Posttranslational Modifications of PPAR‐γ: Fine‐tuning the Metabolic Master Regulator

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors, which comprise a subgroup of three closely homologous genes, PPAR-α (NR1C1), PPAR-β/δ (NR1C2), and PPAR-γ (NR1C3) (refs. 1,2). All PPARs bind to PPAR responsive elements in the promoter regions of target genes as obligate heterodimers with retinoic acid X receptors. Similar to other nuclear receptors, PPARs consist of distinct functional domains including an N-terminal transactivation domain (AF1), a highly conserved DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD) that contains a ligand-dependent transactivation function (AF2; Figure 1a). Ligand binding stabilizes the active conformation of the LBD, thereby serving as a “molecular switch” between activation and repression functions of the receptor (3). On the promoters of some target genes, unliganded PPARs recruit corepressors such as N-CoR and SMRT, which are part of multiprotein complexes containing histone deacetylase activity that repress gene transcription (4). Upon ligand binding, these corepressor complexes are exchanged for activating co-activator complexes, including the SRC1/CBP and TRAP/DRIP/ARC complexes (5). Besides ligand-mediated regulation, the transcriptional activity of nuclear receptors can also be regulated via the ligand-independent AF1 domain. In common with other NRs (6), the AF1 domains of the different PPAR isotypes probably lack a stable secondary structure in aqueous solution, only to adopt a stable conformation upon interaction with other proteins. Posttranslational modifications of PPAR-γ. (a) Schematic representation of the PPAR-γ2 protein. Indicated are the N-terminal activation function 1 (AF1), the DNA-binding domain (DBD), and the C-terminal activation function 2 (AF2), as well as the unique phoshorylation site at position serine 112 and the SUMO sites at position lysine 107 and lysine 395. (b) The sumoyl-phospho switch motif or phosphorylation-dependent sumoylation motif in PPAR-γ1/2 is conserved between species ranging from fish to man. The nuclear receptors ERRα, ERRβ, and ERRγ harbor a similar motif. (c) Phosphorylation of S112 of PPAR-γ by ERK1/2 kinases upon growth factor or prostaglandin PGF2α treatment, or by p38/JNKs after UV or stress results in the inhibition of the adipogenic gene program. The adapter protein Dok1 can modulate MAPK activation. (d) Phosphorylation of S112 of PPAR-γ by the kinases cdk7 (as part of the TFIIH complex) or cdk9 (as part of the P-TEFb complex) results in the stimulation of the adipogenic gene program. ERK, extracellular signal-regulated kinase; ERR, estrogen-related receptor; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PPAR, peroxisome proliferator-activated receptor; P-TEFb, positive transcription elongation factor b; SUMO, small ubiquitin-like modifier; TFIIH, general transcription factor II H. Although all PPAR nuclear receptors play a part in lipid homeostasis and energy metabolism, the different PPAR proteins exhibit different physiological roles due to (i) distinct expression patterns, (ii) specific activation by different ligands, and (iii) intrinsic functional differences between the different isotype proteins. Alternative splicing and differential promoter usage results in two PPAR-γ isoforms, PPAR-γ1 and PPAR-γ2, with the latter harboring a 30-residue extension at its N-terminus (1,2). PPAR-γ1 is expressed in several tissues, including the lower intestine, macrophages, and white adipose tissue (WAT), whereas PPAR-γ2 expression is almost exclusively restricted to WAT. The endogenous ligands for PPAR-γ are not firmly established, although some natural compounds, such as polyunsaturated fatty acids, prostaglandin J2 derivatives (15-deoxy-δ12,14-PGJ2), and nitro-oleic acid, have been shown to be able to activate PPAR-γ (7,8). Synthetic PPAR-γ agonists include the thiazolidinediones (TZDs), which ameliorate insulin resistance and lower blood glucose levels in patients with type 2 diabetes (9). A huge amount of data indicates that PPAR-γ is one of the key players in the differentiation of fibroblast-like mesenchymal stem cells into adipocytes, a process known as adipogenesis. For example, PPAR-γ+/– mice lack most adipose tissue (10,11,12), whereas in vitro differentiation of fibroblasts into mature adipocytes can be induced by the introduction of PPAR-γ (13). In addition, PPAR-γ is also essential for the maintenance of adipose tissue, because conditional knock-out of the Pparg gene resulted in reduced in vivo survival of mature adipocytes (14). Besides its role in adipocyte differentiation and maintenance, PPAR-γ directly regulates the expression of a number of genes involved in net lipid partitioning into mature adipocytes, underscoring the importance of PPAR-γ in glucose and lipid homeostasis. Compelling genetic evidence for this view comes from human familial partial lipodystrophy subtype 3 (MIM 604367) patients, harboring heterozygous mutations in the PPARG gene, as they are characterized by aberrant fat distribution and metabolic disturbances, including insulin resistance and dyslipidemia (15). Interestingly, PPAR-γ was recently shown to be involved in the inhibition of osteoblast differentiation and osteoclastogenesis (16), indicating that novel biological functions of this transcription factor remain to be identified beyond its role in lipid and glucose homeostasis. Although the three PPAR isotypes display a high degree in primary amino acid sequence homology in their LBD and DBD, both the N-terminal AF1 region and the hinge region are less well conserved between isotypes. The idea that these regions are therefore likely to contribute to the isotype-specific responses was recently supported experimentally. Using chimeric PPAR-γ-PPAR-β/δ proteins, the AF1 region of PPAR-γ was shown to be essential for adipogenesis (17,18). Furthermore, gene expression profiling revealed that the AF1 regions of the different PPAR family members are the main determinants of isotype-selective gene expression (18). It seems plausible that, in analogy with the AF2 domain, the activity of the AF1 region is dictated by the set of proteins with which this domain interacts. Only two AF1-interacting proteins have been identified so far which display isotype-specific interactions: PPAR-γ co-activator 2/SCAN domain protein 1 (PGC-2/SDP1), a PPAR-γ-specific co-activator, which promotes adipogenesis (17), and Tip60, an adipogenic acetyltransferase, which stimulates the activity of PPAR-γ but not PPAR-α or PPAR-β/δ ((19) and O. van Beekum and E. Kalkhoven, unpublished data). Interestingly, the AF1 region of PPAR-γ is also subject to various posttranslational modifications (PTMs), and it seems likely that such PTMs regulate interactions with coregulators, and/or vice versa, ultimately controlling the transcriptional output of this nuclear receptor. In this review, we will therefore summarize the various PTMs reported for PPAR-γ, evaluate the (possible) effects on coregulator interactions, and discuss the (potential) relevance for in vivo PPAR-γ signaling. As the first PTM reported for PPAR-γ2, phosphorylation has been studied extensively over the years. Phosphorylation of PPAR-γ2 was mapped to serine 112 (S112; S82 in PPAR-γ1), a conserved mitogen-activated protein kinase (MAPK) consensus site within the AF1 region, which is the only phosphorylation site identified in PPAR-γ so far (Figure 1b). Activation of the MAPKs extracellular signal-regulated kinase 1/2 by growth factors (epidermal growth factor, platelet-derived growth factor, transforming growth factor-β, insulin) or the prostaglandin PGF2α was indeed found to result in increased phosphorylation of S112 (refs. 20,21,22,23,24,25). In addition, the activation of MAPKs, c-Jun N-terminal kinase 1/2 and p38, by stress (UV, anisomysin) also resulted in hyperphosphorylation of S112 (refs. 25,26; Figure 1c). Phosphorylation of PPAR-γ by these treatments resulted in decreased transcriptional activity in reporter assays, whereas the mutation of the phosphorylation site by changing S112 into alanine led to increased transcriptional activity (20,22,25,26,27). Furthermore, several laboratories have shown that the overexpression of PPAR-γ S112A in mouse fibroblasts resulted in increased adipogenesis compared with cells overexpressing wild-type PPAR-γ (20,23,25,28,29,30). Growth factor- or stress-induced phosphorylation of S112 of PPAR-γ, therefore, in general, represses its transcriptional and adipogenic functions. Treatment of cells with insulin, however, has been associated with reduced (20) or increased PPAR-γ activity (24). This discrepancy may be explained by the modulation of the signaling cascade through adapter molecules such as Dok1 (Figure 1c; (31)). Although Ras/MAPK activation by insulin normally represses PPAR-γ activity (20), high levels of Dok1 in adipose tissue, as induced by excessive caloric intake, were found to counteract this repression (31). Phosphorylation of PPAR-γ represses its activity probably through multiple molecular mechanisms. Adams et al. showed that the phosphorylation affected both ligand-independent and ligand-dependent transcription, based on reporter assays using full-length PPAR-γ proteins (25). In addition, their experiments with fusions of the AF1 region to a heterologous DBD (Gal4DBD) also revealed reduced transcriptional activity of the S112A mutant, suggesting either the recruitment of a repressor protein to the AF1 region or the release of an activator in a phosphorylation-dependent manner (25). Subsequent studies showed that PPAR-γ phosphorylation affected ligand binding, and hence reduced transcriptional activity, indicative for AF1-LBD interdomain communication (28). Finally, phosphorylation of S112 has been associated with a repressive PTM named sumoylation, as will be discussed later. In contrast to growth factor- or stress-induced phosphorylation of S112, which results in reduced transcriptional activity, modification of the same residue by the cyclin-dependent kinases, cdk7 and cdk9, was recently found to increase PPAR-γ activity (Figure 1d; (32,33)). The cdk7 kinase is a subunit of the basal transcription factor general transcription factor II H complex, which plays important roles in DNA repair and transcription (34). Disruption of the general transcription factor II H complex, for example, by mutations in the xeroderma pigmentosum complementation group D (XPD) subunit as observed in xeroderma pigmentosum patients, results in predisposition for carcinogenesis caused by increased photosensitivity. PPAR-γ is a bona fide substrate for the cdk7 kinase, because (i) cdk9 could directly phosphorylate PPAR-γ on S112 in vitro; (ii) PPAR-γ was found to be hypophosphorylated in XPD-deficient mice, which lack a functional cdk7-containing general transcription factor II H complex; and (iii) the reintroduction of XPD proteins into these cells restored PPAR-γ phosphorylation levels. However, in contrast to previous studies showing that the phosphorylation of S112 was inhibitory (20,22,25,26,27), cdk7-mediated phosphorylation stimulated PPAR-γ transcriptional activity, as PPAR-γ target gene expression levels were lower in XPD-deficient mice when compared with wild-type mice (32). It should be noted that this effect was specific for brown adipose tissue (BAT), because XPD-deficient mice displayed increased PPAR-γ target gene expression in WAT, especially in the absence of exogenously added ligand (32). Together with the hypoplasia of adipose tissue observed in XPD patients and XPD-deficient mice, these findings suggest that the phosphorylation of PPAR-γ may stimulate its transcriptional activity in certain tissues (e.g., BAT) or under certain conditions. In agreement with this hypothesis, Iankova et al. recently reported that cdk9, which together with cyclin T1/2 forms the positive transcription elongation factor b complex (35), can phosphorylate S112 of PPAR-γ (Figure 1d; (33)). The p55 isoform of cdk9, which is strongly upregulated during adipogenesis, was found to interact with the AF1 region of PPAR-γ and phosphorylate the protein on S112 in vitro (33). Furthermore, the overexpression of cdk9 enhanced PPAR-γ-mediated transcription and stimulated adipocyte differentiation (33). Taken together, these findings indicate that the net result of PPAR-γ phosphorylation may be either inhibition or stimulation of transcriptional activity, depending on the cellular background and kinases involved (Figure 1c,d). Recently, several research groups have reported a PTM that adds another layer of modulating PPAR activity: sumoylation (36,37,38,39). The covalent attachment of small ubiquitin-like modifier (SUMO) peptides (SUMO-1, −2, −3 in mammals) to lysine residues involves an activating enzyme (SAE1/SAE2), a conjugating enzyme (Ubc9), and an E3 ligase (e.g., PIAS1) (ref. 40). This modification occurs on consensus sumoylation motifs ψKXE/D in the substrate proteins, where ψ represents a large hydrophobic residue followed by a lysine, which is the SUMO acceptor site, and X may be any residue. Mutation of either the lysine residue or the acidic residue at position +2 has been shown to ablate sumoylation on these sites (40). Ligation of SUMO peptides, which are ∼100 amino acids long, is linked to various cellular processes, including nuclear-cytoplasmatic transport, apoptosis, and transcriptional regulation (40). Sumoylation of transcriptional regulators mostly correlates with the inhibition of transcription (40). Two functional sumoylation sites have been identified for PPAR-γ, lysine 107 in the AF1 region and lysine 395 in the AF2 region (lysine 77 and 365 in PPAR-γ1, respectively). Conjugation of SUMO-1 or −2 to lysine 107 by the E3 SUMO ligases PIAS1 or PIASxβ modulates PPAR-γ activity in a negative manner, because (i) mutation of K107 itself or distortion of the sumoylation consensus motif by mutating glutamic acid at position 109 into alanine (E109A) increased PPAR-γ activity, and (ii) the overexpression of a dominant-negative form of the SUMO E2-ligase Ubc9 had the same effect. These data indicate that sumoylation of the AF1 domain induces active repression. Although K107 sumoylation has been reported to decrease the stability of the PPAR-γ protein (37), this finding has not been supported by other studies (36,38,39). The exact molecular mechanism behind sumoylation-mediated repression of PPAR-γ therefore remains to be established. Possibly, the repressive effect involves specific binding of a repressor complex, because (i) the deletion of the AF1 region results in increased transcriptional activity (13,19), and (ii) Gal4DBD-AF1 fusion proteins, which rule out DNA- and ligand-binding effects, show increased transcriptional activity upon mutation of K107 (ref. 36). Interestingly, K107 sumoylation is to some extent linked to S112 phosphorylation. K107 and S112 are part of a so-called sumoyl-phospho switch (41) or phosphorylation-dependent sumoylation motif (42), a conserved motif present in at least 80 different proteins including several transcription factors and co-activator s. The consensus site consists of the following motif: ψKxExxSP, in which ψ is a hydrophobic residue, K is the sumo acceptor lysine, x is any amino acid, and SP forms a part of the downstream phosphorylation site. Between the different PPAR members, this motif is only found in the AF1 region of PPAR-γ and conserved from fish to humans, as shown in Figure Mutation of the serine 112 to which lysine 107 sumoylation the for the PPAR-γ2 which increased sumoylation A similar between phosphorylation and sumoylation was recently reported for the nuclear receptors estrogen-related and estrogen-related which also a switch (Figure 1b). can be as to the between phosphorylation and sumoylation may result in transcriptional repression (Figure phosphorylation of S112 may in a site for a specific SUMO E3 which in lysine in recruitment of a repressor complex Figure the modification of S112 and K107 may a site for a repressor complex Figure that are to gene promoters in a manner include the transcriptional repressor protein the protein (ref. and the repressor complex but research is to any of these proteins or another to be identified repressor protein is involved in repression of PPAR-γ Sumoylation of PPAR-γ. (a) Phosphorylation of PPAR-γ S112 may stimulate repression by phosphorylation-dependent recruitment of a SUMO E3 in sumoylation of K107 and binding of a transcriptional repressor complex phosphorylation and sumoylation may be to a site for a transcriptional repressor complex (b) Sumoylation of PPAR-γ of the transcriptional repressor complex from gene in the ligand-dependent repression of the gene program. DBD, DNA-binding PPAR, peroxisome proliferator-activated receptor; SUMO, small ubiquitin-like In contrast to lysine 107 sumoylation, of SUMO-1 to lysine is not involved in the regulation of PPAR-γ target genes, but in the of genes by PPAR-γ in macrophages, the gene Treatment with ligand results in sumoylation of which in PPAR-γ2 to corepressor complexes that are to target genes activation (Figure These complexes are from the promoter PPAR-γ2 is in repression. A similar mechanism was recently reported for the of target genes by the nuclear receptors and although in this or and not SUMO-1 was to the receptor is the covalent attachment of a to lysine residues in the substrate protein. Although of linked through lysine proteins for attachment of or with lysine is linked to different cellular Similar to sumoylation, an activating enzyme a conjugating enzyme and an E3 ligase or A of evidence indicates that the II and the are linked studies were by the and showed that the of transcription factors is an essential in the regulation of its target genes, by the of protein complexes at the promoter region The PPAR-γ protein has a and was found to be and by the PPAR-γ2 and are strongly linked to ligand binding and as these processes, whereas the mutation of glutamic acid to both activation and The of adipocytes to the as may upon of into adipose tissue was also found to increase and of PPAR-γ indicating that phosphorylation is not the only PTM that can be regulated by Interestingly, as for sumoylation, also to be regulated by phosphorylation. The hypophosphorylated form of PPAR-γ, which increased transcriptional activity (as was found to be the protein a between protein and transcriptional activity It is the phosphorylation of PPAR-γ may regulate its and It should be noted that acceptor have been identified for the PPAR-γ protein in however, the that sites will be identified in the a the in vivo relevance of PTMs of PPAR-γ has not been experimentally. In an et al. showed that PPAR-γ S112A mice are to insulin resistance when a Although the S112A mutation was shown to PPAR-γ into a of adipogenesis in fibroblasts the S112A not or in mice, because differences in or fat were between and wild-type It was that the mechanism for the from insulin resistance in decreased adipocyte and Interestingly, heterozygous mutation of to as similar to the S112A mutation results in a form of PPAR-γ, was reported as a genetic in the In contrast to the S112A mice, with this mutation were all with three out of patients type 2 diabetes on insulin The PPAR-γ was a of adipogenesis compared with the wild-type protein in with the of the S112A protein by several laboratories (20,23,25,28,29,30). first the data from S112A mice and patients to be in with suggesting differences between mice and humans, as well as between in vitro and in vivo However, this is probably the mouse and human data is by the that et al. characterized S112A mice whereas human mutations are heterozygous after the first on human and a was in contrast to the previous this displayed high insulin levels and insulin resistance These findings indicate that the heterozygous mutation is not to in humans, but that other genetic and/or factors are The of the however, of this et al. found patients in a of and and reported one and were found in two and or a of studies on and heterozygous S112A and mice in different genetic and under different may therefore be a to these the physiological relevance of PTMs of PPAR-γ is at only the in vivo effects of mutating S112 in PPAR-γ were studied which has in PPAR-γ It will therefore be essential for of PPAR-γ to such to other et al. reported in that PPAR-γ was a (20), studies have followed showing that PPAR-γ is subject to different which its transcriptional output (Figure Although the and substrate amino acids were identified in most it is these modifications the function of the PPAR-γ protein. Two molecular which are not are most likely to a PTM could have an as was shown for serine 112 phosphorylation of PPAR-γ that ligand binding (28). the effect of a PTM on PPAR-γ function could be either the recruitment of novel proteins or the release of proteins that were to the receptor. This of so-called proteins to forms of PPAR-γ represents an molecular but the proteins remain to be Posttranslational modifications the transcriptional output of PPAR-γ. (a) Phosphorylation of S112 together with sumoylation of K107 results in reduced to activate the adipogenic gene program. (b) Phosphorylation of PPAR-γ S112 can also result in the activation of the receptor. In this sumoylation is to (c) is an important in transcriptional activation of the adipogenic gene by PPAR-γ. (d) Sumoylation of plays an important role in ligand-dependent repression of the gene program. least two phosphorylation of S112 stimulates K107 sumoylation whereas the phosphorylation of S112 DBD, DNA-binding PPAR, peroxisome proliferator-activated receptor. has so far been to the that all the PTMs for PPARs can probably by specific cellular have been identified which can or PPAR-γ, although these factors may play an important role in the transcriptional that is and is a PTM the expression of all PPAR-γ target genes or a specific of target may be the PPAR-γ-mediated repression of the gene, which an S112 residue but it is not general this such as and may to the between PTMs and target gene Finally, a to be to between the different PTMs (Figure may be as shown for the so-called switch motif (41) or phosphorylation-dependent sumoylation motif in PPAR-γ2, phosphorylation of S112 sumoylation of K107 (ref. 36). has been for which is by S112 phosphorylation through an mechanism A different type of negative in which two for the same substrate residue, has been for proteins In the of PPAR-γ, this may on lysine which has been shown to be and also van Beekum and E. Kalkhoven, unpublished data). between PTMs the of multiple signaling the of different extracellular and could to a specific of PPAR-γ which is by the proteins, ultimately the transcriptional output (Figure and type 2 diabetes are in of The physiological roles of PPAR-γ in lipid and glucose have led to the of PPAR-γ ligands of the for the of insulin Treatment of patients with PPAR-γ ligands of the has been linked to effects such as and increased of These effects may be due to the of high of PPAR-γ suggesting that in may be a This may be through the of partial so-called PPAR modulating the PTMs on PPAR-γ may present a novel in this (Figure of PPAR the and the proteins, the specific transcriptional and the cellular and in vivo effects can be of for the of novel to the various in which the PPAR-γ protein plays a and for the and and of for to could only be due to The of