Interplay of Posttranslational Modifications in Sp1 Mediates Sp1 Stability during Cell Cycle Progression

doi:10.1016/j.jmb.2011.09.027

J. Mol. Biol. (2011) 414, 1–14 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Interplay of Posttranslational Modifications in Sp1 Mediates Sp1 Stability during Cell Cycle Progression Yi-Ting Wang 1 , Wen-Bin Yang 3 , Wen-Chang Chang 1, 2, 3, 4 and Jan-Jong Hung 1, 3, 4 ⁎ 1

Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan 3 Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan 4 Center for Infectious Disease and Signal Transduction Research, National Cheng Kung University, Tainan 701, Taiwan 2

Received 28 January 2011; received in revised form 8 July 2011; accepted 14 September 2011 Available online 28 September 2011 Edited by M. Yaniv Keywords: sumoylation; RNF4; ubiquitin E3 ligase; phosphorylation

Although Sp1 is known to undergo posttranslational modifications such as phosphorylation, glycosylation, acetylation, sumoylation, and ubiquitination, little is known about the possible interplay between the different forms of Sp1 that may affect its overall levels. It is also unknown whether changes in the levels of Sp1 influence any biological cell processes. Here, we identified RNF4 as the ubiquitin E3 ligase of Sp1. From in vitro and in vivo experiments, we found that sumoylated Sp1 can recruit RNF4 as a ubiquitin E3 ligase that subjects sumoylated Sp1 to proteasomal degradation. Sp1 mapping revealed two ubiquitination-related domains: a small ubiquitinlike modifier in the N-terminus of Sp1(Lys16) and the C-terminus of Sp1 that directly interacts with RNF4. Interestingly, when Sp1 was phosphorylated at Thr739 by c-Jun NH2-terminal kinase 1 during mitosis, this phosphorylated form of Sp1 abolished the Sp1–RNF4 interaction. Our results show that, while sumoylated Sp1 subjects to proteasomal degradation, the phosphorylation that occurs during the cell cycle can protect Sp1 from degradation by repressing the Sp1–RNF4 interaction. Thus, we propose that the interplay between posttranslational modifications of Sp1 plays an important role in cell cycle progression and keeps Sp1 at a critical level for mitosis. © 2011 Elsevier Ltd. All rights reserved.

*Corresponding author. Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan. E-mail address: [email protected]. Abbreviations used: SUMO, small ubiquitin-like modifier; JNK, c-Jun NH2-terminal kinase; STUbL, SUMO-targeted ubiquitin ligase; SIM, SUMO interaction motif; PML, promyelocytic leukemia; NEM, N-ethyl maleimide; GST, glutathione S-transferase; GFP, green fluorescent protein; wt, wild type; shRNA, short hairpin RNA; DMEM, Dulbecco's modified Eagle's medium; RIPA, radioimmune precipitation assay; TBST, Trisbuffered saline/Tween 20.

Introduction The specificity protein/Krüppel-like factor family is composed of transcription factors containing a combination of three conserved Cys2His2 zinc fingers that form the DNA-binding domain. 1 Sp1, a ubiquitously expressed transcription factor in mammalian cells, is important in a variety of physiological processes, including cell cycle regulation, apoptosis, and differentiation. 2–5 Previous studies including ours have revealed that posttranslational modification of Sp1 could alter its transcriptional activity, DNA-binding affinity, or protein

0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.

2 stability. 4,6–10 Common posttranslational modifications of Sp1 include phosphorylation, glycosylation, acetylation, ubiquitination, and sumoylation. Numerous studies have documented that Sp1 activity and/or expression levels were elevated in human pancreatic cancer, breast cancer, colorectal cancer, gastric carcinoma, hepatocellular carcinoma, and thyroid carcinoma. 11–13 In addition to several oncogenes such as vascular endothelial growth factor, urokinase plasminogen activator, urokinase plasminogen activator receptor, and epithelial growth factor receptor, many tumor suppressor genes, such as p27 kip1, p21 WAF1/CIP1, p16 INK4 , and pp2a-c, are also induced by Sp1. 14–20 Further, many pro-apoptotic gene promoters, including fas, fas ligand, fax, and caspase 3, have been found to contain Sp1-binding elements. 21–23 Previous studies have revealed that upregulation of Sp1 transcriptional activity leads to Sp1 accumulation during tumorigenesis. Our earlier studies specifically indicated that Sp1 stability might be an important factor for Sp1 accumulation in most tumors. Our results demonstrated that modification of Sp1 by small ubiquitin-like modifier (SUMO)-1 at Lys16 could induce proteasome-dependent degradation. 9 In addition, Sp1 could be phosphorylated by c-Jun NH2-terminal kinase (JNK) 1 at Thr278/Thr739 during mitosis, thus protecting Sp1 from degradation by the proteasome-dependent degradation pathway. 10 Studies have identified a crucial mechanism for regulating the cellular levels of SUMO conjugates. 24–26 Intriguingly, this mechanism uses a novel family of ubiquitin E3 ligases that specifically target SUMOconjugated proteins known as SUMO-targeted ubiquitin ligases (STUbLs). 27–29 The STUbL recruitment to sumoylated proteins was mediated through tandem SUMO interaction motifs (SIMs). The SIMs contain a consensus sequence (V/L/I, V/L/I, X, V/L/I or V/L/I, X, V/L/I, V/L/I). As such, these STUbLs challenge the current view that SUMO does not promote degradation of its target proteins. 26 A human ortholog of the yeast SUMOactivated ubiquitin E3 ligase (RNF4) was recently identified. 27 RNF4 is a RING-domain-containing ubiquitin E3 ligase that targets and regulates sumoylated proteins, including promyelocytic leukemia (PML), PML-retinoic acid receptor α, poly (ADP-ribose) polymerase 1, and hypoxia-inducible factor-2α. 30–35 In addition to the carboxy-terminal-located RING finger motif, RNF4 contains two putative nuclear localization signals and acidic amino acid stretches that are similar to the activation domains of some transcription factors. 36 RNF4 was primarily identified as a bridging factor that regulated steroid-receptordependent transcription. 37,38 However, RNF4 may participate in Sp1-dependent transcription and cooperate with androgen receptor and Sp1. 39,40 RNF4 was

Modifications in Sp1 during Cell Cycle Progression

able to stimulate the rat luteinizing hormone-β promoter by interacting with Sp1 and steroidogenic factor-1 and then protect it from androgen suppression. 41 Several RNF4-interacting partners have been identified, including androgen receptor, Sp1, POZ–AT hook–zinc finger protein, stromelysin-1 platelet-derived growth-factor-responsive element binding protein, TATA-binding protein, trichorhinophalangeal syndrome type 1 protein, steroidogenic factor-1, nuclear factor-Y, and PML. 30,32,36,38,40–42 In vitro ubiquitination assays have demonstrated that RNF4 functions as a ubiquitin E3 ligase and that its E3 ligase activity is closely linked to its transcription regulatory functions. 37,43 The same group also demonstrated that SUMO-1 promotes the association between RNF4 and PML nuclear bodies. 44 Moreover, RNF4 recruitment to sumoylated proteins was mediated through tandem SIMs within the N-terminus of RNF4. 27,29 Data have shown that arsenic-triggered PML sumoylation was targeted at ubiquitin-mediated proteolysis via a SUMO-triggered RNF4/ubiquitin-mediated pathway. 30,32,33 A previous study reported that conserved cysteine residues (C136/C139) located within the RING finger domains of RNF4 and acted as a functional unit of the ubiquitin E3 ligase activity. 43 Therefore, RNF4 functioned as a ubiquitin E3 ligase and targeted a sumoylated protein for ubiquitin-mediated proteolysis. In the present study, we clearly mapped the interacting regions of Sp1 and RNF4, determined RNF4 was the ubiquitin E3 ligase of Sp1, and it modulated Sp1 level via sumoylation and phosphorylation.

Results Sp1 interacts with RNF4 Our earlier studies reported that both sumoylation and phosphorylation could modulate Sp1 stability and determine its overall level under different conditions such as tumorigenesis and the cell cycle. 9,10 Here, we attempted to identify the ubiquitin E3 ligase of Sp1. HeLa cells were treated with or without N-ethyl maleimide (NEM) for 4 h. The Sp1– RNF4 interaction was analyzed by immunoprecipitation with anti-Sp1 or anti-RNF4 antibodies (Fig. 1a-a and a-b). Results show that Sp1, sumoylated Sp1, and RNF4 levels were increased after NEM treatment and that treatment significantly increased the frequency of Sp1–RNF4 interactions (Fig. 1a-a and a-b). Next, we performed a glutathione S-transferase (GST) pulldown assay using truncated Sp1 fragments to identify the Sp1 region involved in its association with RNF4 (Fig. 1b). After purification and verification by Coomassie blue staining, GST, full-length GST-Sp1 (1–785 aa), and three truncated Sp1 fragments [GST-

Modifications in Sp1 during Cell Cycle Progression

Sp1(8–618 aa), GST-Sp1(8–290 aa), and GST-Sp1(618– 785 aa)] were incubated with green fluorescent protein (GFP)-RNF4-overexpressed HeLa cell lysates. Results indicated that full-length GST-Sp1(1–785 aa) and C-terminal GST-Sp1(619–785 aa) interacted well with GFP-RNF4, but not GST-Sp1(8–290 aa) or GSTSp1(8–618 aa). The truncated Sp1 fragments were incubated with Flag-RNF4mt-overexpressed HeLa cell lysates to perform the GST pull-down assay. Results indicated that RNF4 containing all mutated SIMs interacted with full-length GST-Sp1(1–785 aa)

3 and C-terminal GST-Sp1(619–785 aa) (Supplementary Fig. 1). To further explore whether the association between Sp1 and RNF4 is a direct one, we carried out a GST pull-down assay using GST, GST-Sp1(619– 785 aa), and His-RNF4 (Fig. 1c). The assay results revealed that GST-Sp1(619–785 aa) was able to pull down His-RNF4, indicating that RNF4 interacts directly with the C-terminal region of Sp1 (Fig. 1c and Supplementary Fig. 2). Taken together, these results suggest that the C-terminal region of Sp1 contributes to the Sp1–RNF4 interaction. Sumoylation of Sp1 facilitates the interaction between Sp1 and RNF4 Studies have shown that RNF4 is recruited to sumoylated proteins through tandem SIMs within the N-terminus of RNF4. Previous studies have demonstrated that RNF4 with all SIMs mutated did not bind to SUMO, suggesting that SIMs of RNF4 are critical for its binding to sumoylated proteins. 30,33,45 To further delineate the effect of sumoylation on Sp1– RNF4 interaction, we co-transfected HA-Sp1-myc, HA-SUMO-1-Sp1-myc, and HA-Sp1(K16R)-myc with GFP-RNF4 to perform the immunoprecipitation assay using anti-HA antibodies (Fig. 2a). The assay results indicated that the intracellular level of HA-SUMO-1Sp1-myc was lower than that of HA-Sp1-myc or HASp1(K16R)-myc. This finding is consistent with that of our earlier study. 9 In this study, we found that small amounts of SUMO-1-Sp1 could recruit more RNF4 than Sp1 wild type (wt) or the sumoylation-deficient mutant Sp1(K16R) (Fig. 2a-a). To test whether the SIMs of RNF4 are involved in the interaction with sumoylated Sp1, we constructed RNF/SIM that deletes all SIMs (residues 30–76). The assay results indicated that there were no significant differences in the interaction between RNF4/SIM and Sp1, Sp1 (K16R), or SUMO-1-Sp1, implying that the Sp1 sumoylation plays a role in the SIMs contribution to Sp1–RNF4 interaction (Fig. 2a-b). To directly examine the effect of sumoylation of Sp1 on its interaction with RNF4 or RNF/SIM, we used the Escherichia coli Fig. 1. Sp1–RNF4 interaction. (a) HeLa cells were treated with NEM for 4 h, harvested for the immunoprecipitation assay using anti-Sp1 (a-a) and anti-RNF4 (a-b) antibodies, and then analyzed by immunoblotting with anti-Sp1 and anti-RNF4 antibodies. Long and short Sp1 exposures are shown in (a). (b) GST-Sp1(8–290 aa), GSTSp1(8–618 aa), and GST-Sp1(619–785 aa) were synthesized from E. coli. After purification, the proteins were analyzed using Coomassie blue staining (b-a). The GST pull-down assay was carried out using GFP-RNF4-overexpressed cell lysates. The samples were then analyzed by immunoblotting with anti-GFP antibodies (b-b). (c) GST, GST-Sp1(619– 785 aa), and His-RNF4 were purified from E. coli (c-a), and a GST pull-down assay was performed. The samples were then analyzed by immunoblotting with anti-His and antiGST antibodies (c-b).

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Modifications in Sp1 during Cell Cycle Progression

Fig. 2. Effect of sumoylation on Sp1–RNF4 interaction. (a) HA-Sp1-myc, HA-SUMO-1-Sp1-myc, or HA-Sp1(K16R)myc was co-transfected with GFP-RNF4 (a-a) or GFP-RNF4/SIM (a-b) in HeLa cells. Cells were harvested for the immunoprecipitation assay using anti-HA antibodies and were then analyzed by immunoblotting with anti-GFP and anti-HA antibodies. After three independent experiments, the relative level of RNF4/SIM recruitment was quantified. (b) The pT-E1/E2/His-SUMO-1 plasmid (b-a) was co-transformed with GST-Sp1 into E. coli to synthesize His-SUMO-1-GSTSp1. After purification, the presence of His-SUMO-1-GST-Sp1, GST-Sp1, and His-SUMO-1 was confirmed by Coomassie blue staining (b-b). These purified proteins were used for the GST pull-down assay incubated with Flag-RNF4-expressed or Flag-RNF4mt-expressed HeLa cell lysates. The samples were then analyzed with an immunoblotting assay using antiFlag antibodies (b-c).

sumoylated system pT-E1/E2/His-SUMO-1 to obtain pure SUMO-1-Sp1 (Fig. 2b). pT-E1/E2/HisSUMO-1 and GST-Sp1 were co-transformed into E. coli, and His-SUMO-1 was attached to Lys16 of GSTSp1 (Fig. 2b-a). The respective purities of His-SUMO1, GST-Sp1, and His-SUMO-1-GST-Sp1 were verified using Coomassie blue staining (Fig. 2b-b). Purified His-SUMO-1, GST-Sp1, and His-SUMO-1-GST-Sp1 were then incubated with Flag-RNF4 or Flag-RNF4mt to perform the GST pull-down assay. The assay results revealed that more RNF4 could be recruited by His-SUMO-1-GST-Sp1 than by GST-Sp1. When RNF4 with all mutated SIMs was used, no difference in RNF4mt recruitment by GST-Sp1 or by His-SUMO-1GST-Sp1 was observed. In conclusion, based on the data shown in Figs. 1 and 2, the C-terminus of Sp1 directly interacts with RNF4, and sumoylation enhances Sp1–RNF4 interaction in an SIM-dependent manner.

overexpressed in HeLa cells, Sp1 and SUMO-1-Sp1 levels decreased in an RNF4-dose-dependent manner (Fig. 3a-a). RNF4 was knocked down by short hairpin RNAs (shRNAs), Sp1 was significantly increased, and SUMO-1-Sp1 was slightly increased (Fig. 3a-b). When RNF4 was mutated to RNF4mt or RNF4/CS1, the effects of RNF4 on Sp1 and SUMO-1Sp1 were completely abolished (Fig. 3b). To determine the effect of sumoylation on Sp1 protein levels in RNF4-overexpressing cells, we co-transfected HASUMO-1-Sp1-myc with GFP-RNF4, GFP-RNF4/ CS1, or GFP-RNF4/SIM (Fig. 3c). SUMO-1-Sp1 clearly decreased in the presence of RNF4. However, there was no significant difference in SUMO-1-Sp1 levels in the presence of RNF4/CS1 or RNF4/SIM (Fig. 3c). Taken together, these data suggest that RNF4 affects Sp1 levels in an Sp1-sumoylationdependent manner. RNF4 is a ubiquitin E3 ligase of Sp1

RNF4 reduces Sp1 levels Our data indicated that RNF4 interacts with Sp1 (Fig. 1). To further address the effect of RNF4 on Sp1, we overexpressed or silenced RNF4 and monitored the resulting Sp1 level (Fig. 3a). When RNF4 was

Studies have indicated that RNF4 might be involved in protein degradation. 27,29–32 As shown in Fig. 3, SUMO-1-Sp1 and Sp1 were downregulated by RNF4; thus, we speculated that RNF4 functions as a ubiquitin E3 ligase of Sp1. To test our hypothesis,

Modifications in Sp1 during Cell Cycle Progression

5

Fig. 3. Role of RNF4 in regulating Sp1 protein levels. (a) Different doses of GFP-RNF4 (a-a) and RNF4 shRNA (a-b) were expressed in HeLa cells. HeLa cells were subjected to an immunoblotting assay using anti-Sp1, anti-GFP, anti-RNF4, and anti-actin antibodies. (b) GFP-RNF4/CS1 (b-a) and Flag-RNF4mt (b-b) were expressed in HeLa cells. Samples were analyzed by an immunoblotting assay using anti-Sp1, anti-GFP, anti-Flag, and anti-actin antibodies. (c) GFP-RNF4, GFPRNF4/CS1, and GFP-RNF4/SIM were co-transfected with HA-SUMO-1-Sp1-myc in HeLa cells for 24 h. The cells were harvested for use in an immunoblotting assay with anti-HA, anti-GFP, and anti-actin antibodies.

we examined the ubiquitination signals of SUMO-1Sp1 after RNF4 overexpression and proteasome inhibitor (MG132) treatment (Fig. 4a). The results indicated that RNF4 could induce SUMO-1-Sp1 polyubiquitination in the presence of HA-RNF4 and MG132 (Fig. 4a-a). SUMO-1-Sp1 was decreased upon HA-RNF4 overexpression but was rescued in the presence of MG132 (Fig. 4a-b). To further address the effect of sumoylation on Sp1 levels, we co-transfected the sumoylation-deficient mutants Sp1(K16R) and HA-RNF4 into HeLa cells and then treated them with MG132 for 4 h. No significant difference was found in Sp1(K16R) levels in RNF4overexpressing cells (Fig. 4b). In addition, when RNF4 was silenced by RNF4 shRNA, the Sp1 ubiquitination signal decreased in an RNF4-dosedependent manner (Fig. 4c). GFP-RNF4 and the mutants GFP-RNF4/CS1 and GFP-RNF4/SIM were co-transfected with myc/His-ubiquitin into HeLa cells to characterize whether RNF4 functions as a ubiquitin E3 ligase of Sp1. Cell lysates were harvested to perform the immunoprecipitation

assay with anti-ubiquitin (Fig. 4d). The assay results indicated that ubiquitinated Sp1 level was increased in RNF4-overexpressing cells, but no difference was observed in RNF4/CS1- or RNF4/SIM-overexpressing cells (Fig. 4d). These data suggest that RNF4 decreases Sp1 levels by increasing its degradation via a ubiquitin-dependent proteasome pathway. We performed an in vitro ubiquitination assay to further characterize RNF4 as a ubiquitin E3 ligase of Sp1 (Fig. 5). Equal amounts of Sp1 immunoprecipitated from HeLa cell lysates were incubated with ubiquitin ligase E1, ubiquitin ligase E2, purified His-RNF4 (ubiquitin ligase E3), ubiquitin, and an adenosine triphosphate regeneration system (Fig. 5a). The resulting data indicated that Sp1 ubiquitination signals were enhanced in the presence of RNF4. Purified His-RNF4, His-RNF4/CS1, and His-RNF4/SIM were also used to perform the in vitro ubiquitination assay. As shown in Fig. 5b, RNF4 increased Sp1 ubiquitination, but no such increase was seen in the presence of RNF4/CS1 or RNF4/SIM. To study whether Sp1 sumoylation

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Modifications in Sp1 during Cell Cycle Progression

Fig. 4. RNF4 mediates Sp1 degradation. (a) HA-SUMO-1-Sp1-myc and HA-RNF4 were co-transfected into HeLa cells for 24 h. After M132 treatment for 4 h, cell lysates were harvested for immunoblotting with anti-HA, anti-myc, and antiactin antibodies (a-a and a-b). After three independent experiments, the relative level of HA-SUMO-1-Sp1-myc was quantified (a-c). (b) HA-Sp1(K16R)-myc and HA-RNF4 were co-transfected into HeLa cells for 24 h. After M132 treatment for 4 h, the cell lysates were harvested for immunoblotting with anti-HA, anti-myc, and anti-actin antibodies. (c) The RNF4 shRNAs (RNF4-1 and RNF4-2) and scramble shRNA were transfected into HeLa cells for 48 h. The cells were immunoblotted with anti-Sp1, anti-RNF4, and anti-actin antibodies. (d) Different doses of GFP-RNF4, GFP-RNF4/CS1, or GFP-RNF4/SIM were co-transfected with myc/His-ubiquitin for 24 h. The cell lysates were then subjected to immunoprecipitation with anti-ubiquitin antibodies. The samples were then analyzed by immunoblotting with anti-Sp1, anti-ubiquitin, anti-GFP, and anti-actin antibodies.

affects its ubiquitination, we overexpressed HASp1-myc and HA-SUMO-1-Sp1-myc in HeLa cells and immunoprecipitated them with anti-HA antibodies to perform the in vitro ubiquitination assays (Fig. 5c). Data revealed that Sp1 sumoylation could increase ubiquitination signals in the presence of RNF4. Taken together, these data indicate that RNF4 may act as a ubiquitin E3 ligase of Sp1 and that sumoylation could facilitate RNF4-mediated degradation.

Since Sp1 was immunoprecipitated from HeLa cells, many co-precipitated proteins may have affected the in vitro ubiquitination assay. In order to directly examine the role of RNF4 in Sp1 degradation, we used pT-E1/E2/His-SUMO-1 to obtain pure SUMO-1-Sp1 protein. In vitro ubiquitination assays were carried out using purified GST-Sp1 or His-SUMO-1-GST-Sp1 (Fig. 5d). We found that RNF4 could increase Sp1 ubiquitination, and the increased level of ubiquitinated Sp1 in His-SUMO-1-GST-Sp1 was significantly

Modifications in Sp1 during Cell Cycle Progression

7

Fig. 5. RNF4 functions as a ubiquitin E3 ligase of Sp1. (a) Sp1 immunoprecipitated with anti-Sp1 antibodies was used as a substrate for the in vitro ubiquitination assay. Sp1 was incubated with E1, E2, ubiquitin, and purified His-RNF4 and was analyzed by immunoblotting with anti-Sp1 antibodies. (b) Sp1 immunoprecipitated with anti-Sp1 antibodies was incubated with ubiquitin ligase E1, ubiquitin ligase E2, or purified His-RNF4, His-RNF4/CS1, or His-RNF4/SIM for the in vitro ubiquitination assay. Samples were then analyzed by immunoblotting with anti-Sp1 and anti-His antibodies. (c) HA-Sp1-myc or HA-SUMO-1-Sp1-myc were overexpressed in HeLa cells and immunoprecipitated with anti-HA antibodies. Sp1 or SUMO-1-Sp1 from the cells lysates was used as a substrate and was incubated with E1, E2, ubiquitin, or purified His-RNF4 for the in vitro ubiquitination assay. Samples were then analyzed by immunoblotting with antiHA and anti-His antibodies. (d) The E. coli sumoylated system pT-E1/E2/His-SUMO-1 was utilized to obtain pure HisSUMO-1-GST-Sp1. After purification, GST-Sp1 or His-SUMO-1-GST-Sp1 was used as a substrate and incubated with ubiquitin ligase E1, ubiquitin ligase E2, ubiquitin, or His-RNF4 for the in vitro ubiquitination assay. Samples were then analyzed by immunoblotting with anti-Sp1 and anti-RNF4 antibodies.

higher than that of GST-Sp1. These data strongly suggest that RNF4 is the ubiquitin E3 ligase of Sp1 and that Sp1 sumoylation facilitates RNF4-mediated degradation efficiency. Phosphorylation modulates RNF4-mediated Sp1 degradation Our earlier study indicated that Sp1 was phosphorylated by JNK1 at Thr278/Thr739 during

mitosis and that the phosphorylation shielded Sp1 from the ubiquitin-dependent degradation pathway. 10 In the present study, we found that RNF4 directly interacts with the C-terminus of Sp1 (619–785 aa) (Fig. 1). Therefore, we hypothesized that JNK-induced Sp1 phosphorylation might affect Sp1–RNF4 interaction. First, we examined the protein levels of Sp1-wt and Sp1(T739D), which mimics the phosphorylated form of Sp1, in the presence of RNF4 (Fig. 6a). RNF4 was co-transfected

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Modifications in Sp1 during Cell Cycle Progression

Fig. 6. Sp1 phosphorylation affects Sp1–RNF4 interaction. (a) GFP-Sp1-wt or GFP-Sp1(T739D) was co-transfected with GFP-RNF4 into HeLa cells, and the cell lysates were analyzed by immunoblotting with anti-GFP and anti-actin antibodies (a-a).(b) The GFP-Sp1-wt, GFP-Sp1(T739D), GFP-Sp1(T278D/T739D), and GFP-Sp1(T278D) plasmids were individually transfected into HeLa cells for 24 h. The cell lysates were analyzed by immunoblotting with anti-GFP or anti-Sp1 antibodies (b-a). After three independent experiments, the levels of Sp1 and its mutants recruited by His-RNF4 were individually quantified and normalized to their input levels. A statistical t-test was performed (**p b 0.01; b-b). (c) HeLa cells were synchronized with nocodazole treatment for 16 h in mitosis (M), and cells in interphase (I) were lysed using 2 × sample buffer. The samples were analyzed by immunoblotting with anti-Sp1, anti-His, anti-cyclin B1, and anti-actin antibodies. The cells in mitosis and interphase were also lysed using RIPA buffer for use in the His-RNF4 pull-down assay. Samples were then analyzed by immunoblotting with anti-Sp1 and anti-His antibodies. (d) Sp1 immunoprecipitated from cells in interphase or in the mitotic stage was used as a substrate (d-a) and was incubated with purified His-RNF4 for the in vitro ubiquitination assay (d-b). The samples were then analyzed by immunoblotting with anti-Sp1 anti-cyclin B1 or anti-His antibodies.

into HeLa cells with GFP-Sp1-wt or GFP-Sp1 (T739D). Sp1-wt protein levels decreased due to RNF4, while the Sp1(T739D) protein level remained

stable in RNF4-overexpressing cells (Fig. 6a). We performed a His pull-down assay to address the effect of Sp1 phosphorylation on the Sp1–RNF4

Modifications in Sp1 during Cell Cycle Progression

interaction (Fig. 6b). GFP-Sp1, GFP-Sp1(T278D), GFP-Sp1(T739D), and GFP-Sp1(T278D/T739D) were transfected into HeLa cells, and the cell lysates were harvested and incubated with purified HisRNF4 proteins. The interaction between RNF4 and Sp1(T739D) or Sp1(T278D/T739D) was drastically decreased compared to that with Sp1-wt, while the interaction between RNF4 and the phosphorylated form Sp1(T278D) was similar to that with Sp1-wt (Fig. 6b-a). We further investigated the Sp1 sumoylation status during cell cycle progression (Fig. 6c). Our data indicated that Sp1 sumoylation was slightly increased during mitosis as compared to that during interphase. The molecular weight of SUMO-1-Sp1 was similar during mitosis and interphase, implying that hyperphosphorylation of Sp1 during mitosis might block its sumoylation. To further assess the effect of Sp1 phosphorylation on the Sp1–RNF4 interaction, we collected Sp1 from HeLa cells in the interphase and mitotic stage to perform the His pull-down assay (Fig. 6d). The assay results indicated that the Sp1–RNF4 interaction is nearly abolished during mitosis. We also found that the SUMO-1-Sp1 level was slightly increased during mitosis. We then performed an in vitro ubiquitination assay to directly characterize the effect of phosphorylation on RNF4-mediated Sp1 degradation. Sp1 from HeLa cells in the interphase and mitotic stage were used as the substrates and were then incubated with ubiquitin ligase E1, ubiquitin ligase E2, purified His-RNF4, ubiquitin, and an adenosine triphosphate regeneration system (Fig. 6e). As shown in Fig. 6e, we found that RNF4 could induce Sp1 ubiquitination during the interphase, while a lower ubiquitination signal was present during mitosis. Taken together, these data show that highly phosphorylated Sp1 during mitosis is more resistant to RNF4-mediated ubiquitination than during interphase, and this strongly suggests that the high phosphorylation of Sp1 during mitosis retain its protein levels by preventing it from interacting with the ubiquitin E3 ligase RNF4.

Discussion The present study was the first to clarify the interplay of intra-posttranslational Sp1 modifications consisting of ubiquitination, sumoylation, and phosphorylation (Fig. 7). We found that RNF4 is recruited to SUMO-1-Sp1, and it then downregulates the protein level of Sp1. We identified RNF4 as the ubiquitin E3 ligase for Sp1 and determined that sumoylation is a critical factor for RNF4mediated Sp1 degradation. We also showed that Sp1 phosphorylation in mitosis repressed the Sp1– RNF4 interaction, protecting Sp1 from degradation (Fig. 7). Therefore, the interplay between Sp1

9

Fig. 7. Model for modulating Sp1–RNF4 interaction. The schematic diagram shown here illustrates the posttranslational modifications of Sp1, such as sumoylation and phosphorylation, which could modulate Sp1 stability by affecting Sp1–RNF4 interaction.

sumoylation and phosphorylation that controls Sp1–RNF4 interaction is important for maintaining Sp1 protein levels during cell cycle progression. Sp1 is an important transcriptional factor that regulates the expression of many genes, including those involved in cell growth, apoptosis, and development. 2,3 One study revealed that sumoylation has a negative effect on the transcriptional activity of Sp1. 46 In our earlier study, we found that Sp1 can be more strongly polyubiquitinated in normal cervical tissues than in cervical tumor tissues. 10 We determined that both transcriptional activity and protein stability contribute to Sp1 accumulation in most cancer strains. 9 We found that Sp1 sumoylation at Lys16 can increase ubiquitination and then increase Rpt6 recruitment to degrade Sp1 in a proteasomedependent pathway. 9 As seen in Fig. 2a, the expression patterns of Sp1, Sp1(K16R), and SUMO1-Sp1 were quite variable. The expression level of Sp1 (K16R) was very high, but SUMO-1-Sp1 expression was very low. This finding is consistent to that of our earlier study 9 and may due to Sp1 sumoylation be prone to degradation. In addition, one band below the HA-Sp1(K16A)-myc was found in earlier studies. 9,45 To identity this band, we constructed HA-Sp1-myc, an HA tag in the N-terminus and a myc tag in the C-terminus of Sp1. However, we used anti-HA and anti-myc antibodies for the immunoblot assay, and this band was always present, implying that it was not a truncated fragment of Sp1 (data not shown). When HA-SUMO-1-Sp1-myc was overexpressed in HeLa cells and immunoblotted using anti-HA antibodies, a cleaved form of Sp1 was observed (Fig. 4a-b). This cleaved form of Sp1 was not observed when

10 immunoblotting was performed using anti-myc antibodies. Therefore, cleavage of the C-terminal Sp1 might take place under this condition, although the detailed mechanism remains unclear. In addition, phosphorylation is the most common type of Sp1 modification. Many residues have shown that Sp1 is phosphorylated by different kinases such as extracellular signal-regulated kinases 1 and 2 and JNK1. 10,47 Furthermore, phosphorylation-induced modification at different Sp1 residues can positively or negatively affect its transcriptional activity, protein stability, and DNA-binding affinity. 48 For example, JNK1-phosphorylated Sp1 at Thr739 increases Sp1 protein stability. 10 This study provides the first evidence of the interplay between ubiquitination, sumoylation, and phosphorylation. Our data also revealed that the C-terminus of Sp1 can interact directly with RNF4. As shown in Fig. 5, our data also indicate that Sp1 can be ubiquitinated by the ubiquitin E3 ligase RNF4. Finally, Sp1 phosphorylation at Thr739 can suppress the Sp1–RNF4 interaction and shield it from degradation (Fig. 6). These data support that intraposttranslational modification of Sp1 controls its protein stability, which might be a critical factor for Sp1 accumulation during tumorigenesis. Although phosphorylation affected Sp1–RNF4 interaction, it did not alter the Sp1 sumoylation level (Fig. 6). Figure 1 shows that RNF4 interacts with the C-terminus of Sp1 and the SUMO-1 modifier within the N-terminus. Therefore, phosphorylation at Thr739 localized within the C-terminal of Sp1 might change the conformation of Sp1, resulting in the inhibition of RNF4 recruitment. As a result, the C-terminus of Sp1 might interact specifically with RNF4. This interaction might also explain why RNF4 is not the only ubiquitin E3 ligase for all sumoylated proteins. In addition, RNF4 is related to SUMO-2 conjugation, 30–33,45 but this study and earlier studies indicated that Sp1 is modified by SUMO-1 and not by SUMO-2 (Supplementary Fig. 3). 9,46 Common posttranslational modifications of Sp1 include phosphorylation, glycosylation, acetylation, ubiquitination, and sumoylation. We identified the two modulators sumoylation and phosphorylation that may contribute to Sp1 stability. Our earlier studies showed that Sp1 accumulates during tumorigenesis via sumoylation repression and that Sp1 levels are maintained via hyperphosphorylation during mitosis. 9,10 Data from other studies have suggested that phosphorylation at the N-terminal Sp1 might affect its further modification. 49 The presence of different phosphorylation residues affecting Sp1 cleavage might reflect the different states of cell cycle progression, such as interphase or mitosis. A relationship between sumoylation and phosphorylation also occurs in other proteins. For example, phosphorylation of heat shock factor 1, heat shock factor 4b, signal transducer and activator of transcription 1 protein, and globin transcription factor 1 could

Modifications in Sp1 during Cell Cycle Progression

upregulate sumoylation. 50 Studies have also shown that phosphorylation of PML protein, c-Jun, and p53 leads to desumoylation. 51–53 In the present study, our data also suggest the importance of posttranslational modifications affecting the intermolecular cross talk between Sp1 and RNF4. We found that the interaction between hyperphosphorylated Sp1 and RNF4 is downregulated in mitosis, leading to lower ubiquitination levels (Fig. 6). In vivo and in vitro experiments were carried out to prove that RNF4 is the ubiquitin E3 ligase of Sp1. However, studies have demonstrated that reduced O-glycosylation of Sp1 was associated with increased proteasome susceptibility and that a E3 ligase SCF (β-transducin repeat-containing protein) targeted Sp1 for proteasomal degradation in response to glucose starvation. 54,55 Therefore, different ubiquitin E3 ligases may modulate Sp1 stability under the different physical conditions; the detail mechanism needs further address. It is also interesting to note that no upshift bands were found in sumoylated Sp1 during mitosis (Fig. 6c). This result might be explained by intra-posttranslational regulations of Sp1 occurring between sumoylation and phosphorylation. The Sp1–RNF4 interaction may depend on the associated levels of Sp1 sumoylation and phosphorylation. However, the exact mechanism involved here awaits further investigation.

Materials and Methods Cell culture and transfection Human epidermoid carcinoma HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 μg/mL streptomycin sulfate, and 100 units/mL penicillin G sodium in an incubator maintained at 37 °C with an atmosphere of 5% CO2. Cells at 90% confluence were used for the transfection. Plasmids were transfected into cells by lipofection using Lipofectamine according to the manufacturer's instructions with slight modification. Cells were replated 24 h before transfection at an optimal cell density in 6 mL of fresh culture in a 10-cm dish. For the transfection, 4 μL of Lipofectamine was added to 4 μg of the indicated plasmid, as described in each experiment, in 0.4 mL of Opti-MEM medium for 30 min at room temperature. The culture medium was then changed to 3 mL of Opti-MEM medium containing the plasmid and Lipofectamine followed by incubation at 37 °C in an atmosphere of 5% CO2 for 6 h. The medium was replaced with 6 mL of fresh DMEM, and the cells were incubated for an additional 18 h. Antibodies and reagents Polyclonal antibodies against Sp1 were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Anti-HA antibody was obtained from Roche Molecular Biochemicals (Indianapolis, IN, USA). Anti-RNF4 was purchased from

11

Modifications in Sp1 during Cell Cycle Progression

Sigma-Aldrich (St. Louis, MO, USA), and anti-actin and anti-ubiquitin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal antibodies against GST and cyclin B1 were purchased from Santa Cruz Biotechnology, those against Flag and His were obtained from Sigma-Aldrich, and that against GFP was purchased from Biomol International, L.P. (Butler Pike, PA, USA). Lipofectamine 2000, DMEM, and Opti-MEM medium were obtained from Invitrogen Life Technologies (Grand Island, NY, USA). MG132 was purchased from Tocris Bioscience (Missouri, MO, USA). NEM, isopropyl1-β-D-thiogalactopyranoside (IPTG), and nocodazole were purchased from Sigma-Aldrich. The fetal bovine serum was obtained from HyClone Laboratories (Logan, UT, USA). The Fraction II HeLa Cell Lysate Conjugation Kit was purchased from Boston Biochem (Cambridge, MA, USA). Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA, USA). RNF4 expression was ablated with two individual shRNAs, which were purchased from National RNAi Core Facility (Academia Sinica, Taipei, Taiwan). The target sequences of the RNF4 shRNAs were 5′-CATACTCCCAGAAACGCCAGG-3′(shRNA RNF4-1) and 5′CGGGCTTCTGACTGCTCCA TA-3′ (shRNA RNF4-2). All other chemical reagents used were of the highest purity obtainable. Plasmids All of the full-length Sp1 constructs including the wt and mutants used in this study consisted of 785 aa. The expression plasmids containing HA-Sp1-myc, HA-SUMO1-Sp1-myc, HA-Sp1(K16R)-myc, HA-Sp1, HA-SUMO-1Sp1, HA-Sp1(K16R), HA-Sp1(E18A), GST-Sp1, pT-E1/ E2/His-SUMO-1, myc/His-ubiquitin, and wt and mutant GFP-Sp1 have been described previously. Full-length RNF4 cDNA was inserted into the GFP fusion vector pEGFP-C1 (Clontech, CA, USA) and the pcDNA3.0-HA vector to create pEGFP-RNF4 and HA-RNF4, respectively. Plasmids EGFP-RNF/SIM and His-RNF4/SIM, from which we deleted all of the SIMs (residues 30–76), were constructed, and the amplified sequences were verified. Polymerase chain reaction-based mutagenesis was used to generate the RNF4 RING finger mutant (RNF4/CS1), which has been reported to be devoid of ubiquitin E3 ligase activity. 43 Flag-RNF4 and RNF4 that contains all the mutated SIMs (Flag-RNF4mt) were gifts from Dr. Ronald T. Hay (University of Dundee, UK). 30,33,56 Protein synthesis in E. coli GST-Sp1(1–785 aa), GST-Sp1(8–290 aa), GST-Sp1(8– 618 aa), and GST-Sp1(619–785 aa) were purified from transformed E. coli BL21(DE3) culture at the mid-log phase in 200 mL of LB medium containing ampicillin (50 mg/mL). For His-SUMO-1-GST-Sp1(1–785 aa), the GST-Sp1(1–785 aa) and pT-E1/E2/His-S1 plasmids were co-transformed into E. coli BL21(DE3) in 200 mL of LB medium containing ampicillin (50 mg/mL) and chloramphenicol (10 mg/mL). For the fusion proteins His-RNF4, His-RNF4/SIM, and His-RNF4/CS1, the pET28b-RNF4, pET28b-RNF4/CS1, or pET28b-RNF4/SIM plasmids, respectively, were

transformed into E. coli BL21(DE3) in 200 mL of LB medium containing kanamycin (50 mg/mL). IPTG was then added to the cultures to reach a final concentration of 1 mM. After a 4-h IPTG treatment, the E. coli cultures were collected and lysed in phosphate-buffered saline buffer containing protease inhibitor cocktails. Each E. coli lysate was homogenized by sonication for 4 min and then centrifuged at 13,000 rpm for 20 min at 4 °C. The supernatant was incubated with 0.2 mL of glutathione Sepharose™ 4B beads (GE Healthcare, Buckinghamshire, UK) or Ni-NTA agarose (Qiagen). The beads that conjugated the fusion proteins were washed with the lysis buffer and eluted with the elution buffer. After elution, the fusion proteins were desalted for use in subsequent experiments. Immunoprecipitation assay Cell lysates were prepared using a radioimmune precipitation assay (RIPA) buffer {50 mM Tris (pH 7.5); 150 mM NaCl; 5 mM ethylenediaminetetraacetic acid; 0.1% Nonidet P-40; and 10 μg/mL leupeptin, aprotinin, and 4-[2-aminoethyl]benzenesulfonyl fluoride}. An equal amount of protein was used in each experiment, and it was pre-incubated with protein A/G-Sepharose for 30 min at 4 °C and centrifuged for pellet removal. The supernatants were then incubated with anti-HA, antiRNF4, anti-ubiquitin, or anti-Sp1 antibodies at a dilution of 1:200 at 4 °C for 1 h. The immunoprecipitated pellets were subsequently incubated with protein A/G-Sepharose, washed five times with RIPA buffer, and subjected to Western blotting analysis. Western blotting Cell lysates were fractionated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer's protocol (GE Healthcare). After incubation with 3% nonfat milk in Tris-buffered saline/Tween 20 [TBST; 10 mM Tris (pH 8.0), 150 mM NaCl, and 0.5% Tween 20] for 60 min, the membranes were washed once with TBST and then incubated with antibodies against Sp1 (1:5000), GST (1:1000), GFP (1:1000), cyclin B1 (1: 3000), ubiquitin (1:1000), tubulin (1:10,000), actin (1:10,000), HA (1:2000), His (1:2000), Flag (1:2000), myc (1:2000), or RNF4 (1:1000) at room temperature for 16 h. Membranes were washed three times for 10 min and incubated with a 1:3000 dilution of horseradish-peroxidase-conjugated antimouse, anti-rabbit, anti-rat, or anti-goat antibody for 2 h. Blots were washed with TBST three times and were developed using an ECL system (Pierce Chemical, Rockford, IL, USA) according to the manufacturer's protocol. Pull-down assay For the His pull-down assay, His-RNF4 was incubated with various plasmid-overexpressed cell lysates as described in the figure legends. For the GST pull-down assay, GST, GST-Sp1(8–618 aa), GST-Sp1(8–290 aa), GSTSp1(619–785 aa), His-SUMO-1, His-SUMO-1-GST-Sp1,

12 and GST-Sp1 were incubated with the cell lysates in the presence of Flag-RNF4 or Flag-RNF4mt at 4 °C for 1 h in 5 mL of binding buffer [20 mM Tris–HCl (pH 7.5), 300 mM NaCl, 50 μM ZnCl2, 0.5% Nonidet P-40, 1 mM dithiothreitol, and 1 mg/mL bovine serum albumin]. The beads were washed six times with binding buffer, and the bound proteins were analyzed by SDS-PAGE and recognized using individual antibodies. In vitro ubiquitination assay For studying RNF4-mediated degradation, we precipitated Sp1, HA-Sp1-myc, HA-SUMO-1-Sp1-myc, GSTSp1, or His-SUMO-1-Sp1 from transfected HeLa cells using anti-Sp1 or anti-HA antibodies. Purified His-RNF4, His-RNF4/CS1, or His-RNF4/SIM was incubated in 5 μL ubiquitin solution, 4 μg/μL Fraction II HeLa, 5 μM MG132, and 4 μM ubiquitin aldehyde. After incubation at 37 °C for 1 h, the reaction products were then subjected to Western blotting analysis. In vivo ubiquitination assay The in vivo ubiquitination assay was performed as described previously. HeLa cells were transfected with myc/His-ubiquitin, GFP-RNF4, GFP-RNF4/CS1, or GFPRNF4/SIM. After the transfection, the cells were treated with MG132 (10 μM) for 4 h. The cells were then lysed using RIPA buffer and immunoprecipitated with antiubiquitin. The supernatants were then subjected to Western blotting analysis using anti-Sp1 antibodies.

Acknowledgements This work was supported by the National Cheng Kung University project of the Program for Promoting Academic Excellence and Developing World Class Research Centers and by the National Science Council, Taiwan (grants NSC 97-2311-B-006-002MY3, NSC 97-2320-B-006-016-MY3, and DOH100TD-PB-111TM014).

Supplementary Data Supplementary data to this article can be found online at doi:10.1016/j.jmb.2011.09.027

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