Abstract
The receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is considered a master regulator of adipocyte differentiation and promotes glucose and lipid metabolism in mature adipocytes. We recently identified the yeast Sterile 20 (Ste20) protein kinase ortholog, Map4k4, in an RNA interference-based screen as an inhibitor of PPARgamma expression in cultured adipocytes. Here, we show that RNA interference-mediated silencing of Map4k4 elevates the levels of both PPARgamma1 and PPARgamma2 proteins in 3T3-L1 adipocytes without affecting PPARgamma mRNA levels, suggesting that Map4k4 regulates PPARgamma at a post-transcriptional step. PPARgamma degradation rates are remarkably rapid as measured in the presence of cycloheximide (t(1/2) = 2 h), but silencing Map4k4 had no effect on PPARgamma degradation. However, depletion of Map4k4 significantly enhances [(35)S]methionine/cysteine incorporation into proteins, suggesting that Map4k4 signaling decreases protein translation. We show a function of Map4k4 is to inhibit rapamycin-sensitive mammalian target of rapamycin (mTOR) activity, decreasing 4E-BP1 phosphorylation. In addition, our results show mTOR and 4E-BP1 are required for the increased PPARgamma protein expression upon Map4k4 knockdown. Consistent with this concept, adenovirus-mediated expression of Map4k4 decreased PPARgamma protein levels and mTOR phosphorylation. These data show that Map4k4 negatively regulates PPARgamma post-transcriptionally, by attenuating mTOR signaling and a 4E-BP1-dependent mechanism.
Highlights
Sion of genes required for triglyceride synthesis and storage
PPAR␥ is activated by lipid ligands [5] and plays an important role in glucose homeostasis and regulation of lipid metabolism in mature adipocytes
To test if Map4k4-mediated increases in PPAR␥ protein synthesis are due to regulation of other translation initiation factors, we examined the changes in the phosphorylation of eIF2-␣ by SDS-PAGE and immunoblotting using phospho-eIF2-␣ antibody (Ser-51)
Summary
PPAR␥, peroxisome proliferator-activated receptor ␥; siRNA, small interfering RNA; ANOVA, analysis of variance; mTOR, mammalian target of rapamycin; HA, hemagglutinin. One of the first steps of initiation of protein synthesis and a rate-limiting step is the formation of a “ternary complex” by Met-tRNA and GTP-bound eIF2-␣. Another means of translation regulation involves modulation of eIF4e (cap-binding protein) availability that is achieved by eIF4e-binding proteins called 4e-BPs. Hypophosphorylated 4e-BPs bind to eIF4e and prevent interaction between the 43 S preinitiation complex and mRNA that leads to inhibition of translation initiation. Mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase and a major regulator of cell growth and proliferation via modulating protein synthesis in response to environmental cues. In this study we have examined the role of Map4k4 in regulating PPAR␥ protein and embarked on identifying the players that are regulated by Map4k4 that potentially would regulate PPAR␥ protein expression
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