MINIREVIEW SREBPs: protein interaction and SREBPs Ryuichiro Sato Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan Introduction The sterol regulatory element-binding protein (SREBP) family members SREBP-1 and SREBP-2 are localized on the endoplasmic reticulum (ER) as membrane proteins after being synthesized. Once the intracellular cholesterol level is decreased, the SREBPs subsequently move in vesicles to the Golgi complex, where they are processed sequentially by two proteases. These cleavage steps release the mature forms of SREBPs, which enter the nucleus and activate genes related to cholesterol and fatty acid metabolism [1,2]. In both the cytoplasm and nucleus, SREBPs associate with a variety of proteins. This interaction determines their intracellular translo- cation and stability, and also regulates their activities as transcriptional factors. Protein interaction on the ER and in the cytosol SREBPs are localized on the ER membrane, associat- ing with another ER membrane protein, SREBP cleav- age-activating protein (SCAP) (Fig. 1). SCAP has two distinct domains. The N-terminal domain has eight transmembrane helices, which include the so-called ste- rol-sensing domain. This domain resembles sequences in three other proteins that are postulated to interact with sterols: HMG-CoA reductase, the Niemann–Pick C1 protein, and Patched [3]. The C-terminal domain of SCAP contains five WD repeats, which are sequences of  40 amino acids found in many proteins involved in protein–protein interactions. The WD repeat domain is the region of SCAP that forms a complex with the C-terminal domain of SREBPs [4]. When cells Keywords ATF6; HNF-4; importin; LRH-1; PGC-1; S1P; S2P; SCAP; SREBPs Correspondence R. Sato, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan Fax: +81 3 5841 5136 Tel: +81 3 5841 8029 E-mail: aroysato@mail.ecc.u-tokyo.ac.jp (Received 6 August 2008, revised 22 October 2008, accepted 24 October 2008) doi:10.1111/j.1742-4658.2008.06807.x Sterol regulatory element-binding proteins (SREBPs) are tightly controlled by various mechanisms, including intracellular localization, protein process- ing, limited proteolysis, post-translational modifications and interaction with associated proteins. Here, I review the regulatory mechanisms of SREBP activity through the interaction with various kinds of protein. Abbreviations AFF6, activating transcription factor-6; ARC, activator-recruited co-factor; CBP, CREB-binding protein; ER, endoplasmic reticulum; HNF-4, hepatocyte nuclear factor-4; LDL, low-density lipoprotein; LRH-1, liver receptor homolog-1; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1, peroxisome proliferator-activated receptor-c coactivator-1; S1P, site 1 protease; S2P, site 2 protease; SCAP, SREBP cleavage- activating protein; SREBP, sterol regulatory element-binding protein; SUMO, small ubiquitin-like modifier. 622 FEBS Journal 276 (2009) 622–627 ª 2008 The Author Journal compilation ª 2008 FEBS become depleted in cholesterol, SCAP escorts SREBPs from the ER to the Golgi apparatus, where two proteases, designated site 1 protease (S1P) and site 2 protease (S2P), reside. In the Golgi apparatus, S1P, a membrane-bound serine protease, cleaves SREBPs in the luminal loop between their two membrane-span- ning segments. The N-terminal domain is then released from the membrane by S2P, a membrane-bound zinc metalloproteinase. In the cytoplasm, the N-terminal cleaved forms of SREBPs interact with importin-b,an escort protein of nuclear proteins, and thereafter are transported into the nucleus [5]. It is a quite character- istic transport pathway, in that the nuclear import occurs in the absence of importin-a. Furthermore, the dimerization of SREBPs via the leucine zipper domain is required for the interaction with importin-b [6]. In the nucleus, SREBPs detach from importin-b, and their transcription factor activities are regulated through interaction with a variety of nuclear proteins. Interaction with the ubiquitous transcription factors Sp1 and NF-Y in the nucleus SREBPs were first discovered as transcription factors that stimulate low-density lipoprotein (LDL) receptor gene expression [7]. In the promoter of the LDL recep- tor gene, a pair of essential elements exists to which a ubiquitous transcription factor, Sp1, binds. An SREBP-binding site, SRE, is closely located between these two Sp1-binding sites, and all of these sites are required for full activation of the LDL receptor pro- moter. Similarly, a number of promoters of the SREBP target genes contain the SREs and certain proximal elements to which another ubiquitous tran- scription factor, NF-Y, binds [8–10]. SREBPs physi- cally interact with both Sp1 and NF-Y, thereby synergistically augmenting the transcription of the target genes [11,12]. Coactivators directly bind to SREBPs, thereby inducing the transcription of target genes Transcription factors, which bind to specific nucleotide sequences in the promoter region, require coactivators that facilitate access of the transcriptional machinery to a nucleosomal template. One of the well-known coactivators, CREB-binding protein (CBP), interacts with the N-terminal region of SREBP-1 [13], thereby supporting high levels of synergistic activation by SREBPs. CBP possesses acetyltransferase activity, and therefore is thought to be involved in the acetylation of histones and alteration of chromatin structure. A recent study revealed that the activator-recruited co-factor (ARC)–mediator coactivator complex, a large complex associating with RNA polymerase II, interacts with SREBPs through structurally related motifs in both CBP and the ARC105 subunit of the ARC–mediator coactivator complex [14]. How SREBPs recruit both of these coactivators on target genes remains unclear. The peroxisome proliferator-activated receptor-c coactivator-1 (PGC-1) family of coactivators is of particular importance in the control of liver metabo- lism. PGC-1a stimulates mitochondrial biogenesis and Fig. 1. SREBPs interact with SCAP or importin-b in the cytoplasm. SREBPs local- ized on the ER membrane associate with another ER membrane protein, SCAP. This complex is transported to the Golgi appara- tus, where SREBPs are processed sequen- tially by two proteases, S1P and S2P. The cleaved forms of SREBPs, as homodimers, interact with importin-b, which escorts them to the nucleus. R. Sato Protein interaction and SREBPs FEBS Journal 276 (2009) 622–627 ª 2008 The Author Journal compilation ª 2008 FEBS 623 respiration, and modulates hepatic gluconeogenesis. In contrast, PGC-1b, a transcriptional coactivator closely related to PGC-1a, is highly induced in response to short-term high-fat feeding in mice. PGC-1b interacts with SREBPs, thereby inducing a broad program of lipid metabolism, including de novo lipogenesis and lipoprotein secretion [15]. This suggests, at least in part, a mechanism through which dietary saturated fats stimulate hyperlipidemia and atherogenesis. SREBPs dock on PGC-1b at a domain that has no counterpart in PGC-1a, and hence, PGC-1a does not coactivate the SREBPs. Protein modification of SREBPs regulates their activities In the nucleus, SREBPs are unstable and rapidly degraded by the ubiquitin–proteasome pathway [16]. The rapid turnover of nuclear SREBPs is not affected by the intracellular sterol levels, and the half-life is estimated to be  3 h. In the presence of proteasome inhibitors, nuclear SREBPs become stable and enhance the transcription of endogenous target genes. Polyubiq- uitination is the rate-limiting step in protein degrada- tion, and involves a three-step cascade of ubiquitin transfer reactions. The ubiquitin ligase (E3) required for the final reaction of SREBP ubiquitination is a complex consisting of Skp1, Cul1, Rbx1, and one of a family of F-box proteins, Fbw7 [17]. Glycogen syn- thase kinase-3b phosphorylates serine and ⁄ or threonine residues near the ubiquitination site in SREBP-1 and SREBP-2, and SREBP ubiquitination is enhanced in a manner dependent on the phosphorylation. SREBPs do interact with the Fbw7 protein when the phosphor- ylation is enhanced by glycogen synthase kinase-3b. SREBPs are modified by another protein, small ubiquitin-like modifier (SUMO). SUMO-1 is a 101 amino acid protein having 18% identity with ubiquitin, but with a remarkably similar secondary structure. With the increase in the number of proteins modified by SUMO, it has become obvious that the effects of SUMO conjugation are diverse and largely depend on the function of the protein targeted for sumoylation. Sumoylation of transcription factors, including SREBPs, is prone to result in attenuation of their tran- scriptional activities [18–20]. Sumoylation requires a multistep reaction similar to that of ubiquitination, but the specific enzymes are distinct from those involved in ubiquitination. Ubc9 is a SUMO-conjugating enzyme (E2) that directly interacts with most sumoylated pro- teins, including SREBPs [20]. In some cases, ubc9 itself plays, to a certain extent, a SUMO E3-like role in the absence of any E3 ligases. Unlike ubiquitination, which requires phosphorylation near the ubiquitination site, sumoylation competes with the phosphorylation near the sumoylation site, which occurs in response to growth factor stimuli [21]. This implies that growth factor stimuli interfere with sumoylation, thereby enhancing SREBP transcriptional activities, and lipid synthesis required for cell growth. Sumoylated SREBPs recruit a corepressor complex containing his- tone deacetylase 3 to suppress their transcriptional activities [21]. Histone deacetylase 3 is unable to directly interact with SREBPs, but a certain subunit in the corepressor complex, which is not yet identified, is considered to be involved in the interaction. SREBPs interact with activating transcription factor-6 (ATF6) and nuclear receptors to regulate their transcriptional activities ATF6 is an ER membrane-bound transcription factor activated in response to ER stress. During the quies- cent state, the C-terminus of ATF6 resides in the ER lumen, with its N-terminus projecting into the cytosol. Once unfolded or misfolded proteins accumulate in the ER, ATF6 moves from the ER to the Golgi, where both ATF6 and SREBPs are cleaved by S1P and S2P. Such proteolytic cleavage causes the nuclear localiza- tion of the N-terminal leucine zipper transcription factor to direct the transcriptional activation of the chaperone molecules and enzymes essential for protein folding [22,23]. Overexpression of the cleaved form of ATF6, and also glucose depletion, which causes ATF6 cleavage, suppresses the transcription of SREBP target genes, suggesting that the interaction between ATF6 and SREBPs inhibits SREBP-mediated transcription. Indeed, ATF6 interacts with SREBP-2 via its leucine zipper domain, thereby reducing the transcriptional activity of SREBP through the recruitment of histone deacetylase 1 [24]. In the liver, several types of nuclear receptor orches- trate glucose and lipid metabolism. Among them, hepatocyte nuclear factor-4 (HNF-4), which was ini- tially identified as a transcription factor essential for liver-specific gene expression, activates the expression of the gluconeogenic genes encoding phosphoenolpyr- uvate carboxykinase (PEPCK) and glucose-6-phospha- tase. At the same time, HNF-4 also regulates the expression of certain crucial genes for lipid metabo- lism, including the genes encoding apolipoprotein B and microsome triglyceride transfer protein [25]. Over- expression of SREBP-1 severely reduces the hepatic expression level of PEPCK in SREBP-1a transgenic and SREBP-1c adenovirus-infected mice. SREBP-1 Protein interaction and SREBPs R. Sato 624 FEBS Journal 276 (2009) 622–627 ª 2008 The Author Journal compilation ª 2008 FEBS does not directly bind to the PEPCK promoter, but the HNF-4-binding site is responsible for the SREBP-1 inhibition. SREBPs and HNF-4 physically interact through the N-terminal transactivation domain of SREBP and the C-terminal ligand-binding domain of HNF-4 [26]. HNF-4 recruits a coactivator, PGC-1a, for its transcriptional activation. SREBPs interfere with this recruitment of PGC-1a. Under fasting condi- tions, HNF-4 and PGC-1a vigorously activate the expression of gluconeogenic genes. In contrast, under feeding conditions, SREBP-1c, the expression of which is highly enhanced by insulin, might negatively regulate HNF-4 transcriptional activity by competing with PGC-1a, leading to a reduction of gluconeogenesis. In contrast to the above findings, the transcriptional activity of SREBPs is augmented by HNF-4 [27]. Overexpression of HNF-4 enhances the expression of SREBP target genes in culture cells, but not through the direct binding of HNF-4 to the promoters. HNF-4 interaction with SREBPs probably augments their transcriptional activities due to HNF-4-mediated recruitment of several coactivators, which are not recruited by SREBPs alone, including PGC-1a. In the liver and intestine, where lipid biosynthesis is quite active and HNF-4 is exclusively expressed, the syner- gistic activity of SREBPs and HNF-4 might cause lipids to be distributed to other tissues that do not have the capacity to biosynthesize sufficient lipids on their own. In a study aimed at identifing other nuclear receptor family members affecting SREBP transcriptional activ- ities, liver receptor homolog-1 (LRH-1) was found to suppress them [28] (Fig. 2). Unlike other nuclear recep- tor family members, LRH-1 acts as a monomer transcription factor to regulate the expression of genes related to cholesterol metabolism, including the genes encoding CYP7A1, a rate-limiting enzyme for bile acid synthesis, and apolipoprotein A-1, a high-density lipo- protein protein. The basic helix–loop-helix leucine zipper domain in SREBPs binds to the ligand-binding domain in LRH-1, thereby reciprocally suppressing their transcriptional activities. SREBPs interfere with the recruitment of a coactivator of LRH-1, PGC-1a, resulting in the inhibition of LRH-1 activity. When human hepatoma HepG2 cells are cultured with an HMG-CoA reductase inhibitor, statin, the reduction of intracellular cholesterol levels activates SREBPs, and eventually suppresses the expression of LRH-1 target genes, including the genes encoding CYP7A1, apolipo- protein A-I, and the small heterodimer partner. Although most nuclear receptors are activated only when their specific ligands are present, HNF-4 and LRH-1 appear to be exceptional, in that they are con- stitutively active in the abundance of their endogenous ligands, acyl-CoA and phospholipids, respectively [29,30]. The small heterodimer partner, one of the nuclear receptor family members, acts as a negative regulator of both HNF-4 and LRH-1 by suppressing their transcriptional activities. SREBPs might consti- tute another group of inhibitory nuclear factors modu- lating the activity of these nuclear receptors in response to a wide variety of physiological changes. Conclusions SREBPs are translocated from the ER to the Golgi complex, where they are processed, and then trans- ported into the nucleus. In this pathway, two interact- Fig. 2. HNF-4 stimulates and LRH-1 suppresses the transcriptional activities of SREBP-1a and SREBP-2. HEK293 cells were transfected with either 0.1 lg of pGAL4–SREBP1a (Gal4–DBD–SREBP1a) or pGAL4–SREBP2 (Gal4–DBD–SREBP2), 0.2 lg of pG5Luc containing five copies of the Gal4-binding sites, and 10 ng of phRL-TK, together with increasing amounts of an expression vector for HNF-4a or LRH-1 (0.2 and 0.6 lg); they were then cultured in a medium containing 10% fetal bovine serum for 48 h. Luciferase assays were performed. The promoter activities in the absence of pGAL4–SREBP1a or pGAL4–SREBP2 are represented as 1. All data are presented as means ± SD. R. Sato Protein interaction and SREBPs FEBS Journal 276 (2009) 622–627 ª 2008 The Author Journal compilation ª 2008 FEBS 625 ing proteins, SCAP and importin-b, play an important role in determining the fate of SREBPs. In the nucleus, multiple nuclear proteins form a complex with SREBPs on their target gene promoters to regulate the transcriptional activity. In addition, SREBPs interact with ubiquitin- or SUMO-transfer enzymes, thereafter being rapidly degraded or inactivated, respectively. Some nuclear receptors and transcription factors also associate with SREBPs in the nucleus. This association exerts considerable physiological influence on the expression of their target genes. Further studies will be required to elucidate the more complex network among the numerous transcription factors that regu- late lipid and energy metabolism. Acknowledgements The author is grateful to K. Boru of Pacific Edit for reviewing the manuscript. 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Sato Protein interaction and SREBPs FEBS Journal 276 (2009) 622–627 ª 2008 The Author Journal compilation ª 2008 FEBS 625 ing proteins, SCAP and importin-b,