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A Phosphorylation Cascade Controls the Degradation of Active SREBP1

2009; Elsevier BV; Volume: 284; Issue: 9 Linguagem: Inglês

10.1074/jbc.m807906200

1083-351X

Maria T. Bengoechea-Alonso, Johan Ericsson,

Peroxisome Proliferator-Activated Receptors

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes. Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes. The sterol regulatory element-binding protein (SREBP) 2The abbreviations used are: SREBP, sterol regulatory element-binding protein; GSK-3, glycogen synthase kinase-3; HMG, 3-hydroxy-3-methylglutaryl; LDL, low density lipoprotein; siRNA, small interference RNA; PI3K, phosphatidylinositol 3-kinase; mTor, mammalian target of rapamycin; shRNA, short hairpin RNA; WCE, whole cell extract. family of transcription factors controls cholesterol and lipid metabolism and plays critical roles during adipocyte differentiation and insulin signaling (1Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. 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Ericsson J. Cell Metab. 2005; 1: 379-391Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 16Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. Chem. 2001; 276: 36431-36437Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). It has been suggested that nuclear SREBP molecules are, at least in part, ubiquitinated and degraded as a functional consequence of their transcriptional activity (12Punga T. Bengoechea-Alonso M.T. Ericsson J. J. Biol. Chem. 2006; 281: 25278-25286Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 15Sundqvist A. Ericsson J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13833-13838Crossref PubMed Scopus (64) Google Scholar). GSK-3-mediated phosphorylation of Thr-426 and Ser-430 in the C terminus of mature SREBP1 creates a docking site for Fbw7 (also known as human CDC4), the substrate recognition component of a specific SCF ubiquitin ligase (9Sundqvist A. Bengoechea-Alonso M.T. Ye X. Lukiyanchuk V. Jin J. Harper J.W. Ericsson J. Cell Metab. 2005; 1: 379-391Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Fbw7 is a tumor suppressor (19Welcker M. Clurman B.E. Nat. Rev. Cancer. 2007; 20: 83-93Google Scholar) and targets cyclin E (20Koepp D.M. Schaefer L.K. Ye X. Keyomarsi K. Chu C. Harper J.W. Elledge S.J. Science. 2001; 294: 173-177Crossref PubMed Scopus (659) Google Scholar, 21Strohmaier H. Spruck C.H. Kaiser P. Won K.A. Sangfelt O. Reed S.I. Nature. 2001; 413: 316-322Crossref PubMed Scopus (502) Google Scholar), c-Myc (22Yada M. Hatakeyama S. Kamura T. Nishiyama M. Tsunematsu R. Imaki H. Ishida N. Okumura F. Nakayama K. Nakayama K.I. EMBO J. 2004; 23: 2116-2125Crossref PubMed Scopus (612) Google Scholar, 23Welcker M. Orian A. Jin J. Grim J.E. Harper J.W. Eisenman R.N. Clurman B.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9085-9090Crossref PubMed Scopus (681) Google Scholar), Notch (24Oberg C. Li J. Pauley A. Wolf E. Gurney M. Lendahl U. J. Biol. 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Genes Dev. 2008; 22: 252-264Crossref PubMed Scopus (147) Google Scholar) and SRC-3 (30Wu R.C. Feng Q. Lonard D.M. O'Malley B.W. Cell. 2007; 129: 1125-1140Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) for degradation in a phosphorylation-dependent manner. Fbw7 interacts with nuclear SREBP1 and enhances its ubiquitination and degradation in a manner dependent on the phosphorylation of Thr-426 and Ser-430. The phosphorylation of these two residues is enhanced in response to DNA binding and GSK-3β is recruited to the promoters of SREBP target genes in vivo (12Punga T. Bengoechea-Alonso M.T. Ericsson J. J. Biol. Chem. 2006; 281: 25278-25286Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Consequently, Fbw7 is recruited to SREBP target promoters and induces the ubiquitination and degradation of SREBP1. In the current study, we identify a novel serine residue, Ser-434, in the C terminus of mature SREBP1 that is phosphorylated by GSK-3. Our results demonstrate that the phosphorylation of Ser-434 in SREBP1 is required for the GSK-3-dependent phosphorylation of Thr-426 and Ser-430 in its phosphodegron. Consequently, the phosphorylation of Ser-434 regulates the interaction between SREBP1 and Fbw7, thereby controlling the degradation of mature SREBP1. The phosphorylation of Ser-434 is also enhanced in response to DNA binding, and mutation of Ser-434 blocks the phosphorylation of Thr-426 and Ser-430 during this process, suggesting that the phosphorylation of Ser-434 could control the degradation of active SREBP1 molecules. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain, and the phosphorylation of Ser-434 could function as a molecular switch to control these processes. Cell Culture—All tissue culture media and antibiotics were obtained from Invitrogen and Sigma. HEK293, HEK293T, HepG2, U2OS, and HeLa cells were from ATCC. Fbw7-positive and Fbw7-negative HCT116 cells were provided by B. Vogelstein (31Rajagopalan H. Jallepalli P.V. Rago C. Velculescu V.E. Kinzler K.W. Vogelstein B. Lengauer C. Nature. 2004; 428: 77-81Crossref PubMed Scopus (479) Google Scholar). Reagents and Antibodies—Anti-FLAG antibody (M5), cycloheximide, and standard chemicals were from Sigma. Monoclonal anti-Myc (9E10), anti-SREBP1 (2A4), anti-tubulin (TU-02), anti-GST (B-14), rabbit anti-SREBP1 (H-160), and anti-phosphorylated (Ser-9/Ser-21) GSK-3 (sc-11757) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GSK-3α/β was from Cell Signaling, and recombinant GSK-3β was from New England Biolabs. Generation of Phosphorylation-specific SREBP1 Antibodies—Synthetic phosphopeptides corresponding to residues 431-437 (Ser-434 phosphorylated) in human SREBP1a were coupled to keyhole limpet hemocyanin before being injected into rabbits. The phosphopeptides and the corresponding non-phosphorylated peptides, as well as phospho-Ser, were coupled to Sulfolink (Pierce) and used as affinity matrices to purify the antibodies from rabbit sera (9Sundqvist A. Bengoechea-Alonso M.T. Ye X. Lukiyanchuk V. Jin J. Harper J.W. Ericsson J. Cell Metab. 2005; 1: 379-391Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 11Bengoechea-Alonso M.T. Ericsson J. Cell Cycle. 2006; 5: 1708-1718Crossref PubMed Scopus (53) Google Scholar). Plasmids and Transfections—The expression vectors for FLAG-SREBP1a and SREBP1c (amino acid residues 2-490 and 2-466, respectively) have been described (15Sundqvist A. Ericsson J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13833-13838Crossref PubMed Scopus (64) Google Scholar). Point mutants were generated by site-directed mutagenesis (QuikChange, Stratagene). The HMG-CoA synthase (SYNSRE-luc), LDL receptor (LDLr-luc), and fatty acid synthase (FAS-luc) promoter-reporter constructs have been described (32Ericsson J. Jackson S.M. Edwards P.A. J. Biol. Chem. 1996; 271: 24359-24364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). All other expression vectors have been described previously (20Koepp D.M. Schaefer L.K. Ye X. Keyomarsi K. Chu C. Harper J.W. Elledge S.J. Science. 2001; 294: 173-177Crossref PubMed Scopus (659) Google Scholar, 23Welcker M. Orian A. Jin J. Grim J.E. Harper J.W. Eisenman R.N. Clurman B.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9085-9090Crossref PubMed Scopus (681) Google Scholar). Transient transfections were performed using the MBS transfection kit (Stratagene). The Fbw7, GSK-3α/β, and control siRNA were from Ambion and have been described before (18Lipford J.R. Deshaies R.J. Nat. Cell Biol. 2003; 5: 845-850Crossref PubMed Scopus (156) Google Scholar, 35Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1774) Google Scholar). Immunoprecipitations and Immunoblotting—Cells were lysed in buffer A (50 mm HEPES (pH 7.2), 150 mm NaCl, 1 mm EDTA, 20 mm NaF, 2 mm sodium orthovanadate, 10 mm β-glycerophosphate, 1% (w/v) Triton X-100, 10% (w/v) glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 mm sodium butyrate, 1% aprotinin, 0.1% SDS, and 0.5% sodium deoxycholate) and cleared by centrifugation. For co-immunoprecipitations, cell lysates were prepared in the absence of SDS and sodium deoxycholate. Cell lysates and immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Millipore). To ensure that equal amounts of protein were loaded in each well, the levels of α-tubulin in the samples were estimated by Western blotting. Determination of Protein Half-life—Cells were treated with cycloheximide to stop protein synthesis and incubated for the indicated times. Total cell lysates were prepared, and the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. SREBP1 was visualized by Western blotting followed by quantitation on a charge-coupled device camera (Fuji) and image analysis software (Aida Image Analyzer 3.10). Luciferase and β-Galactosidase Assays—Cells were transiently transfected with the indicated promoter-reporter genes in the absence or presence of expression vectors for SREBP1, either wild-type or the indicated mutants. After 36 h, luciferase activities were determined in duplicate samples as described by the manufacturer (Promega, Madison, WI). The pCH110 vector encoding the β-galactosidase gene under the control of the SV40 promoter (Amersham Biosciences) was used as an internal control for transfection efficiency. Luciferase values (relative light units) were calculated by dividing the luciferase activity by the β-galactosidase activity. The data represent the average ± S.D. of three independent experiments performed in duplicates. Reverse Transcription-PCR Assays—RNA was extracted with TRIzol reagent (Invitrogen). Total RNA was subjected to reverse transcription with oligo(dT), followed by PCR with target-specific primers. The PCR reactions, using Invitrogen High Fidelity DNA polymerase, were optimized for the individual target genes. The PCR programs and primer sequences for the human LDL receptor, HMG-CoA synthase, fatty acid synthase, and glyceraldehyde-3-phosphate dehydrogenase genes are available on request. GSK-3 Phosphorylates Ser-434 in SREBP1—We recently identified two phosphorylated residues in the C terminus of mature SREBP1a, Thr-426 and Ser-430 (9Sundqvist A. Bengoechea-Alonso M.T. Ye X. Lukiyanchuk V. Jin J. Harper J.W. Ericsson J. Cell Metab. 2005; 1: 379-391Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). GSK-3 is involved in the phosphorylation of both Thr-426 and Ser-430. However, kinase assays with recombinant GSK-3β and SREBP1a followed by phosphopeptide mapping indicated that the C terminus of mature SREBP1a contained additional residues phosphorylated by GSK-3 (data not shown). The C-terminal domains of mature SREBP1a and SREBP1c are identical. Data base searches suggested that Ser-434 in SREBP1a (Ser-410 in SREBP1c) could be phosphorylated by GSK-3. To determine if SREBP1a was phosphorylated on Ser-434, we generated a phospho-Ser-434-specific anti-SREBP1 antibody (pS434). This antibody recognized wild-type SREBP1a following expression in HEK293T cells, whereas it failed to recognize the S434A mutant (Fig. 1A). Wild-type SREBP1c was also recognized by the antibody, whereas the S410A mutant was not (Fig. 1A), demonstrating that both isoforms of SREBP1 are phosphorylated on this serine residue. In addition, the recognition of wild-type SREBP1a was competed with a peptide containing phospho-Ser-434, but not with peptides in which Thr-426 or Ser-430 was phosphorylated (supplemental Fig. S1), supporting the specificity of the antibody. Our phosphopeptide mapping suggested that Ser-434 could be phosphorylated by GSK-3β in vitro. Indeed, in vitro kinase assays demonstrated that GSK-3β phosphorylates Ser-434 in SREBP1a (Fig. 1B). To test whether endogenous GSK-3 could phosphorylate Ser-434 in endogenous SREBP1, HeLa cells were treated with lithium, a pharmacologic GSK-3 inhibitor. The pS434 antibody only recognized the mature form of SREBP1 and not the membrane-associated precursor, suggesting that phosphorylation of Ser-434 is specific for the nuclear form of the protein. Inhibition of GSK-3 reduced the phosphorylation of Ser-434 (Fig. 1C), indicating that this residue is phosphorylated by GSK-3 in vivo. Lithium also reduced the phosphorylation of Ser-434 in HepG2 (Fig. 1D). Similar results were obtained with the GSK-3 inhibitor SB 415286 (Fig. 1E). The reduction in Ser-434 phosphorylation was specific for GSK-3 inhibitors, because inhibitors of mitogen-activated protein kinase (UO126) and cyclin-dependent kinases (roscovitine) failed to affect the phosphorylation of this residue in HeLa cells (Fig. 1F). Interestingly, prolonged treatment of HeLa cells with lithium and SB 415286 increased the steady-state levels of mature SREBP1 (Fig. 2A), supporting the notion that GSK-3-dependent phosphorylation of mature SREBP1 promotes its degradation (9Sundqvist A. Bengoechea-Alonso M.T. Ye X. Lukiyanchuk V. Jin J. Harper J.W. Ericsson J. Cell Metab. 2005; 1: 379-391Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). In support of this possibility, we found that nuclear SREBP1 phosphorylated on Ser-434 accumulated in HeLa cells treated with the proteasome inhibitor MG-132 (Fig. 2B), indicating that nuclear SREBP1 phosphorylated on this residue is rapidly turned over by proteasome-mediated degradation. Similar results were also obtained in HepG2 cells (supplemental Fig. S2). Furthermore, siRNA-mediated inactivation of GSK-3 reduced the phosphorylation of Ser-434 in endogenous SREBP1 in HeLa cells (Fig. 2C), as well as HepG2 and U2OS cells (supplemental Fig. S2), confirming that GSK-3 contributes to the phosphorylation of Ser-434 in SREBP1. siRNA-mediated inactivation of both GSK-3α and -β in HeLa cells resulted in a small reduction of the levels of mature SREBP1 (Fig. 2C). This could be explained, at least in part, by a significant reduction in the expression of the precursor form of SREBP1 in response to prolonged inactivation of GSK-3 (data not shown). Growth factors, including insulin, negatively regulate the activity of GSK-3 through Akt-mediated phosphorylation. Thus, we speculated that insulin-dependent inhibition of GSK-3 could attenuate the phosphorylation of Ser-434 in SREBP1. As illustrated in Fig. 2D, a short treatment of HepG2 cells with insulin enhanced the inhibitory phosphorylation of GSK-3. Consequently, the phosphorylation of SREBP1 on Ser-434 was reduced in response to insulin treatment, resulting in the accumulation of nuclear SREBP1. These data indicate that the phosphorylation of Ser-434 in SREBP1 requires endogenous GSK-3 activity and that these processes are regulated by insulin signaling. Phosphorylation of Ser-434 Destabilizes SREBP1—We have previously demonstrated that the mature form of SREBP1 is destabilized following GSK-3-dependent phosphorylation of Thr-426 and Ser-430 (9Sundqvist A. Bengoechea-Alonso M.T. Ye X. Lukiyanchuk V. Jin J. Harper J.W. Ericsson J. Cell Metab. 2005; 1: 379-391Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). To determine if phosphorylation of Ser-434 could be involved in regulating the stability of SREBP1, cells were transfected with mature SREBP1a, either wild-type or the phosphorylation-deficient S434A mutant. The steady-state levels of the S434A mutant were enhanced compared with the wild-type protein (Fig. 3A), indicating that phosphorylation of Ser-434 destabilizes SREBP1. Similar results were obtained when the corresponding residue (Ser-410) was mutated in SREBP1c (Fig. 3A), suggesting that phosphorylation of this residue regulates the stability of both SREBP1 isoforms. The S434A mutant of SREBP1a was also stabilized in HEK293, HeLa, and HepG2 cells (supplemental Fig. S3). Interestingly, the steady-state levels of a phosphorylation-mimetic mutant of SREBP1a (S434D) were slightly reduced compared with the wild-type protein (Fig. 3B), further supporting the notion that phosphorylation of Ser-434 destabilizes SREBP1. The mRNA levels of the constructs in Fig. 3 (A and B) were similar (supplemental Fig. S4), suggesting that the expression of these mutants was affected at the protein level. To confirm that the enhanced abundance of the S434A mutant resulted from reduced degradation, we measured the half-life of mature SREBP1a, either wild-type or the S434A mutant, in transfected HEK293T cells. As illustrated in Fig. 3C, the turnover of the mutant protein was reduced compared with wild-type SREBP1a. The accumulation of the S434A mutant should lead to an enhanced expression of SREBP target genes. To test this hypothesis, HepG2 cells were transfected with an SREBP-responsive promoter-reporter gene in the absence or presence of mature SREBP1a, either wild-type, S434A, or S434D. In support of our hypothesis, the transcriptional activity of the S434A mutant was enhanced, whereas the activity of the S434D mutant was reduced compared with the wild-type protein (Fig. 3D). Mutation of the corresponding residue in SREBP1c (S410) also enhanced its transcriptional activity (Fig. 3D), indicating that the phosphorylation of Ser-434 and its effect on the stability of SREBP1 influences the biological function of SREBP1. This possibility was supported when we analyzed the expression of endogenous target genes in HEK293 cells transfected with mature SREBP1a. The expression of both the LDL receptor and HMG-CoA synthase genes was higher in cells expressing the S434A mutant compared with cells expressing wild-type SREBP1a (Fig. 3E). If our hypothesis was correct, activation of GSK-3 should induce the degradation of mature SREBP1. To test this, cells were treated with wortmannin and LY294002, two phosphatidylinositol 3-kinase (PI3K) inhibitors. Both inhibitors reduced the inhibitory phosphorylation of GSK-3 and resulted in reduced steady-state levels of mature SREBP1 (Fig. 4A). This effect was specific for the PI3K inhibitors, because the mTor inhibitor rapamycin failed to affect SREBP1 levels. Interestingly, both PI3K inhibitors failed to affect the levels of mature SREBP1 in the presence the proteasome inhibitor MG-132 (Fig. 4B), suggesting that activation of GSK-3 results in enhanced degradation of mature SREBP1. To test this hypothesis, HepG2 cells were transfected with mature SREBP1a, either wild-type, S434A, or the double mutant T426A/S430A and treated with LY294002. As seen in Fig. 4C, LY294002 reduced the levels of wild-type SREBP1a, whereas the degradation of the S434A mutant was attenuated and the double mutant was resistant to LY294002 treatment, suggesting that phosphorylation of Ser-434 and the phosphodegron in SREBP1 is important for this effect. As expected, inhibition of PI3K in HepG2 cells attenuated the accumulation of mature SREBP1 in response to a short pulse of insulin (Fig. 4D). Again, this effect was specific for PI3K, because rapamycin only had a marginal effect, suggesting that PI3K signaling, but not mTor, is important for the acute response to insulin signaling in these cells. Ser-434 Regulates the Phosphorylation of Thr-426 and Ser-430—Our data indicate that phosphorylation of Ser-434 promotes the degradation of SREBP1. The active form of SREBP1 is targeted for degradation following phosphorylation of Thr-426 and Ser-430. One possibility was therefore that the phosphorylation of Ser-434 could influence the subsequent phosphorylation of Thr-426 and Ser-430. To test this possibility, HEK293T cells were transfected with mature SREBP1a, either wild-type or the T426A, S430A, or S434A mutants, and the phosphorylation of Thr-426, Ser-430, and Ser-434 was determined following immunoprecipitation of the various proteins. As illustrated in Fig. 5A, all three residues were phosphorylated when wild-type SREBP1a was expressed in cells. Mutation of Thr-426 did not only block the phosphorylation of this residue but also significantly reduced the phosphorylation of Ser-430, whereas the phosphorylation of Ser-434 was only somewhat reduced in the T426A mutant. Mutation of Ser-430 attenuated the phosphorylation of Thr-426 and Ser-434. Importantly, mutation of Ser-434 drastically reduced the phosphorylation of Ser-430 and also resulted in a significant attenuation of the phosphorylation of Thr-426. Thus, both Thr-426 and Ser-434 regulate the phosphorylation of Ser-430, and Ser-434 regulates the phosphorylation of both Thr-426 and Ser-430. Therefore, phosphorylation of Ser-434 could potentially regulate the phosphorylation of the phosphodegron in SREBP1, thereby affecting the stability of mature SREBP1. This possibility is in agreement with our observation that the S434A mutant of SREBP1 is stabilized (Fig. 3, A-C). The results in Fig. 5A suggest that Ser-434 regulates the GSK-3-dependent phosphorylation of Thr-426 and Ser-430. To test this possibility, we used recombinant SREBP1, either wild-type or the S434A mutant, in kinase assays with recombinant GSK-3β. As seen in Fig. 5B, the GSK-3β-dependent phosphorylation of Thr-426 was significantly reduced, and the phosphorylation of Ser-430 completely lost in the S434A mutant, confirming that Ser-434 plays an important role in the phosphorylation of both these residues. Interestingly, mutation of Ser-430 reduced the GSK-3-dependent phosphorylation of Thr-426 (Fig. 5C), suggesting that Ser-430 is a priming site for the phosphorylation of Thr-426. SREBP1 has a serine residue (Ser-438 in SREBP1a) four residues downstream of Ser-434 that could function as a priming site for the phosphorylation of Ser-434. However, mutation of this residue (S438A) failed to affect the phosphorylation of Ser-434, Ser-430, or Thr-426 and failed to affect the steady-state levels of transfected mature SREBP1a (supplemental Fig. S5). Taken together, our results suggest that the GSK-3-dependent phosphorylation of Thr-426 is dependent on the phosphorylation of Ser-430, which in turn is dependent on the phosphorylation of Ser-434. We recently found that GSK-3β is recruited to SREBP target genes and that the GSK-3β-mediated phosphorylation of Thr-426 and Ser-430 in SREBP1 is enhanced in response to DNA binding (12Punga T. Bengoechea-Alonso M.T. Ericsson J. J. Biol. Chem. 2006; 281: 25278-25286Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). To determine if the phosphorylation of Ser-434 is enhanced in response to DNA binding, we performed in vitro kinase assays using recombinant mature SREBP1a and HeLa nuclear