The structural determinants responsible for c-Fos protein proteasomal degradation differ according to the conditions of expression

Abstract

c-fos gene is expressed constitutively in a number of tissues as well as in certain tumor cells and is inducible, in general rapidly and transiently, in virtually all other cell types by a variety of stimuli. Its protein product, c-Fos, is a short-lived transcription factor that heterodimerizes with various protein partners within the AP-1 transcription complex via leucine zipper/leucine zipper interactions for binding to specific DNA sequences. It is mostly, if not exclusively, degraded by the proteasome. To localize the determinant(s) responsible for its instability, we have conducted a genetic analysis in which the half-lives of cFos mutants and chimeras made with the stable EGFP reporter protein were compared under two experimental conditions taken as example of continous and inducible expression. Those were constitutive expression in asynchronously growing Balb/C 3T3 mouse embryo fibroblasts and transient induction in the same cells undergoing the G0/G1 phase transition upon stimulation by serum. Our work shows that c-Fos is degraded faster in synchronousthan in asynchronous cells. This difference in turnover is primarily accounted for by several mechanisms. First, in asynchronous cells, a unique C-terminal destabilizer is active whereas, in serum-stimulated cells two destabilizers located at both extremities of the protein are functional. Second, heterodimerization and/or binding to DNA accelerates protein degradation only during the G0/G1 phase transition. Adding another level of complexity to turnover control, phosphorylation at serines 362 and 374, which are c-Fos phosphorylation sites largely modified during the G0/G1 phase transition, stabilizes c-Fos much more efficiently in asynchronous than in serum-stimulated cells. In both cases, the reduced degradation rate is due to inhibition of the activity of the C-terminal destabilizer. However, in serum-stimulated cells, this effect is partially masked by the activation of the N-terminal destabilizer and basic domain/leucine zipper-dependent mechanisms. Taken together, our data show that multiple degradation mechanisms, differing according to the conditions of expression, may operate on c-Fos to ensure a proper level and/or timing of expression. Moreover, they also indicate that the half-life of c-Fos during the G0/G1 phase transition is determined by a delicate balance between opposing stabilizing and destabilizing mechanisms operating at the same time.

Proline-mediated Proteasomal Degradation of the Prostate-specific Tumor Suppressor NKX3.1

“…However, our studies have demonstrated that C21 deletion did not alter polyubiquitination, suggesting that C21 degron activity is independent of E3-ubiquitin ligase recognition or interaction. Distinct pathways activate alternative degradation mechanisms for proteins such as Rb and the Fos-family members upon viral infection, oxygen depletion, or other physiological conditions (27,45). The data presented here demonstrate the presence of additional, context-dependent NKX3.1 turnover mechanism.…”

“…The enhanced green fluorescence protein-expressing vector has been described previously (27). Codon-optimized oligonucleotides containing wild-type or mutant degron sequences were ligated in reading frame using EcoRI and KpnI at the C-terminal multiple cloning site of pEGFP-C1 4 vector (Clontech).…”

Heterodimerization with Fra-1 cooperates with the ERK pathway to stabilize c-Jun in response to the RAS oncoprotein

“…The inhibition of JNK or ERK activity was monitored by immunoblotting, using phospho-specific antibodies (Cell Signaling) recognizing c-Jun (Ser73), c-Jun (Ser73) and the active phosphorylated isoforms of ERK1/2 and JNK1/2, as in panels a and b. Ferrara et al, 2003). Consistently, GFP-aFOS hardly decayed during the CHX chase (Figure 2b), suggesting that stabilization of c-Jun in GFP-aFOS cells could be due to heterodimerization with the stable GFP-aFOS protein.…”

Physical and functional cooperation between AP-1 and β-catenin for the regulation of TCF-dependent genes

“…We next mapped the regions of the c-Jun and c-Fos proteins that interacted with b-catenin by using [ 35 S] methionine-labeled fragments of the two proteins (Figure 2a and b). The various c-Jun and c-Fos deletion fragments have been previously described (Kardassis et al, 1999;Ferrara et al, 2003). Functional cooperation between b-catenin and AP-1 We next determined whether AP-1 has a functional implication in the transcriptional regulation of two b-catenin-dependent target genes, cyclin D1 and c-myc (He et al, 1998;Sasaki et al, 2003), by using transient transfection experiments of their cognate promoter reporter together with c-Jun and/or c-Fos expression plasmids in HepG2 cells.…”

“…The expression vectors pcDNA/c-Jun(1-331), pcDNA/c-Jun(D3-122) and pcDNA/c-Jun(226-331) with deletion in the N-terminal region, pcDNA/c-Jun DBDmut, mutated in the DNA-binding domain, and pcDNA/c-JunDLZ, deleted of the leucine zipper domain have been previously described (Kardassis et al, 1999). The pcDNA3/c-Fos, pcDNA3/c-Fos-DLZ, pcDNA3/ c-Fos-DDBD, pcDNA3/c-Fos-DN and pcDNA3/c-Fos-DC encoding the wild or mutated human c-Fos proteins were the generous gift of M Piechaczyk (Ferrara et al, 2003). Expression vectors for TCF4 and TCF4DN were a gift from C Perret.…”