Publications

Sequence analysis of the human genome and the association of genetic aberrations with diseases have provided a rough framework whereby the impact of individual genotypes can be assessed. To fully understand the effect of individual and co-occurring genetic aberrations, as well as their individual and collected contribution to the development of diseases, it is critical to analyse the matching proteome and to determine how the organisation, expression level and function of protein networks are affected. Sensitive mass spectrometric platforms in combination with innovative workflows allow qualitative and quantitative analyses of the cellular as well as the extracellular proteome. Importantly, in addition to specifically identifying the content of the proteome, several aspects of the proteomic organisation can be analysed including protein complexes, protein modifications, enzymatic activities and subcellular/organelle localisation. Together, these measurements will provide novel insight into the biological effect of disease-causing mutations ultimately coupling genotype and phenotype.

Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs (Rb-/- MEFs).

MS (mass spectrometry) techniques are rapidly evolving to high levels of performance and robustness. This is allowing the application of these methods to the interrogation of signalling networks with unprecedented depth and accuracy. In the present review we discuss how MS-based multiplex quantification of kinase activities and phosphoproteomics provide complementary means to assess biological signalling activity. In addition, we discuss how a wider application of these analytical concepts to quantify kinase signalling will result in a more comprehensive understanding of normal and disease biology at the system level.

Cold adaptation elicits a paradoxical simultaneous induction of fatty acid synthesis and beta-oxidation in brown adipose tissue. We show here that cold exposure coordinately induced liver X receptor alpha (LXRalpha), adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein-1c (SREBP1c) and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC1alpha) in brown and inguinal white adipose tissues, but not in epididymal white adipose tissue. Using in vitro models of white and brown adipocytes we demonstrate that beta-adrenergic stimulation induced expression of LXRalpha, ADD1/SREBP1c and PGC1alpha in cells with a brown-like adipose phenotype. We demonstrate that ADD1/SREBP1c is a powerful inducer of PGC1alpha expression via a conserved E box in the proximal promoter and that beta-adrenergic stimulation led to recruitment of ADD1/SREBP1c to this E box. The ability of ADD1/SREBP1c to activate the PGC1alpha promoter exhibited a striking cell type dependency, suggesting that additional cell type-restricted factors contribute to ADD1/SREBP1c-mediated activation. In conclusion, our data demonstrate a novel role of ADD1/SREBP1c as a regulator of PGC1alpha expression in brown adipose tissue.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR gamma, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differentiation has remained enigmatic. Previously, we showed that lipoxygenase (LOX) activity is involved in activation of PPAR gamma during the early stages of adipocyte differentiation. Of the seven known murine LOXs, only the unconventional LOX epidermis-type lipoxygenase 3 (eLOX3) is expressed in 3T3-L1 preadipocytes. Here, we show that forced expression of eLOX3 or addition of eLOX3 products stimulated adipogenesis under conditions that normally require an exogenous PPAR gamma ligand for differentiation. Hepoxilins, a group of oxidized arachidonic acid derivatives produced by eLOX3, bound to and activated PPAR gamma. Production of hepoxilins was increased transiently during the initial stages of adipogenesis. Furthermore, small interfering RNA-mediated or retroviral short hairpin RNA-mediated knockdown of eLOX3 expression abolished differentiation of 3T3-L1 preadipocytes. Finally, we demonstrate that xanthine oxidoreductase (XOR) and eLOX3 synergistically enhanced PPAR gamma-mediated transactivation. Collectively, our results indicate that hepoxilins produced by the concerted action of XOR and eLOX3 may function as PPAR gamma activators capable of promoting the early PPAR gamma-dependent steps in the conversion of preadipocytes into adipocytes.

Protein phosphorylation dynamically regulates cellular activities in response to environmental cues. Sequence conservation analysis of recent proteome-wide phosphorylation data revealed that many previously unidentified phosphorylation sites are not well-conserved leading to the proposal that many are non-functional. However, this is based on the assumption that protein phosphorylation modulates protein function through specific position on protein sequence. Based on emerging understanding on phospho-regulation of cellular activities, we argue, with examples, that non-positionally conserved phosphorylation sites can very well be functional. We previously identified phosphorylation events that need not be conserved at same positions across orthologous proteins but are likely maintained by evolutionary conserved signaling networks through orthologous kinases. We found that proteins with such conserved phosphorylation patterns are statistically over-represented with protein and DNA-binding annotation. Here, we further correlated these proteins with protein-protein interaction data from an independent systematic study and observed they indeed interact frequently with other proteins. Hence, we speculate that non-positionally conserved phosphorylation site could be modulating biomolecular association of phosphorylated proteins possibly through fine-tuning protein's bulk electrostatic charge and through creating binding sites for phospho-binding interaction domains. We, therefore, advocate the development of complementary evolutionary approaches to interpret physiological important sites.

Signaling events are frequently described in textbooks as linear cascades. However, in reality, input cues are processed by dynamic and context-specific networks, which are assembled from numerous signaling molecules. Diseases, such as cancer, are typically associated with multiple genomic alterations that likely change the structure and dynamics of cellular signaling networks. To assess the impact of such genomic alterations on the structure of signaling networks and on the ability of cells to accurately translate environmental cues into phenotypic changes, we argue studies must be conducted on a network level. Advances in technologies and computational approaches for data integration have permitted network studies of signaling events in both cancer and normal cells. Here we will review recent advances and how they have impacted our view on signaling networks with a specific angle on signal processing in cancer.

Protein kinases enable cellular information processing. Although numerous human phosphorylation sites and their dynamics have been characterized, the evolutionary history and physiological importance of many signaling events remain unknown. Using target phosphoproteomes determined with a similar experimental and computational pipeline, we investigated the conservation of human phosphorylation events in distantly related model organisms (fly, worm, and yeast). With a sequence-alignment approach, we identified 479 phosphorylation events in 344 human proteins that appear to be positionally conserved over approximately 600 million years of evolution and hence are likely to be involved in fundamental cellular processes. This sequence-alignment analysis suggested that many phosphorylation sites evolve rapidly and therefore do not display strong evolutionary conservation in terms of sequence position in distantly related organisms. Thus, we devised a network-alignment approach to reconstruct conserved kinase-substrate networks, which identified 778 phosphorylation events in 698 human proteins. Both methods identified proteins tightly regulated by phosphorylation as well as signal integration hubs, and both types of phosphoproteins were enriched in proteins encoded by disease-associated genes. We analyzed the cellular functions and structural relationships for these conserved signaling events, noting the incomplete nature of current phosphoproteomes. Assessing phosphorylation conservation at both site and network levels proved useful for exploring both fast-evolving and ancient signaling events. We reveal that multiple complex diseases seem to converge within the conserved networks, suggesting that disease development might rely on common molecular networks.

Cells have self-organizing properties that control their behavior in complex tissues. Contact between cells expressing either B-type Eph receptors or their transmembrane ephrin ligands initiates bidirectional signals that regulate cell positioning. However, simultaneously investigating how information is processed in two interacting cell types remains a challenge. We implemented a proteomic strategy to systematically determine cell-specific signaling networks underlying EphB2- and ephrin-B1-controlled cell sorting. Quantitative mass spectrometric analysis of mixed populations of EphB2- and ephrin-B1-expressing cells that were labeled with different isotopes revealed cell-specific tyrosine phosphorylation events. Functional associations between these phosphotyrosine signaling networks and cell sorting were established with small interfering RNA screening. Data-driven network modeling revealed that signaling between mixed EphB2- and ephrin-B1-expressing cells is asymmetric and that the distinct cell types use different tyrosine kinases and targets to process signals induced by cell-cell contact. We provide systems- and cell-specific network models of contact-initiated signaling between two distinct cell types.

Eph receptors constitute the largest family of receptor tyrosine kinases in the human genome. EphA2 is one prominent member that is overexpressed and functionally altered in many invasive cancers, including pancreatic cancer. Dasatinib, which is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, has recently been shown to have significant activity against EphA2. As selective small molecule EphA2 inhibitors are not currently available, we investigated the therapeutic potential to target EphA2 by dasatinib in pancreatic cancer cell lines. Using in vitro kinase assays, we found that EphA2 receptor tyrosine kinase was inhibited directly by dasatinib in a dose-dependent manner. Stimulation with ephrinA1 produced rapid increases of EphA2 phosphorylation that were inhibited by dasatinib, although the effects on activation of downstream signalling differed among the pancreatic cancer cell lines. Dasatinib also inhibited ligand-induced binding of EphA2 to the ubiquitin ligase Cbl, and the internalisation and degradation of EphA2, suggesting that these processes are dependent on kinase activity. Treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src.

Familial clustering of colorectal cancer occurs in 15-20% of cases, however recognized cancer syndromes explain only a small fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected. Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity, with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer.

Protein serine/threonine phosphatase 4 (PP4c) is an essential polypeptide involved in critical cellular processes such as microtubule growth and organization, DNA damage checkpoint recovery, apoptosis, and tumor necrosis factor alpha signaling. Like other phosphatases of the PP2A family, PP4c interacts with regulatory proteins, which specify substrate targeting and intracellular localization. The identification of these regulatory proteins is, therefore, key to fully understanding the function of this enzyme class. Here, using a sensitive affinity purification/mass spectrometry approach, we identify a novel, stable cytosolic PP4c interacting partner, KIAA1622, which we have renamed PP4R4. PP4R4 displays weak sequence homology with the A (scaffolding) subunit of the PP2A holoenzyme and specifically associates with PP4c (and not with the related PP2Ac or PP6c phosphatases). The PP4c.PP4R4 interaction is disrupted by mutations analogous to those abrogating the association of PP2Ac with PP2A A subunit. However, unlike the PP2A A subunit, which plays a scaffolding role, PP4R4 does not bridge PP4c with previously characterized PP4 regulatory subunits. PP4c.PP4R4 complexes exhibit phosphatase activity toward a fluorogenic substrate and gammaH2AX, but this activity is lower than that associated with the PP4c.PP4R2.PP4R3 complex, which itself is less active than the free PP4c catalytic subunit. Our data demonstrate that PP4R4 forms a novel cytosolic complex with PP4c, independent from the complexes containing PP4R1, PP4R2.PP4R3, and alpha4, and that the regulatory subunits of PP4c have evolved different modes of interaction with the catalytic subunit.

Shc family proteins serve as phosphotyrosine adaptor molecules in various receptor-mediated signaling pathways. In mammals, three distinct Shc genes have been described that encode proteins characterized by two phosphotyrosine-interaction modules, an amino-terminal phosphotyrosine binding (PTB) domain and a carboxy-terminal Src homology 2 domain. Here, we report the analysis of an uncharacterized fourth Shc family protein, ShcD/Shc4, that is expressed in adult brain and skeletal muscle. Consistent with this expression pattern, we find that ShcD can associate via its PTB domain with the phosphorylated muscle-specific kinase (MuSK) receptor tyrosine kinase and undergo tyrosine phosphorylation downstream of activated MuSK. Interestingly, additional sites of tyrosine phosphorylation, including a novel Grb2 binding site, are present on ShcD that are not found in other Shc family proteins. Activation of MuSK upon agrin binding at the neuromuscular junction (NMJ) induces clustering and tyrosine phosphorylation of acetylcholine receptors (AChRs) required for synaptic transmission. ShcD is coexpressed with MuSK in the postsynaptic region of the NMJ, and in cultured myotubes stimulated with agrin, expression of ShcD appears to be important for early tyrosine phosphorylation of the AChR. Thus, we have characterized a new member of the Shc family of docking proteins, which may mediate a specific aspect of signaling downstream of the MuSK receptor.

12R-lipoxygenase (12R-LOX) and epidermis-type LOX-3 (eLOX-3) are novel members of the multigene family of mammalian LOX. A considerable gap exists between the identification of these enzymes and their biologic function. Here, we present evidence that 12R-LOX and eLOX-3, acting in sequence, and eLOX-3 in combination with another, not yet identified LOX are critically involved in terminal differentiation of keratinocytes and adipocytes, respectively. Mutational inactivation of 12R-LOX and/or eLOX-3 has been found to be associated with development of an inherited ichthyosiform skin disorder in humans and genetic ablation of 12R-LOX causes a severe impairment of the epidermal lipid barrier in mice leading to post-natal death of the animals. In preadipocytes, a LOX-dependent PPARgamma activating ligand is released into the cell supernatant early upon induction of differentiation and available evidence indicates that this ligand is an eLOX-3-derived product. In accordance with this data is the observation that forced expression of eLOX-3 enhances adipocyte differentiation.

Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, respectively. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.

Adipocyte precursor cells give raise to two major cell populations with different physiological roles: white and brown adipocytes. Here we demonstrate that the retinoblastoma protein (pRB) regulates white vs. brown adipocyte differentiation. Functional inactivation of pRB in wild-type mouse embryo fibroblasts (MEFs) and white preadipocytes by expression of simian virus 40 large T antigen results in the expression of the brown fat-specific uncoupling protein 1 (UCP-1) in the adipose state. Retinoblastoma gene-deficient (Rb-/-) MEFs and stem cells, but not the corresponding wild-type cells, differentiate into adipocytes with a gene expression pattern and mitochondria content resembling brown adipose tissue. pRB-deficient MEFs exhibit an increased expression of the Forkhead transcription factor Foxc2 and its target gene cAMP-dependent protein kinase regulatory subunit RIalpha, resulting in increased cAMP sensitivity. Suppression of cAMP-dependent protein kinase activity in Rb(-/-)MEFs blocked the brown adipocyte-like gene expression pattern without affecting differentiation per se. Immunohistochemical studies revealed that pRB is present in the nuclei of white but not brown adipocyte precursor cells at a developmental stage where both cell types begin to accumulate lipid and brown adipocytes express UCP-1. Furthermore, pRB rapidly undergoes phosphorylation upon cold-induced neodifferentiation and up-regulation of UCP-1 expression in brown adipose tissue. Finally, down-regulation of pRB expression accompanies transdifferentiation of white into brown adipocytes in response to beta3-adrenergic receptor agonist treatment. We propose that pRB acts as a molecular switch determining white vs. brown adipogenesis, suggesting a previously uncharacterized function of this key cell cycle regulator in adipocyte lineage commitment and differentiation.

Arachidonic acid inhibits adipocyte differentiation of 3T3-L1 cells via a prostaglandin synthesis-dependent pathway. Here we show that this inhibition requires the presence of a cAMP-elevating agent during the first two days of treatment. Suppression of protein kinase A activity by H-89 restored differentiation in the presence of arachidonic acid. Arachidonic acid treatment led to a prolonged activation of extracellular signal-regulated kinases I and 2 (ERK1/2), and suppression of ERK1/2 activity by the addition of U0126 rescued differentiation. Upon induction of differentiation, expression of cyclooxygenase-2 (COX-2) was transiently induced and then declined, whereas COX-1 expression declined gradually as differentiation progressed. Treatment with arachidonic acid led to sustained expression of COX-1 and COX-2. Omission of a cAMP-elevating agent or addition of H-89 or U0126 prevented sustained expression of COX-2. Unexpectedly, we observed that selective COX-1 or COX-2 inhibitors rescued adipocyte differentiation in the presence of arachidonic acid as effectively as did the nonselective COX-inhibitor indomethacin. De novo fatty acid synthesis, diacylglycerol acyltransferase (DGAT) activity, and triacylglycerol accumulation were repressed in cells treated with arachidonic acid. Indomethacin restored DGAT activity and triacylglycerol accumulation without restoring de novo fatty acid synthesis, resulting in an enhanced incorporation of arachidonic acid into cellular triacylglycerols.-Petersen, R. K., C. Jorgensen, A. C. Rustan, L. Froyland, K. Muller-Decker, G. Furstenberger, R. K. Berge, K. Kristiansen, and L. Madsen. Arachidonic acid-dependent inhibition of adipocyte differentiation requires PKA activity and is associated with sustained expression of cyclooxygenases.

Adipocytes play a central role in whole-body energy homoeostasis. Complex regulatory transcriptional networks control adipogensis, with ligand-dependent activation of PPARgamma (peroxisome proliferator-activated receptor gamma) being a decisive factor. Yet the identity of endogenous ligands promoting adipocyte differentiation has not been established. Here we present a critical evaluation of the role of LOXs (lipoxygenases) during adipocyte differentiation of 3T3-L1 cells. We show that adipocyte differentiation of 3T3-L1 preadipocytes is inhibited by the general LOX inhibitor NDGA (nordihydroguaiaretic acid) and the 12/15-LOX selective inhibitor baicalein. Baicalein-mediated inhibition of adipocyte differentiation was rescued by administration of rosiglitazone. Treatment with baicalein during the first 4 days of the differentiation process prevented adipocyte differentiation; supplementation with rosiglitazone during the same period was sufficient to rescue adipogenesis. Accordingly, we demonstrate that adipogenic conversion of 3T3-L1 cells requires PPARgamma ligands only during the first 4 days of the differentiation process. We show that the baicalein-sensitive synthesis of endogenous PPARgamma ligand(s) increases rapidly upon induction of differentiation and reaches a maximum on days 3-4 of the adipocyte differentiation programme. The conventional platelet- and leucocyte-type 12(S)-LOXs and the novel eLOX-3 (epidermis-type LOX-3) are expressed in white and brown adipose tissue, whereas only eLOX-3 is clearly expressed in 3T3-L1 cells. We suggest that endogenous PPARgamma ligand(s) promoting adipocyte differentiation are generated via a baicalein-sensitive pathway involving the novel eLOX-3.

Arachidonic acid inhibits adipocyte differentiation of 3T3-L1 cells via a prostaglandin synthesis-dependent pathway. Here we show that this inhibition requires the presence of a cAMP-elevating agent during the first two days of treatment. Suppression of protein kinase A activity by H-89 restored differentiation in the presence of arachidonic acid. Arachidonic acid treatment led to a prolonged activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 activity by the addition of U0126 rescued differentiation. Upon induction of differentiation, expression of cyclooxygenase-2 (COX-2) was transiently induced and then declined, whereas COX-1 expression declined gradually as differentiation progressed. Treatment with arachidonic acid led to sustained expression of COX-1 and COX-2. Omission of a cAMP-elevating agent or addition of H-89 or U0126 prevented sustained expression of COX-2. Unexpectedly, we observed that selective COX-1 or COX-2 inhibitors rescued adipocyte differentiation in the presence of arachidonic acid as effectively as did the nonselective COX-inhibitor indomethacin. De novo fatty acid synthesis, diacylglycerol acyltransferase (DGAT) activity, and triacylglycerol accumulation were repressed in cells treated with arachidonic acid. Indomethacin restored DGAT activity and triacylglycerol accumulation without restoring de novo fatty acid synthesis, resulting in an enhanced incorporation of arachidonic acid into cellular triacylglycerols.

A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity. Ras-dependent signaling plays a significant, although incompletely understood, role in adipocyte differentiation, because activated Ras has been reported to either promote or inhibit adipogenesis depending on the cellular context. In various cell types activation of Ras leads to activation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein kinase B (PKB)/Akt, which exert opposing effects on adipogenesis, with ERK1/2 inhibiting and PKB/Akt promoting terminal differentiation. Here we report that the levels of activated ERK1/2 and PKB/Akt are significantly increased in pRB-deficient MEFs both before and after the addition of adipogenic inducers. Consistently, we detected higher levels of activated Ras in MEFs lacking pRB. Suppression of ERK1/2 activation by the MEK inhibitor UO126 restored the ability of pRB-deficient MEFs to undergo adipocyte differentiation, as manifested by expression of adipocyte marker genes and lipid accumulation. Furthermore and reflecting the elevated levels of activated PKB/Akt in the pRB-deficient MEFs, differentiation proceeded in an insulin-independent manner. In conclusion, we suggest that pRB plays a pivotal role in adipogenesis by suppressing MAPK activity.

The peroxisome proliferator-activated receptors (PPARs) bind and are activated by a variety of fatty acids and derivatives thereof. Agonist binding enhances PPAR-mediated transactivation via release of corepressors and recruitment of coactivator complexes. Recently, we and others have reported that acyl-CoA esters act as PPAR antagonists in vitro. Here, we show that the binding of the nonhydrolyzable acyl-CoA analogue, S-hexadecyl-CoA, differentially affected conformation and coactivator recruitment of the individual PPAR subtypes. In protease protection assays, S-hexadecyl CoA increased the sensitivity of PPARalpha and PPARdelta towards chymotrypsin, whereas the action of chymotrypsin on PPARgamma was only marginally affected, suggesting distinct subtype-dependent differences in the effects of S-hexadecyl-CoA on conformation of the PPARs. In keeping with these findings, S-hexadecyl-CoA abrogated ligand-induced recruitment of coactivators to PPARalpha and PPARdelta, whereas coactivator recruitment to PPARgamma was unaffected by S-hexadecyl-CoA.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARalpha. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPARalpha conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARalpha-retinoid X receptor alpha heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARalpha bound specifically to immobilized palmitoyl-CoA and Wy14643, but not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the sensitivity of PPARalpha to chymotrypsin digestion, and the S-hexadecyl-CoA-induced sensitivity required a functional PPARalpha ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARalpha but increased recruitment of the nuclear receptor co-repressor NCoR. In cells, the concentration of free acyl-CoA esters is kept in the low nanomolar range due to the buffering effect of high affinity acyl-CoA-binding proteins, especially the acyl-CoA-binding protein. By using PPARalpha expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARalpha-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo.