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Hyperphosphorylation Regulates the Activity of SREBP1 During Mitosis

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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 do- main; 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 ma- ture (M) SREBP1a, either WT or ⌬C, followed by metabolic labeling with 32P and immunoprecipitation. The migration of the WT and ⌬C proteins is indi- cated. 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
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