Isoform-specific SCF(Fbw7) ubiquitination mediates differential regulation of PGC-1α

Abstract

The E3 ubiquitin ligase and tumor suppressor SCF(Fbw7) exists as three isoforms that govern the degradation of a host of critical cell regulators, including c-Myc, cyclin E, and PGC-1α. Peroxisome proliferator activated receptor-gamma coactivator 1α (PGC-1α) is a transcriptional coactivator with broad effects on cellular energy metabolism. Cellular PGC-1α levels are tightly controlled in a dynamic state by the balance of synthesis and rapid degradation via the ubiquitin-proteasome system. Isoform-specific functions of SCF(Fbw7) are yet to be determined. Here, we show that the E3 ubiquitin ligase, SCF(Fbw7), regulates cellular PGC-1α levels via two independent, isoform-specific, mechanisms. The cytoplasmic isoform (SCF(Fbw7β)) reduces cellular PGC-1α levels via accelerated ubiquitin-proteasome degradation. In contrast, the nuclear isoform (SCF(Fbw7α)) increases cellular PGC-1α levels and protein stability via inhibition of ubiquitin-proteasomal degradation. When nuclear Fbw7α proteins are redirected to the cytoplasm, cellular PGC-1α protein levels are reduced through accelerated ubiquitin-proteasomal degradation. We find that SCF(Fbw7β) catalyzes high molecular weight PGC-1α-ubiquitin conjugation, whereas SCF(Fbw7α) produces low molecular weight PGC-1α-ubiquitin conjugates that are not effective degradation signals. Thus, selective ubiquitination by specific Fbw7 isoforms represents a novel mechanism that tightly regulates cellular PGC-1α levels. Fbw7 isoforms mediate degradation of a host of regulatory proteins. The E3 ubiquitin ligase, Fbw7, mediates PGC-1α levels via selective isoform-specific ubiquitination. Fbw7β reduces cellular PGC-1α via ubiquitin-mediated degradation, whereas Fbw7α increases cellular PGC-1α via ubiquitin-mediated stabilization.

FIGURE 1. Differential effect of Fbw7α and Fbw7β on PGC-1α protein steady state levels

A. To examine endogenous PGC-1α, Fbw7^−/− cells were transfected with empty vector, Fbw7α or Fbw7β. 18h later cells were lysed and evaluated via SDS-PAGE and Western blotting for endogenous PGC-1α and actin (upper panel). Quantification of seven independent experiments (lower panel). B. HeLa, Fbw7^−/− or Fbw7^+/+ cells were transfected with PGC-1α with or without Fbw7α or Fbw7β. 18h later cells were lysed and evaluated via SDS-PAGE and Western blotting for PGC-1α and actin (as a loading control) (upper panel). Quantification of six independent experiments (lower panel).

FIGURE 2. Fbw7 isoform specific impact on PGC-1α degradation rate

18h after transfection with the indicated plasmids, Fbw^+/+ (A) or Fbw7^−/− (B, C, D) cells were treated with CHX +/− MG132. Cells were thereafter lysed at 0, 0.5, 1, 2, and 3h and were evaluated for PGC-1α degradation via SDS-PAGE, Western blotting for PGC-1α. The pixels for each band were measured and normalized so that the number of pixels at t=0 was 100%. The log₁₀ of the percent of pixels was plotted versus time for each time point and the t^½ was calculated from the log of 50%.

FIGURE 3. Effect of Fbw7α and Fbw7β on PGC-1α steady state levels in isoform-specific Fbw7 targeted cells

A. Effects of Fbw7α and Fbw7β on endogenous PGC-1α in Fbw7α-targeted and Fbw7β-targeted cells. B. Effects of Fbw7α and Fbw7β on exogenously expressed PGC-1α in Fbw7α-targeted and Fbw7β-targeted cells. Details are provided in legend to Figure 1.

FIGURE 4. Confocal microscopic localization of Fbw7 isoforms in the absence and presence of exogenous PGC-1α

18h after transfection with Fbw7 isoforms alone or cotransfected with PGC-1α, HeLa cells were fixed and localization of Fbw7 isoforms and PGC-1α was visualized with anti-FLAG (Fbw7) (B, C) and anti-PGC-1α via double-label confocal immunofluorescence (C). Panel A diagrams the Fbw7 isoforms examined in B and C and indicates the subcellular localization as shown in Table 2.

FIGURE 5. Effect of Fbw7α chimeric protein mutants on PGC-1α steady state levels

Fbw7^−/− cells were transfected with PGC-1α with or without Fbw7α, Fbw7β, or Fbw7α mutants (Fbw7α-NLS^mut; Fbw7α-NLS^del; Nβ-Fbw7α). 18h later cells were lysed and evaluated via SDS-PAGE and Western blotting as described in Figure 1.

FIGURE 6. Effect of Fbw7 chimeric protein mutants on PGC-1α degradation rate

18h after transfection of the indicated plasmids, Fbw7^−/− cells were treated with CHX and thereafter lysed at 0 or 2h and evaluated for PGC-1α degradation via SDS-PAGE, Western blotting for PGC-1α as described in Figures 2 and 4.

FIGURE 7. Effect of Fbw7α and Fbw7β on ubiquitin conjugates of PGC-1α

Fbw7^−/− cells, +/− MG132, were transfected with PGC-1α, Fbw7α, Fbw7β, Fbw7α mutants and HA-ubiquitin, as noted. 18h later, cells were lysed, aliquots were Western blotted with anti-HA (panel A, B) to detect total cellular ubiquitin-protein conjugates or with anti-PGC-1α (panel C, D) to detect total cellular PGC-1α. Additional aliquots were immunoprecipitated with anti-HA and thereafter Western blotted for PGC-1α (panel E) to determine specific ubiquitin-PGC-1α conjugates. Ubiquitin-PGC-1α conjugates are marked by bracket. Low molecular weight conjugates are seen below ~225 kDa.