As a central regulator in energy metabolism, PGC-1α is modulated by a plethora of mechanisms. Among them is posttranslational modification. Phosphorylation and acetylation/deacetylation of PGC-1α and their functional impacts on systematic metabolism have been well documented (6
). Here, using an unbiased screening technique, we identified RNF34 as an E3 ubiquitin ligase that ubiquitinates PGC-1α protein and controls its turnover. Importantly, through both ectopic expression and knockdown experiments, we showed that RNF34 regulates the endogenous level of PGC-1α protein, PGC-1α target gene expression, and cellular respiration rate in brown fat cells. These effects are not associated with any change of PGC-1α mRNA expression and are not observed when a ligase-defective point mutant of RNF34 is expressed. Thus, our results demonstrate a critical role of this RNF34-mediated modification of PGC-1α in energy expenditure.
Besides RNF34, the SCFFbw7
complex is another E3 ubiquitin ligase for PGC-1α (16
). The distinctions between the two ligases are evident. First, the interaction with and degradation of PGC-1α by Fbw7 require phosphorylation of four residues located in the N terminus of PGC-1α, first by p38 MAPK-mediated priming phosphorylation and second by Gsk3β-mediated phosphorylation (16
). Interestingly, p38 MAPK, by phosphorylating three of the four residues, was shown instead to stabilize PGC-1α (19
). Moreover, this phosphodegron recognition motif is conserved among PGC-1 members, indicating that Fbw7 might be an E3 ligase for PGC-1β as well (16
). On the other hand, RNF34 targets the C-terminal half of PGC-1α for degradation, independently of the phosphorylation of these four residues, and RNF34 does not regulate PGC-1β turnover. Second, Fbw7 is a haploinsufficient tumor suppressor that targets multiple substrates for degradation, including the proto-oncoproteins Myc, Jun, Notch, and cyclin E and the lipogenic regulator SREBP (32
). Third, in contrast to RNF34, Fbw7 expression in the brown fat cells is not regulated by environmental temperature or by β3-adrenergic receptor signaling. Fourth, the functional relevance of Fbw7-mediated ubiquitination of PGC-1α in PGC-1α target gene expression and energy metabolism is unknown. Liver-specific Fbw7 knockout mice display massive hepatic deposition of triglycerides and increased levels of SREBP protein but no increase of PGC-1α protein levels (17
). Thus, the metabolic axis for Fbw7 in the liver appears to be the regulation of lipogenesis through SREBP. We found that overexpression of Fbw7 in brown fat cells slightly decreases the endogenous PGC-1α protein level (see Fig. S4 in the supplemental material). Whether Fbw7 is involved in brown fat metabolism remains to be determined.
An intriguing observation is that cold exposure suppresses RNF34 expression in the brown fat cells. This suppression is mediated by β3-adrenergic receptor signaling. Among numerous regulators or modifiers of PGC-1α, RNF34 is the only one reported to date to be regulated by environmental temperature. The results indicate that ubiquitination of PGC-1α by RNF34 is likely to be coordinated with environmental cues and strongly suggest an in vivo
physiological function of RNF34 in thermogenesis. This expression pattern presents a striking contrast with that of PGC-1α, whose transcription is induced by the presence of either cold exposure or β3-adrenergic signaling. Thus, in the cold, PGC-1α activity is maximized not only by induction of its mRNA but also by possible stabilization of its protein, ensuring sufficient heat production to maintain body temperature. Currently, the detailed mechanism underlying the temperature modulation of RNF34 expression is unclear. The modulation might not be cell-autonomous, as treatment of brown fat cells in vitro
with the β3-adrenergic receptor agonist has no effect on RNF34 expression (our unpublished data). Interestingly, Cidea, a lipid droplet protein that also negatively regulates brown fat metabolism (4
), displays a similar pattern of regulation. Cidea expression in the brown fat cells is suppressed by cold exposure through β3-adrenergic signaling (27
) but not in brown fat cells in vitro
(our unpublished data).
Phosphorylation and acetylation/deacetylation of PGC-1α are critical for PGC-1α-regulated energy metabolism in liver and skeletal muscle. Here we identify ubiquitination of PGC-1α by RNF34 as a regulatory module operating in brown fat cells. It should be interesting to investigate whether this RNF34-mediated ubiquitination coordinates with and/or affects the dynamics of other modifications of PGC-1α. Although our studies were performed using isolated brown fat cells, the regulation of RNF34 expression by environmental temperature strongly indicates a potential role of this E3 ligase in brown fat metabolism in vivo, which is planned to be fully investigated using tissue-specific RNF34 knockout mice in the future. Given the inverse correlation between brown fat activity and human obesity, inhibition of RNF34 ligase activity might be useful to promote energy expenditure and counteract obesity.