deficient cells. Moreover, we find that activation of the PI3K/Akt pathway by insulin-like growth factor-1 alters Mdm2-mediated GRK2 degradation, leading to enhanced GRK2 stability and increased kinase levels. These data put forward a novel mechanism for controlling GRK2 expression in physiological and pathological conditions.     

DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair.

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC.HR23B, TFIIH, XPF.ERCC1 and XPG, up to 17-fold for CPDs and approximately 2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors.     

CRAF autophosphorylation of serine 621 is required to prevent its proteasome-mediated degradation

The CRAF protein kinase regulates proliferative, differentiation, and survival signals from activated RAS proteins to downstream effectors, most often by inducing MEK/ERK activation. A well-established model of CRAF regulation involves RAS-mediated translocation of CRAF to the plasma membrane, where it is activated by a series of events including phosphorylation. Here we have discovered a new mode of regulation that occurs prior to this step. By creating a kinase-defective version of CRAF in mice or by use of the RAF inhibitor sorafenib, we show that CRAF must first undergo autophosphorylation of serine 621 (S621). Autophosphorylation occurs in cis, does not involve MEK/ERK activation, and is essential to ensure the correct folding and stability of the protein. In the absence of S621 phosphorylation, CRAF is degraded by the proteasome by mechanisms that do not uniquely rely on the E3 ubiquitin ligase CHIP.     

Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.     

Pen-2 is sequestered in the endoplasmic reticulum and subjected to ubiquitylation and proteasome-mediated degradation in the absence of presenilin

The gamma-secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. gamma-Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature gamma-secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the gamma-secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.     

Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for “histone code” reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi.     

The cellular localization pattern of Varicella-Zoster virus ORF29p is influenced by proteasome-mediated degradation

Varicella-zoster virus (VZV) open reading frame 29 (ORF29) encodes a single-stranded DNA binding protein. During lytic infection, ORF29p is localized primarily to infected-cell nuclei, whereas during latency it appears in the cytoplasm of infected neurons. Following reactivation, ORF29p accumulates in the nucleus. In this report, we analyze the cellular localization patterns of ORF29p during VZV infection and during autonomous expression. Our results demonstrate that ORF29p is excluded from the nucleus in a cell-type-specific manner and that its cellular localization pattern may be altered by subsequent expression of VZV ORF61p or herpes simplex virus type 1 ICP0. In these cases, ORF61p and ICP0 induce nuclear accumulation of ORF29p in cell lines where it normally remains cytoplasmic. One cellular system utilized by ICP0 to influence protein abundance is the proteasome degradation pathway. Inhibition of the 26S proteasome, but not heat shock treatment, resulted in accumulation of ORF29p in the nucleus, similar to the effect of ICP0 expression. Immunofluorescence microscopy and pulse-chase experiments reveal that stabilization of ORF29p correlates with its nuclear accumulation and is dependent on a functional nuclear localization signal. ORF29p nuclear translocation in cultured enteric neurons and cells derived from an astrocytoma is reversible, as the protein's distribution and stability revert to the previous states when the proteasomal activity is restored. Thus, stabilization of ORF29p leads to its nuclear accumulation. Although proteasome inhibition induces ORF29p nuclear accumulation, this is not sufficient to reactivate latent VZV or target the immediate-early protein ORF62p to the nucleus in cultured guinea pig enteric neurons.     

Cytoplasmically "sequestered" wild type p53 protein is resistant to Mdm2-mediated degradation.

The Mdm2 oncoprotein mediates p53 degradation at cytoplasmic proteasomes and is the principal regulator for maintaining low, often undetectable levels of p53 in unstressed cells. However, a subset of human tumors including neuroblastoma constitutively harbor high levels of wild type p53 protein localized to the cytoplasm. Here we show that the abnormal p53 accumulation in such cells is due to a profound resistance to Mdm2-mediated degradation. Overexpression of Mdm2 in neuroblastoma (NB)(1) cell lines failed to decrease the high steady state levels of endogenous p53. Moreover, exogenous p53, when introduced into these cells, was also resistant to Mdm2-directed degradation. This resistance is not due to a lack of Mdm2 expression in NB cells or a lack of p53-Mdm2 interaction, nor is it due to a deficiency in the ubiquitination state of p53 or proteasome dysfunction. Instead, Mdm2-resistant p53 from NB cells is associated with covalent modification of p53 and masking of the modification-sensitive PAb 421 epitope. This system provides evidence for an important level of regulation of Mdm2-directed p53 destruction in vivo that is linked to p53 modification.