Hyperphosphorylation Regulates the Activity of SREBP1 During Mitosis

Hyperphosphorylation regulates the activity of SREBP1 during mitosis

Maria T. Bengoechea-Alonso, Tanel Punga, and Johan Ericsson*

Ludwig Institute for Cancer Research, Biomedical Center, Box 595, Husargatan 3, S-751 24 Uppsala, Sweden

Edited by Michael S. Brown, University of Texas Southwestern Medical Center, Dallas, TX, and approved June 29, 2005 (received for review February 22, 2005)

The sterol regulatory element-binding protein (SREBP) family of cholesterol was enhanced in G2͞M-arrested cells. Thus, our results transcription factors controls the biosynthesis of cholesterol and demonstrate that the activity of mature SREBP1 is regulated by other lipids, and lipid synthesis is critical for cell growth and phosphorylation during the cell cycle, suggesting that SREBP1 may proliferation. We were, therefore, interested in the expression and provide a link between lipid synthesis, proliferation, and cell activity of SREBPs during the cell cycle. We found that the expres- growth. sion of a number of SREBP-responsive promoter-reporter genes were induced in a SREBP-dependent manner in cells arrested in Materials and Methods G2͞M. In addition, the mature forms of SREBP1a and SREBP1c were Cell Culture and Treatments. All tissue culture media and antibiotics hyperphosphorylated in mitotic cells, giving rise to a phospho- were obtained from Sigma. HEK293T, HepG2, and HeLa cells epitope recognized by the mitotic protein monoclonal-2 (MPM-2) were from the American Type Culture Collection. To arrest cells in antibody. In contrast, SREBP2 was not hyperphosphorylated in G2͞M, cells were treated with nocodazole (100 ng͞ml), taxol (1 mitotic cells and was not recognized by the MPM-2 antibody. The ␮M), or monastrol (100 ␮M) for 16 h. To arrest cells in G1͞S and MPM-2 epitope was mapped to the C terminus of mature SREBP1, S, cells were treated with hydroxyurea (5 mM) and aphidicolin (2 and the mitosis-speciﬁc hyperphosphorylation of SREBP1 de- ␮g͞ml), respectively, for 16 h. pended on this domain of the protein. The transcriptional and DNA-binding activity of SREBP1 was enhanced in cells arrested in Reagents and Antibodies. Horseradish peroxidase-conjugated anti- G2͞M, and these effects depended on the C-terminal domain of the mouse and anti-rabbit IgG and protein G Sepharose were from protein. In part, these effects could be explained by our observa- Amersham Pharmacia Biosciences. Anti-Flag antibody, nocoda- tion that mature SREBP1 was stabilized in G2͞M. In agreement with zole, taxol, and standard chemicals were from Sigma. Monastrol these observations, we found that the synthesis of cholesterol was was from Alexis, San Diego. Monoclonal anti-SREBP1 (2A4), increased in G2͞M-arrested cells. Thus, our results demonstrate anti-tubulin (TU-02), anti-cyclin B1 (GNS1), anti-cyclin A (BF683), that the activity of mature SREBP1 is regulated by phosphorylation and rabbit anti-SREBP1 (H-160) antibodies were from Santa Cruz during the cell cycle, suggesting that SREBP1 may provide a link Biotechnology. Anti-phospho-Cdk1 (Thr-161) and anti-Cdc25C between lipid synthesis, proliferation, and cell growth. were from Cell Signaling Technology, Beverly, MA. Anti-mitotic protein monoclonal-2 (MPM-2) was from Upstate Biotechnology, cell cycle ͉ cholesterol ͉ phosphorylation ͉ proliferation Lake Placid, NY. The anti-phospho-serine antibody was from Zymed. embers of the sterol regulatory element-binding protein (SREBP) family of transcription factors control cholesterol Plasmids and DNA Transfections. The expression vectors for Flag- M tagged SREBP1a (amino acid residues 2–490), SREBP1a-⌬C and lipid metabolism and play critical roles in insulin signaling and ⌬ during adipocyte differentiation (1). Two genes, srebf1 and srebf2, (amino acid residues 2–417), SREBP1a- TAD (TAD, transacti- encode three different SREBPs (SREBP1a, SREBP1c, and vation domain; amino acids 90–490), SREBP1c (amino acid resi- SREBP2) (2). SREBPs are synthesized as large precursor proteins dues 2–466), and SREBP2 (amino acid residues 2–485) have been that are inserted into the nuclear and endoplasmic reticulum (ER) described (10). The 3-hydroxy-3-methylglutaryl (HMG)-CoA syn- membranes and are transcriptionally inactive. In sterol-depleted thase (SYNSRE-luc), farnesyldiphosphate synthase (FPPS-luc), fatty acid synthase (FAS-luc), and low-density lipoprotein receptor cells, SREBP cleavage-activating protein (SCAP) escorts SREBP ⌬ to the Golgi apparatus where SREBP is processed sequentially by (LDLr) (LDLr-luc and LDLr SRE-luc) promoter-reporter con- two membrane-associated proteases, resulting in the release of the structs have been described (10–12). Transient transfections were performed by using the MBS transfection kit (Stratagene). Label- mature form of the protein (3, 4). This transcriptionally active 32 fragment of SREBP is translocated to the nucleus and binds to the ing of transfected 293T cells with P, phosphoamino acid analysis promoters of SREBP target genes. Most of these genes are involved on immunoprecipitated material and Edman degradation were in the control of lipid biosynthesis and metabolism (5). In sterol- performed as described (13). loaded cells, the SCAP͞SREBP complex is trapped in the ER as a Further information on materials and certain methods used in this study [immunoprecipitations and immunoblotting, analysis of result of sterol-induced binding of SCAP to Insigs, which are ␤ resident proteins of the ER membrane (6, 7). lipid synthesis, luciferase and -gal assays, RT-PCR and chromatin It is well established that the synthesis of membrane lipids is immunoprecipitation (ChIP) assays, double-thymidine block, and FACS analysis] can be found in Supporting Methods, which is

critical for cell growth and proliferation (8, 9). Here, we report that BIOCHEMISTRY the expression of a number of SREBP-responsive promoter- published as supporting information on the PNAS web site. reporter genes are induced in a SREBP-dependent manner in cells ͞ arrested in G2 M. In addition, the mature forms of SREBP1a and This paper was submitted directly (Track II) to the PNAS ofﬁce. SREBP1c are hyperphosphorylated in mitotic cells, whereas ma- Abbreviations: SREBP, sterol regulatory element-binding protein; MPM-2, mitotic protein ture SREBP2 is not. The phosphorylated residues were mapped to monoclonal-2; FAS, fatty acid synthase; LDLr, low-density lipoprotein receptor; FPPS, the C terminus of mature SREBP1. The transcriptional potency of farnesyl diphosphate synthase; HMG, 3-hydroxy-3-methylglutaryl; DBD, DNA-binding domain; TAD, transactivation domain. mature SREBP1 was enhanced in cells arrested in G2͞M, and these effects depended on the C-terminal domain of the protein. In *To whom correspondence should be addressed. E-mail: [email protected]. agreement with these observations, we found that the synthesis of © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501494102 PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 ͉ 11681–11686 Downloaded by guest on September 25, 2021 Fig. 1. Mature SREBP1 is highly phosphorylated. (A) Schematic illustration of mature SREBP1a and the ⌬C mutant, lacking the C-terminal domain. bHLH, basic helix–loop–helix. (B) 293T cells were transfected with Flag-tagged mature (M) SREBP1a, either WT or ⌬C, followed by metabolic labeling with 32P and immunoprecipitation. The migration of the WT and ⌬C proteins is indicated. The band corresponding to WT SREBP1a was excised and used for

phosphoamino acid analysis (Right). The levels of SREBP1a in whole-cell lysates Fig. 2. Mature SREBP1 is hyperphosphorylated in cells arrested in G2͞M. (A) (WCL) were detected by Western blotting. (C) Mature SREBP1a was immuno- HeLa cells were left untreated or treated with nocodazole (Noc) to induce precipitated from transfected 293T cells and incubated in the absence or G2͞M arrest. After immunoprecipitation of SREBP1, the levels and phosphor- presence of ␭-phosphatase, separated on SDS͞PAGE, and visualized by West- ylation (MPM-2) of SREBP1 were determined by Western blotting. The migra- ern blotting. (D) 293T cells were transfected with mature SREBP1a, either WT tion of the precursor (FL) and mature (M) forms of SREBP1 is indicated. (B) HeLa or ⌬C, followed by immunoprecipitation of the Flag-tagged proteins and cells were treated as in A, incubated in the absence or presence of ␭-phos- separation on SDS͞PAGE. The phosphorylation of SREBP1a was monitored phatase in the absence or presence of the phosphatase inhibitor okadaic acid with phospho-serine (pSer, Left) or MPM-2 (Right) antibodies. (OA), and separated by SDS͞PAGE, and the levels and phosphorylation (MPM-2) of SREBP1 were determined by Western blotting. (C) HeLa cells were left untreated or arrested in G2͞M, G1͞S, or S as described in Materials and Results Methods. After immunoprecipitation of SREBP1, the levels and phosphoryla- Mature SREBP1 Is Highly Phosphorylated. Early observations indi- tion (MPM-2) of SREBP1 were determined by Western blotting. Asyn, asyn- cated that SREBP1a is phosphorylated in vivo (14). Subsequently, chronous. (D) HeLa cells were left untreated or treated with nocodazole to induce G ͞M arrest. Where indicated, the nocodazole-treated cells were it has been suggested that both SREBP1 and SREBP2 are phos- 2 washed with PBS and released from the G2͞M arrest in normal media for the phorylated by various protein kinases, both in vivo and in vitro indicated times. After immunoprecipitation of SREBP1, the levels and phos- (15–17). We were interested in mapping the major phosphorylated phorylation (MPM-2) of SREBP1 were determined by Western blotting. (E) residues in SREBP1, especially those found in the Ser͞Thr͞Gly- HepG2 cells were transfected with SYNSRE-luc, LDLr-luc, FAS-luc, FPPS-luc, and rich C terminus of the mature protein (Fig. 1A). To address this CyclinB1-luc. Twenty-four hours after transfection, cells were treated with issue, 293T cells were transfected with plasmids expressing either nocodazole, and luciferase activity was measured. (F) HeLa cells were transfected with SYNSRE-luc, FAS-luc, LDLr-luc, and FPPS-luc. Twenty-four hours full-length mature SREBP1a or a protein lacking the C-terminal after transfection, cells were treated as in C, and luciferase activity was ⌬ 32 domain ( C). After metabolic labeling with P, the proteins were measured. immunoprecipitated and separated by SDS͞PAGE. WT SREBP1a was highly phosphorylated, whereas only little radioactivity was incorporated in the ⌬C protein (Fig. 1B), indicating that the C epitope-specific antibody, whereas it failed to detect the ⌬C protein terminus of mature SREBP1a contains major phosphorylation (Fig. 1D Right). sites. Phospho-amino acid analysis revealed that phosphorylation of serine residues was the dominating modification (Fig. 1B Right). To Mature SREBP1 Is Hyperphosphorylated in G2͞M. The MPM-2 anti- confirm that nuclear SREBP1a was phosphorylated, transfected body recognizes phophorylated Ser͞Thr residues in a subset of mature SREBP1a was immunoprecipited from 293T cells and mitotic phosphoproteins. We wanted, therefore, to determine treated with ␭-phosphatase. Phosphatase-treated mature whether SREBP1 was phosphorylated during mitosis. To address SREBP1a migrated faster in the SDS͞PAGE gel (Fig. 1C), indi- this issue, we analyzed the electrophoretic mobility of endogenous cating that mature SREBP1a is highly phosphorylated in vivo. SREBP1 in asynchronous HeLa cells and cells arrested in G2͞Mby These results were confirmed when the phosphorylation of nocodazole treatment. To monitor cell-cycle arrest, the levels of SREBP1a was monitored with an antibody directed against phos- cyclin B1, phospho-Cdk1, and Cdc25C in the samples were also phorylated serine residues. Mature SREBP1a was recognized by determined. The migration of mature SREBP1 was dramatically this antibody, whereas the antibody failed to detect the protein shifted to higher molecular species in G2͞M-arrested cells (Fig. 2A). lacking the C-terminal domain (Fig. 1D Left). In addition, trans- Mature SREBP1 was also recognized by the MPM-2 antibody in fected mature SREBP1a was recognized by the MPM-2 phospho- G2͞M-arrested cells, and the MPM-2 signal was associated with the

11682 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501494102 Bengoechea-Alonso et al. Downloaded by guest on September 25, 2021 shifted form of the protein. The precursor form of SREBP1 was not shifted in response to nocodazole treatment and was not recognized by the MPM-2 antibody, indicating that this modification is specific for mature SREBP1. Similar results were also obtained in HepG2 and U2OS cells (data not shown). The shift in apparent molecular weight and the SREBP1-specific MPM-2 signal were lost by ␭-phosphatase treatment (Fig. 2B), indicating that mature SREBP1 is hyperphosphorylated in cells arrested in G2͞M. Interestingly, endogenous SREBP2 was not phosphorylated in arrested cells and was not recognized by the MPM-2 antibody (Fig. 7, which is published as supporting information on the PNAS web site). The hyperphosphorylation of SREBP1 and the MPM-2 signal were specific for cells arrested in G2͞M and were not observed in cells arrested in other phases of the cell cycle (Fig. 2C; FACS profiles are Fig. 3. Transcriptional activation of SREBP-responsive promoter-reporter provided in Fig. 8, which is published as supporting information on genes in G2͞M. (A) HeLa cells were transfected with SYNSRE-luc and LDLr-luc. the PNAS web site). The hyperphosphorylation of SREBP1 and the Twenty-four hours after transfection, cells were treated with nocodazole in SREBP1-specific MPM-2 signal were rapidly lost after mitotic exit the absence or presence of sterols (cholesterol and 25-hydroxycholesterol; 50 and reentry into G1 after release from nocodazole arrest (Fig. 2D), and 5.0 ␮g͞ml, respectively) to suppress the activation of endogenous SREBPs, suggesting that the hyperphosphorylation of SREBP1 is a dynamic and luciferase activity was measured. (B) HepG2 cells were transfected with process. SYNSRE-luc and LDLr-luc in the absence or presence of empty expression ⌬ Ϫ͞Ϫ Our data suggest that SREBP1 is highly phosphorylated in vector or dominant-negative SREBP1 ( TAD and DBD ). Twenty-four hours ͞ after transfection, cells were treated with nocodazole, and luciferase activity G2 M. Thus, the transcriptional activity of SREBP1 could poten- was measured. (C) HepG2 cells were transfected with LDLr-luc or LDLr⌬SRE- tially be regulated during this phase of the cell cycle. To test this luc. Twenty-four hours after transfection, cells were treated with nocodazole, idea, HepG2 cells were transfected with various SREBP-responsive and luciferase activity was measured. RLU, relative light units. promoter-reporter genes. After transfection, the cells were either left untreated or treated with nocodazole to induce G2͞M arrest. The expression of all of the reporter genes was significantly induced The Hyperphosphorylation and Activation of Mature SREBP1 Is Spe- ͞ in response to nocodazole (Fig. 2E), indicating that the SREBP cific for Mitotic Cells. Nocodazole arrests cells in G2 M by prevent- pathway is activated in G ͞M. The induction was specific for cells ing tubulin polymerization. To make sure that the hyperphospho- 2 ͞ arrested in G ͞M and was not observed in cells arrested in G ͞Sor rylation of SREBP1 was specific for G2 M arrest, we tested the 2 1 ͞ S phase (Fig. 2F; FACS profiles are provided in Fig. 8). The effect of two other drugs that are known to induce G2 M arrest, SYNSRE-luc, FAS-luc, and FPPS-luc promoter-reporter genes taxol and monastrol. Taxol binds and stabilizes microtubules, whereas monastrol is a specific inhibitor of Eg5, a microtubule- were also induced in G2͞M in HeLa cells stably expressing these reporters (Fig. 9, which is published as supporting information on based motor protein. The mature form of endogenous SREBP1 the PNAS web site). was hyperphosphorylated and recognized by the MPM-2 antibody in HeLa cells treated with all of these drugs (Fig. 4A), demonstrating that the hyperphosphorylation of SREBP1 is specific for G2͞M Transcriptional Activation of SREBP-Responsive Promoter-Reporter ͞ Genes in G ͞M Depends on Functional SREBP. To test whether the arrest. When HeLa cells arrested in G2 M were fractionated by 2 mitotic shake-off, hyperphosphorylated SREBP1 and the activation of SREBP-responsive promoter-reporter genes in G2͞M required SREBP, HeLa cells were transfected with two separate SREBP1-specific MPM-2 signal were recovered in the mitotic cells (Fig. 4B), demonstrating that the hyperphosphorylation of SREBP-responsive promoter-reporter genes and either left un- SREBP1 is specific for M phase. To confirm that the hyperphos- treated or treated with nocodazole to induce G ͞M arrest. The 2 phorylation of mature SREBP1 was specific for mitosis, we ana- nocodazole-dependent induction of the reporter genes was reduced lyzed the expression and phosphorylation of SREBP1 at different in cells treated with sterols to suppress the activation of SREBP intervals after release from a double-thymidine G ͞S-phase block. (Fig. 3A), indicating that the induction, in part, depends on mature 1 The hyperphosphorylation of mature SREBP1 and the SREBP1- SREBP. This hypothesis was strengthened when the experiment specific MPM-2 signal reached their highest points close to the was repeated in the absence or presence of two dominant-negative ⌬ mitotic index peak (Fig. 4C), as measured by FACS analysis (Fig. versions of SREBP1, either lacking a TAD ( TAD) or having a 10, which is published as supporting information on the PNAS web mutation in its DNA-binding domain (DBD) that prevents DNA Ϫ͞Ϫ site). Interestingly, the steady-state levels of mature SREBP1 were binding (DBD ). Expression of the dominant-negative versions enhanced in mitotic cells and peaked at the same time as the of SREBP1 decreased the induction of the promoter-reporter hyperphosphorylation of SREBP1 (Fig. 4C), suggesting that phos- genes in response to nocodazole treatment (Fig. 3B), suggesting phorylation of mature SREBP1 could stabilize the protein during that the induction of the reporter genes depends on the transcrip- mitosis. The increase in steady-state levels of mature SREBP1 was tional activity of SREBP. Similar results were obtained when these even more pronounced when cells were released in nocodazole- experiments were repeated in HeLa cells (data not shown). Tran- containing media, when a majority of cells were in the M phase of scriptional activation of SREBP target genes depends on the the cell cycle (Fig. 11, which is published as supporting information binding of SREBP to specific sterol-responsive elements (SREs) on the PNAS web site). The hyperphosphorylated form of SREBP1 BIOCHEMISTRY within the promoters of these genes. Mutation of the SRE in the and the SREBP1-specific MPM-2 signal were also associated with LDLr promoter-reporter gene attenuated the nocodazole- the mitotic fraction when mitotic cells were isolated by mitotic dependent induction of the promoter gene (Fig. 3C), confirming shake-off from asynchronous HeLa cells in the absence of drug that SREBP is required for the enhanced expression of this reporter treatment (Fig. 4D). To determine whether SREBP was transcrip- in G2͞M. This hypothesis was strengthened by our observation that tionally activated in mitotic cells, HeLa cells were transfected with the nocodazole-dependent induction of the LDLr and HMG-CoA SYNSRE-luc and treated with nocodazole to induce G2͞M arrest, synthase promoter-reporter genes was blocked when cotransfected and the cells were fractionated by mitotic shake-off. The activity of with a vector expressing SREBP1-directed short hairpin RNA the promoter-reporter gene was enhanced in both the nonmitotic (data not shown). and mitotic populations (Fig. 4E). However, the relative increase

Bengoechea-Alonso et al. PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 ͉ 11683 Downloaded by guest on September 25, 2021 nized by the MPM-2 antibody in G2͞M-arrested cells (Fig. 5A), demonstrating that the mature forms of these proteins are targeted by phosphorylation. In agreement with our earlier results, mature SREBP2 was not hyperphosphorylated under these conditions, indicating that the M-phase phosphorylation is specific for the SREBP1 isoforms. Mature SREBP1a and SREBP1c were also hyperphosphorylated and recognized by the MPM-2 antibody in cells arrested in G2͞M by taxol treatment (Fig. 12, which is published as supporting information on the PNAS web site). Interestingly, the levels of SREBP1a and SREBP1c were enhanced in response to nocodazole treatment, whereas the expression of SREBP2 was unaffected, suggesting that phosphorylation of mature SREBP1 could stabilize the protein during mitosis. This hypothesis is in agreement with our results on endogenous SREBP1 (Fig. 4C). In agreement with our previous results, the hyperphosphorylation of transfected SREBP1a was specific for G2͞M, because we observed no change in the migration of the protein in 293T cells arrested in G1͞S or S (Fig. 12). Our initial results indicated that the MPM-2 epitope in SREBP1a resides in the C-terminal domain. To test this hypothesis, 293T cells were transfected with mature SREBP1a or the ⌬C mutant and treated with nocodazole to induce G2͞M arrest. The WT protein was hyperphosphorylated in response to nocodazole treatment, whereas the mutant protein was not (Fig. 5B). In addition, the ⌬C protein was not recognized by the MPM-2 antibody in G2͞M-arrested cells. Similar results were obtained in cells arrested in G2͞M by taxol treatment (Fig. 12). The levels of WT SREBP1a were enhanced in cells arrested in G2͞M, whereas the levels of the nonphosphorylated mutant were slightly reduced (Figs. 5B and 12), indicating that phosphorylation of the C-terminal domain could influence the stability of SREBP1. Thus, Fig. 4. The hyperphosphorylation and activation of SREBP1 is speciﬁc for our data indicate that the C-terminal domain in SREBP1 is mitotic cells. (A) HeLa cells were left untreated or treated with nocodazole targeted by phosphorylation during mitosis. To test whether mitotic (Noc), taxol, or monastrol (Mon) to induce G2͞M arrest. After immunoprecipitation of SREBP1, the levels and phosphorylation (MPM-2) of mature (M) kinases could phosphorylate SREBP1, whole-cell lysates were SREBP1 were determined by Western blotting. (B) HeLa cells were left un- prepared from asynchronous or G2͞M-arrested HeLa cells and ͞ treated or treated with nocodazole to induce G2 M arrest (lanes 1 and 2), and used in kinase assays with recombinant mature SREBP1a. Phos- M-phase cells were separated by mitotic shake-off (lanes 3 and 4). After phorylation of SREBP1a was monitored with the MPM-2 antibody. immunoprecipitation of SREBP1, the levels and phosphorylation (MPM-2) of Incubation of SREBP1a with extracts from asynchronous cells mature SREBP1 were determined by Western blotting. Asyn, asynchronous; failed to generate an SREBP1-specific MPM-2 epitope, whereas non-mit, non-mitotic; Mit, mitotic. (C) HeLa cells were synchronized at the mitotic extracts induced a robust MPM-2 signal (Fig. 5C). The ⌬C G1͞S transition by a double-thymidine treatment. Cells were collected at the indicated time points after release from the second thymidine block. Cell protein was not phosphorylated under the same conditions, indi- lysates were generated and analyzed by Western blotting with antibodies to cating that mitotic kinases target the C-terminal domain in the indicated proteins. The migration of the precursor (FL) and mature (M) SREBP1. Transcriptional activation of SREBP target genes de- forms of SREBP1 is indicated. The relative expression of mature SREBP1 is pends on the DNA-binding activity of SREBP. To test whether indicated at the bottom of Upper. After immunoprecipitation of SREBP1, the phosphorylation of SREBP1 could affect its DNA binding, mature phosphorylation (MPM-2) of mature SREBP1 was determined by Western SREBP1a and the ⌬C mutant were expressed in 293T cells, and the blotting. (D) Asynchronous HeLa cells were left untreated, and M-phase cells ͞ were separated by mitotic shake-off. After immunoprecipitation of SREBP1, cells were arrested in G2 M and whole-cell lysates from the the levels and phosphorylation (MPM-2) of mature SREBP1 were determined transfected cells were used in EMSAs. Interestingly, nocodazole by Western blotting. (E) HeLa cells were transfected with the SYNSRE-luc treatment enhanced the DNA-binding activity of the full-length ͞ promoter-reporter gene and treated with nocodazole to induce G2 M arrest, protein 2-fold, whereas the DNA-binding activity of the nonphos- and M-phase cells were separated by mitotic shake-off, and luciferase activity phorylated ⌬C mutant was slightly reduced (Fig. 5D Left). In was measured. extracts from G2͞M-arrested cells expressing mature SREBP1a, the SREBP1:DNA complex was supershifted with the MPM-2 ⌬ was more pronounced in M phase, indicating that the transcrip- antibody, whereas the complex containing the C protein was not tional potency of SREBP1 is enhanced in mitotic cells. Thus, our (Fig. 5D Right). Interestingly, a major portion of the SREBP1:DNA data demonstrate that mature SREBP1 is specifically hyperphos- complex in extracts from cells expressing WT SREBP1a was shifted phorylated in mitotic cells, possibly involving residues in the C by the MPM-2 antibody, indicating that a majority of the DNA- terminus of the mature protein. associated SREBP1 is phosphorylated on MPM-2 epitopes. To test whether phosphorylation of the C terminus could affect the tran- Phosphorylation of the C Terminus of Mature SREBP1 in Mitotic Cells. scriptional activity of SREBP1, cells were transfected with WT In vivo, mature SREBP1 is produced through proteolytic cleavage SREBP1a or the ⌬C mutant together with the SYNSRE-luc of a membrane-associated precursor protein. To determine promoter-reporter gene and the cells were treated with nocodazole. whether the mature form of SREBP1 in isolation was hyperphos- The transcriptional activity of WT SREBP1a was enhanced in phorylated in mitotic cells, 293T cells were transfected with expres- nocodazole-treated cells, whereas the activity of the ⌬C protein was sion vectors for mature SREBP1a, SREBP1c, and SREBP2, and unaffected (Fig. 5E), indicating that phosphorylation of the C the cells were left untreated or treated with nocodazole. Mature terminus of mature SREBP1 contributes to its enhanced transcrip- SREBP1a and SREBP1c were hyperphosphorylated and recog- tional potency in mitotic cells.

11684 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501494102 Bengoechea-Alonso et al. Downloaded by guest on September 25, 2021 Fig. 5. Phosphorylation of the C terminus of mature SREBP1 in mitotic cells. (A) 293T cells were transfected with mature (M) SREBP1a, SREBP1c, or SREBP2 and left untreated or treated with nocodazole (Noc). After immunoprecipitation, the levels and phosphorylation (MPM-2) of the SREBPs were determined by Western blotting. (B) 293T cells were transfected with mature SREBP1a, either WT or ⌬C, and left untreated or treated with nocodazole. After immunoprecipitation, the levels and phosphorylation (MPM-2) of SREBP1 were determined by Western blotting. (C) Recombinant 6ϫHis- SREBP1a, either WT or ⌬C, was incubated with whole-cell lysates from asynchronous HeLa cells or cells arrested in G2͞M in kinase buffer (10 mM Hepes, pH 7.4͞3.5 mM MgCl2͞0.2 mM DTT͞1mMATP͞10 mM ␤-glycerophosphate). The 6ϫHis- tagged proteins were captured on NiTA-agarose, washed, and resolved by SDS͞PAGE. The phosphorylation of SREBP1a was monitored with the MPM-2 antibody. non-mit, non-mitotic; mit, mitotic. (D) 293T cells were transfected with mature SREBP1a, either WT or ⌬C, and left untreated or treated with nocodazole. Whole-cell lysates were used in EMSAs with a 32P-labeled probe containing the SRE-1 sequence from the LDLr promoter. Where indicated, anti-Flag (Left) or MPM-2 (Right) antibodies were included in the assay. *, Supershifted complexes. (E) HepG2 cells were transfected with SYNSRE-luc or LDLr-luc in the presence of mature SREBP1a, either WT or ⌬C. Twenty-four hours after transfection, cells were treated with nocodazole, and luciferase activity was measured.

The Expression of SREBP Target Genes and Cholesterol Synthesis Are nocodazole treatment. The hyperphosphorylation and transcrip- Enhanced in G2͞M. Our results indicate that the expression of tional activation of SREBP1 depend on its Ser͞Thr-rich C-terminal SREBP-responsive promoter-reporter genes is enhanced in G2͞M. domain that also contains the MPM-2 epitope. Thus, our results To test whether endogenous SREBP target genes were induced in suggest that phosphorylation of mature SREBP1 could represent a ͞ G2 M, we used semiquantitative RT-PCR to analyze the expression novel mechanism to regulate SREBP-dependent transcription dur- of the HMG-CoA synthase and LDLr genes in asynchronous ing the cell cycle. ͞ HepG2 cells or cells arrested in G2 M by nocodazole treatment. Although there is a general repression of transcription during The expression of both genes was enhanced in nocodazole-treated mitosis (18), certain genes retain transcriptionally active complexes cells (Fig. 6A), demonstrating that SREBP target genes are induced on their promoters and are expressed during this stage of the cell ͞ in G2 M. During mitosis, chromosomes are highly condensed, cycle (19–24). Although it remains to be demonstrated that making DNA less accessible to transcription factors. However, SREBP1 is actively involved in transcriptional activation of endog- recent data indicate that mitotic chromosomes are accessible to enous target genes during mitosis, we could demonstrate that transcription factors and chromatin proteins. Using chromatin SREBP1 is associated with target promoters in cells arrested in immunoprecipitation, we found that endogenous SREBP1 was G ͞M and that the expression of the corresponding genes was associated with the promoters of the LDLr and HMG-CoA syn- 2 enhanced at G2͞M. The possibility of an active chromatin structure thase genes in G2͞M (Fig. 6B). The SREBP family of transcription factors regulates genes involved in cholesterol and lipid metabolism, and our data indicate that the transcriptional potency of SREBP1 is enhanced in mitosis. Thus, we speculated that lipid synthesis should be enhanced during this phase of the cell cycle. To test this hypothesis, HeLa cells were treated with nocodazole and the synthesis of cholesterol was monitored in untreated and treated cells. In agreement with our hypothesis, the synthesis of cholesterol was enhanced up to 3-fold in cells arrested in G2͞M (Fig. 6C). Thus, our results demonstrate that the function of mature SREBP1 is regulated by phosphorylation during the cell cycle. Discussion Fig. 6. The expression of SREBP target genes and cholesterol synthesis are In the current study we demonstrate that mature SREBP1 is enhanced in G ͞M. (A) RNA was isolated from asynchronous (Asyn, lane 1) or ͞ 2

hyperphosphorylated in cells arrested in G2 M and show that the nocodazole (Noc)-treated (lane 2) HepG2 cells. Total RNA was used to deter- BIOCHEMISTRY hyperphosphorylated form of the protein is associated with the mine the expression of the LDLr, HMG-CoA synthase (HMG-CoA), SREBP1a, mitotic population. In mitotic cells, mature SREBP1 is recognized and GAPDH genes by semiquantitative RT-PCR. (B) HeLa cells were left un- ͞ by the MPM-2 antibody, an antibody that recognizes a subset of treated or treated with nocodazole to induce G2 M arrest. Cells were pro- M-phase phosphoproteins. These effects are specific for SREBP1a cessed for chromatin immunoprecipitation analysis of the LDLr, HMG-CoA and SREBP1c, because SREBP2 fails to be hyperphosphorylated synthase, and p21 (negative control) genes, using anti-SREBP1 antibodies for immunoprecipitation. (C) HeLa cells were left untreated or treated with in G2͞M. In addition, we demonstrate that the transcriptional nocodazole to induce G2͞M arrest. Two hours before the end of the experi- potency of mature SREBP1 is enhanced when cells are arrested in ment, cells were placed in fresh media supplemented with [14C]acetate. Lipids G2͞M and that the HMG-CoA synthase and LDLr genes, both of were extracted and resolved by TLC. Radioactive products were visualized by which are SREBP target genes, are enhanced in response to PhosphorImage analysis.

Bengoechea-Alonso et al. PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 ͉ 11685 Downloaded by guest on September 25, 2021 at SREBP target genes was supported by our observation that the mature SREBP1 during mitosis. In the case of endogenous overall acetylation of histones H3 and H4 at the LDLr and SREBP1, this hypothesis was confirmed by our observation that the HMG-CoA synthase genes was preserved in nocodazole-arrested steady-state levels of mature SREBP1 were enhanced in mitotic HepG2 cells (data not shown). We could also show that a number cells after release from a double-thymidine block. Interestingly, the of SREBP-responsive promoter-reporter genes were induced in an C terminus of mature SREBP1 contains two PEST motifs, se- SREBP-dependent manner after arrest of cells in G2͞M. Interest- quences that are usually found in unstable proteins. Phosphoryla- ingly, the activation of SREBP1 in G2͞M depended on the C- tion of Ser and Thr residues within PEST motifs have been shown terminal domain of the mature protein, indicating that phosphor- to regulate the degradation of a number of proteins (29), including ylation of the C terminus is required for the transcriptional SREBPs (15). The C terminus of mature SREBP1 contains a total activation. Taken together, our results indicate that it is important of 19 Ser and Thr residues, and it will be important to determine for cells to retain a pool of transcriptionally active SREBP1 during which of these are phosphorylated and how these modifications mitosis, either to activate SREBP-responsive genes during this affects the stability of the protein. Stabilization of mature SREBP1 phase of the cell cycle or at the onset of mRNA synthesis at mitotic during mitosis would ensure that cells maintain a certain amount of exit. transcriptionally potent SREBP1 as they leave mitosis and an active SREBP target genes are involved in the biosynthesis of fatty acids chromatin structure is reformed. It is also possible that phosphor- and cholesterol. It has been suggested that cholesterol is essential ylation of the C terminus of mature SREBP1 could affect inter- for proper mitotic progression (25). This hypothesis is supported by actions with coactivators, components of the basic transcriptional ͞ our observation that cholesterol synthesis was enhanced in G2 M. machinery or other proteins that could regulate its transcriptional De novo synthesis of fatty acids appears to be required for cell activity. Alternatively, phosphorylation could affect the conforma- growth and proliferation (26). Expression of the FAS gene is tion of SREBP1, thereby affecting its transcriptional potential. elevated in a wide variety of human cancers, including prostate and Many proteins containing MPM-2 epitopes are substrates of the breast cancer (27, 28). Although the mechanisms underlying the mitotic regulator Pin1, a peptidyl-prolyl isomerase that is present increased FAS expression in tumors are not fully understood, throughout the cell cycle and that is thought to alter its mitotic several studies indicate that overexpression of FAS is part of a more targets by changing their conformation. Pin1 has been shown to general and coordinated activation of lipogenic gene expression, regulate the activity of a number of transcription factors, including mediated at least in part by activation of the SREBP pathway (27, p53 and c-Myc, in a phosphorylation-dependent manner (30, 31). 28). Thus, it may be important to control the expression levels and Interestingly, Pin1 also regulates the dephosphorylation and deg- activity of SREBP1 during the cell cycle, independent of sterol- radation of c-Myc (30). Thus, the identification of proteins that regulated cleavage of the precursor protein. Our results suggest that ͞ interact with SREBP1 in a phosphorylation-dependent manner phosphorylation of specific Ser and or Thr residues in the C- during mitosis will be important. Our results demonstrate that the terminal portion of mature SREBP1 could be one such mechanism activity of mature SREBP1 is regulated by phosphorylation during of regulation. mitosis, suggesting that SREBP1 may provide a link between lipid We found that SREBP1-dependent transcription was activated synthesis, proliferation, and cell growth. in cells arrested in G2͞M through a mechanisms involving phosphorylation of the C terminus of the mature protein. In part, this We thank V. Lukiyanchuk for technical support. This work was sup- activation could be explained by our observation that the level of ported by grants from the Swedish Research Council and the Novo nuclear SREBP1 was enhanced in response to nocodazole-induced Nordisk Foundation (to J.E.). J.E. is a Research Fellow of the Royal cell-cycle arrest, whereas the expression of the ⌬C protein was not, Swedish Academy of Sciences through a grant from the Knut and Alice indicating that phosphorylation of the C terminus could stabilize Wallenberg Foundation.

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