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Ubiquitin-proteasome system regulation of an evolutionarily conserved RNA polymerase II-associated factor 1 involved in pancreatic oncogenesis. Jannatul Ferdoush, Saswati Karmakar, Priyanka Barman, Amala Kaja, Bhawana Uprety, Surinder K. Batra, and Sukesh R. Bhaumik Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00865 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017
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Biochemistry
Ubiquitin-proteasome system regulation of an evolutionarily conserved RNA polymerase II-associated factor 1 involved in pancreatic oncogenesis. Jannatul Ferdoush1, Saswati Karmakar2, Priyanka Barman1, Amala Kaja1, Bhawana Uprety1, Surinder K. Batra2, and Sukesh R. Bhaumik1,* 1
Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL,USA; 2 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
S Supporting Information ABSTRACT: The evolutionarily conserved RNA polymerase IIassociated factor 1 (Paf1) from yeast to humans regulates transcription and associated processes, and thus, malfunctions/misregulations of Paf1 are associated with cellular pathologies. Indeed, Paf1 (also known as PD2 or pancreatic differentiation 2) is found to be upregulated in poorly differentiated cancer cells, and such upregulation is involved in cellular transformation/oncogenesis. However, the basis for Paf1 upregulation in these cells remains largely unknown. In view of this, we have tested here the idea that the ubiquitin-proteasome system (UPS) regulates cellular abundance of Paf1. In this direction, we analyzed the role of UPS in regulation of Paf1’s abundance in yeast. We find that Paf1 undergoes ubiquitylation and is degraded by the 26S proteasome in yeast, thus deciphering UPS regulation of an evolutionarily conserved factor, Paf1, involved in various cellular processes at the crossroads of the cancer networks. Likewise, Paf1 undergoes proteasomal degradation in well-differentiated, but not poorly differentiated, pancreatic cancer cells, hence pointing to the UPS in upregulation of Paf1 in poorly differentiated cancers. Collectively, our results reveal UPS regulation of Paf1, and suggest downregulation of UPS in elevating Paf1’s abundance in poorly differentiated cancers.
RNA polymerase II-associated factor 1 (Paf1) complex (Paf1C), which is conserved among eukaryotes, was first identified and characterized in Saccharomyces cerevisiae via its interaction with RNA polymerase II (1). It consists of Paf1, Cdc73 (Cell Division Cycle 73), Ctr9 (Cln Three Requiring 9), Leo1 (Left Open Reading Frame 1) and Rtf1 (Restores TBP Function 1) in yeast. Likewise, these proteins form Paf1C in human cells (1). However, human Paf1C contains an additional protein, namely Ski8/Wdr61 (WD repeat-containing protein 61) that is known to regulate mRNA decay as a bona-fide component of the Ski (Superkiller) complex (1, 2). The functional and structural integrities of Paf1C are regulated by the abundances of its individual components. For example, increased abundance of Paf1 or Cdc73 enhances the stabilities of other Paf1C components, while depletion of Paf1 decreases the cellular levels of a set of Paf1C components by ~10-fold (1, 3). Similarly, depletion of Cdc73 or Ctr9 reduces the stabilities of other components of Paf1C (1, 4). Further, Paf1 has been implicated to be a scaffold protein in Paf1C (5), and thus, altered abundance of Paf1 has dramatic effects on the functional and structural integrities of Paf1C (1, 3, 4). Paf1C plays important roles in regulation of transcription of RNA polymerase II genes (1). In addition, it also controls transcription of RNA polymerase I genes (1, 6). Thus, malfunction or misregulation of Paf1/Paf1C would impair cellular functions, leading to pathological states. Indeed, Paf1/Paf1C is found to be associated with a number of diseases (1, 7-13). Importantly, Paf1 (also known as PD2 or pancreatic differentiation 2) has been shown to be upregulated in poorly differentiated cancers such as
pancreatic, endocrine and ovarian cancers (8, 12-15). Likewise, it is also upregulated in poorly differentiated or undifferentiated cancer stem cells (15-17). Further, high abundance of Paf1 has been shown to cause cellular transformation and oncogenesis (8, 12-15). However, it remains largely unknown how Paf1 is upregulated in poorly differentiated cancers or cancer stem cells. Such knowledge would be valuable in cancer pathogenesis, and hence therapeutic development. In view of this, we first carried out experiments to analyze the regulation of Paf1 abundance/level using yeast (Saccharomyces cerevisiae) as a model eukaryote, since Paf1 is conserved from yeast to humans (1). Subsequently, we extended our results to well-differentiated and poorly differentiated pancreatic cancer cells. The results of these experiments are described below.
Figure 1: Paf1 undergoes ubiquitylation. (A) Schematic diagram for Ni2+-NTA-based ubiquitylation assay. His6, hexahistidine; WCE, whole cell extract; WB, western blot; and Ni2+-NTA, Ni2+nitrilotriacetic acid. (B) Ubiquitylation analysis of Paf1. Yeast strains expressing Myc-tagged Paf1 and His6-tagged ubiquitin were grown in synthetic medium without uracil at 30°C to an OD600 of 0.7, and were then treated with CuSO4 at a final concentration of 0.1 mM for 6 hr. Precipitation was carried out by the use of Ni2+-NTA–agarose beads, and WB analysis was performed using an anti-Myc antibody against Myc-tagged Paf1. It is well-established that protein stability is controlled by targeted degradation by the 26S proteasome via ubiquitylation or non-targeted degradation by proteases (or proteasome via unfolding, but not ubiquitylation) (18). To determine whether Paf1 abundance in cell is regulated by ubiquitylation and subsequent proteasomal degradation (or ubiquitin-proteasome system, UPS), we first analyzed its ubiquitylation status, using Ni2+-NTA-based ubiquitylation assay. In this direction, we introduced a plasmid expressing hexahistidine (His6)-tagged ubiquitin under the CUP1 promoter (that is induced in the presence of Cu2+) in the yeast strain expressing Myc-tagged Paf1. Using this strain, we performed the ubiquitylation assay as described previously (19-21) and schematically shown in Figure 1A. Ubiquitin and ubiquitylated-proteins were precipitated from the WCE (whole cell extract) using Ni2+-NTA agarose beads that bind to His6 tag attached to ubiquitin. The precipitate was analyzed by western blot assay for the presence of Paf1 using an anti-Myc antibody against Myctagged Paf1. We observed Paf1 in the precipitate (Lane 2 in Fig-
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ure 1B), thus supporting the ubiquitylation of Paf1. As a control, we performed similar experiments using the yeast strain that did not have the plasmid expressing His6-tagged ubiquitin (but expressed Myc-tagged Paf1). We did not find Paf1 in the precipitate (Lane 1 in Figure 1B). These results support that cellular Paf1 is ubiquitylated, and hence Ni2+-NTA agarose beads pulled down Paf1 via its interaction with His6-tagged ubiquitin covalently attached to Paf1. Importantly, we observed a smear of ubiquitylated-Paf1 above ~ 100 kDa (Figure 1B). Such smear indicates that Paf1 is not mono-ubiquitylated, but rather poly-ubiquitylated. Further, the lowest part of the strong smear of ubiquitylated-Paf1 is about ~ 30 kDa above the molecular weight of Paf1. This observation indicates that ubiquitylated-Paf1 has 4 or more ubiquitin molecules, as the molecular weight of each ubiquitin is ~ 7.5 kDa. Taken together, our results support the poly-ubiquitylation of Paf1. However, faint bands/smear below ~ 100 kDa indicate a small population of mono-, di- or tri-ubiquitylated-Paf1 that are in dynamic equilibrium with higher order poly-ubiquitylated forms of Paf1.
Figure 2: Paf1’s abundance is increased following MG132 treatment. (A and B) WB analysis of Paf1's abundance in the presence and absence of MG132 or DMSO. Yeast cells expressing Myctagged Paf1 with a null mutation of PDR5 were grown in YPD (yeast extract, peptone plus 2% dextrose) at 30°C to an OD600 of 0.7, and were then treated with MG132 (75 µM) or DMSO for 2 hr prior to harvesting. -, absence of MG132/DMSO; and +, presence of MG132/DMSO. (C) The results of panels A and B are plotted in the form of a histogram.
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Ubiquitin is a 76-amino-acid-long peptide, and gets attached to the particular lysine (K) residue(s) of the target protein. Such modification of the target protein can be mono-, di-, trior poly-ubiquitylated (4 or more ubiquitin). If the ubiquitin chain of the target protein has 4 or more ubiquitin attached via K48 of ubiquitin, such ubiquitylation would lead to targeted degradation of the host protein by the 26S proteasome, although exception exists (18, 22). However, when the poly-ubiquitin chain is formed by K63 of ubiquitin, the poly-ubiquitylated-target protein would not be targeted for 26S proteasomal degradation, but rather would participate in intracellular trafficking, autophagy or other processes. Likewise, mono-ubiquitylated proteins are not targeted for 26S proteasomal degradation, but function in transport, transcription and other cellular processes (18, 22). Since cellular Paf1 is polyubiquitylated, it may be degraded by the 26S proteasome complex to maintain the optimal level. To test this, we analyzed the stability of Paf1 in the presence and absence of a peptide aldehyde, MG132 (carbobenzoxy-Leu-Leu-leucinal), that inhibits the proteolytic function of the proteasome. If poly-ubiquitylated-Paf1 is targeted for the 26S proteasomal degradation, pharmacological inhibition of the proteolytic function of the proteasome by MG132 would increase the stability/abundance of Paf1. However, yeast cells can be resistant to MG132, as they are capable of multidrug resistance. For this purpose, we deleted the multidrug resistance gene, PDR5, in the yeast strain expressing Myc-tagged Paf1 and then analyzed the level of Paf1 with or without MG132 treatment, as done previously (21, 23). We found that the stability of Paf1 was significantly increased following MG132 treatment (Figure 2A). However, similar increase in the stability of Paf1 was not observed following the treatment of DMSO (Dimethyl sulfoxide or carrier that was used to prepare MG132 solution) (Figure 2B). As a loading control, we also analyzed the level of actin that is not regulated by the 26S proteasome (24, 25). We found that the actin level was not changed in the presence of MG132 (Figures 2A, 2B and 2C). These results (Figures 2A, 2B and 2C) support that 26S proteasome is involved in degradation of cellular Paf1, and hence, Paf1 abundance is significantly increased following pharmacological inhibition of the proteolytic function of the 26S proteasome by MG132.
Figure 3: Paf1’s abundance is increased in the rpt4-ts (temperature sensitive) mutant. (A) WB analysis of Paf1's abundance in the rpt4-ts and wild type (WT) strains. Both the WT and ts mutant strains expressing HA-tagged Paf1 were grown in YPD at 23°C to an OD600 of 0.85, and were then switched to 39°C for 1 hr before harvesting for WB analysis. (B) The results of panel A are plotted in the form of a histogram.
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Biochemistry
To complement above results genetically, we analyzed the level of Paf1 in the wild type (WT) and temperature-sensitive (ts) mutant strains of Rpt4, an essential component of the 26S proteasome for its proteolytic activity (26, 27). Rpt4 is an ATPase present in the base of the 19S RP (regulatory particle) subunit of the 26S proteasome, and ATPase activity of the base plays important roles to unfold the target protein for entering into the barrel of the 20S CP (core particle) of the 26S proteasome for proteolytic degradation (18). If poly-ubiquitylated-Paf1 is degraded by the 26S proteasome, the stability/abundance of Paf1 would be increased in the ts mutant strain of Rpt4 (also known as sug2-ts; 26) at the non-permissive temperature. In this direction, we tagged Paf1 by HA epitope at its C-terminal in the chromosomal locus in the WT and ts mutant strains of Rpt4. Using these strains, we analyzed the levels of Paf1 in the rpt4-ts mutant and WT strains at the non-permissive temperature. We found that the level of Paf1 was dramatically enhanced in the rpt4-ts mutant strain (Figures 3A and 3B). However, the level of actin (loading control) was not similarly increased in the rpt4-ts mutant strain in comparison to the WT equivalent (Figures 3A and 3B). Thus, Paf1 has much higher turnover than actin, and such turnover is impaired in the rpt4-ts mutant strain. Therefore, our results support that the 26S proteasome is involved in the degradation of Paf1, and hence, the stability/abundance of Paf1 is increased in the rpt4-ts mutant strain. Taken together, pharmacological and genetic inhibitions of the proteolytic function of the 26S proteasome dramatically enhances the abundance/stability of Paf1. These results support that poly-ubiquitylated-Paf1 is targeted for degradation by the 26S proteasome.
differentiated, but not poorly differentiated, pancreatic cancer cells. Collectively, our results support UPS regulation of Paf1, and suggest downregulation of proteasomal degradation in elevating Paf1’s abundance in poorly differentiated cancer. However, it remains to be further elucidated as to how Paf1 undergoes ubiquitylation and proteasomal degradation, and how increased abundance of Paf1 leads to oncogenesis. Further, the enzymes involved in regulation of Paf1’s ubiquitylation (e.g., E3 ligase, E2 conjugase and ubiquitin protease) are yet to be identified, which might be involved in oncogenessis by regulating Paf1’s abundance. Therefore, our results open an important avenue of research in identifying new factors involved in regulation of Paf1’s abundance and delineating how increased abundance of Paf1 is associated with cancer, and such knowledge would have significant impact on cancer pathogenesis (and hence therapeutic development), since Paf1 is present at the crossroads of the cancer networks.
ASSOCIATED CONTENT Supporting Information
Materials and methods. The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected]. Phone: (618) 453-6479.
Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Funding Sources The work in the Bhaumik laboratory was supported by the grants from National Institutes of Health (1R15GM088798-01 and 2R15GM088798-02), American Heart Association (15GRNT25700298), and Southern Illinois University School of Medicine. The work in the Batra laboratory was supported by a grant from National Institutes of Health (NIH RO1 CA210637). Figure 4: Analysis of Paf1’s abundance in pancreatic cancer cell lines, CD18 (well-differentiated) and Panc1 (poorly differentiated). (A) WB analysis of Paf1's abundance in the presence and absence of MG132 (10 µM) in CD18 cell line. (B) WB analysis of Paf1's abundance in the presence and absence of MG132 (10 µM) or DMSO in Panc1 cell line. Our results in yeast suggest that misregulation/malfunction of UPS might be involved in upregulation of Paf1 in undifferentiated or poorly differentiated cancers and cancer stem cells. Indeed, we analyzed the proteasomal regulation of Paf1 in well-differentiated and poorly differentiated pancreatic cancer cells. We find that proteasomal inhibition by MG132 increases the stability or abundance of PAF1 in well-differentiated pancreatic cancer cell, CD18 (Figure 4A). However, we did not observe similar increase of Paf1 abundance in poorly differentiated pancreatic cancer cell, Panc1, following proteasomal inhibition by MG132 (Figure 4B). This could be due to the fact that UPS is impaired or down-regulated in poorly differentiated pancreatic cancer cell, Panc1, and hence, proteasomal inhibition does not further enhance the stability or abundance of Paf1 in Panc1. This possibility remains to be further elucidated. Nonetheless, our results indicate proteasomal regulation of Paf1 in well-
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT We thank Thomas Kodadek and Stephen Johnston for yeast strains; and Daniel Finley for plasmid, pUB221.
ABBREVIATIONS Paf1, RNA polymerase II-associated factor 1; Ub, ubiquitin; PD2, pancreatic differentiation 2; K, lysine; WB, western blot; His6, hexahistine; DMSO, dimethyl sulfoxide; 19S RP, 19S regulatory particle; 20S CP, 20S core particle; WT, wild type; ts, temperature-sensitive; and UPS, ubiquitin-proteasome system.
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