Claire Acquaviva

c-Fos Proto-Oncoprotein Is Degraded by the Proteasome Independently of Its Own Ubiquitinylation In Vivo

ABSTRACT Prior ubiquitinylation of the unstable c-Fos proto-oncoprotein is thought to be required for recognition and degradation by the proteasome. Contradicting this view, we report that, although c-Fos can form conjugates with ubiquitin in vivo, nonubiquitinylatable c-Fos mutants show regulated degradation identical to that of the wild-type protein in living cells under two classical conditions of study: transient c-fos gene expression during the G0/G1 phase transition upon stimulation by mitogens and constitutive expression during asynchronous growth. Moreover, c-Fos destruction during the G0/G1 phase transition is unusual because it depends on two distinct but cumulative mechanisms. We report here that one mechanism involves a C-terminal destabilizer which does not need an active ubiquitin cycle, whereas the other involves an N-terminal destabilizer dependent on ubiquitinylation of an upstream c-Fos breakdown effector. In addition to providing new insights into the mechanisms of c-Fos protein destruction, an important consequence of our work is that ubiquitinylation-dependent proteasomal degradation claimed for a number of proteins should be reassessed on a new experimental basis.

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

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 c-Fos 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 synchronous- than 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.

Complex mechanisms for c-fos and c-jun degradation.

Abstractc-fos and c-jun proto-oncogenes have originally been found in mutated forms in murine and avian oncogenic retroviruses. They both define multigenic families of transcription factors. Both c-jun and c-fos proteins are metabolically unstable. In vivo and in vitro work by various groups suggests that multiple proteolytic machineries, including the lysosomes, the proteasome and the ubiquitous calpains, may participate in the destruction of c-fos and c-jun. The relative contribution of each pathway is far from being known and it cannot be excluded that it varies according to the cell context and/or the physiological conditions. It has been demonstrated that, in certain occurrences, the degradation of both c-fos and c-jun by the proteasome in vivo involves the ubiquitin pathway. However, the possibility that proteasomal degradation can also occur in a manner independent of the E1 enzyme of the ubiquitin cycle remains an open issue.

Differential directing of c-Fos and c-Jun proteins to the proteasome in serum-stimulated mouse embryo fibroblasts

c-Fos and c-Jun proteins are highly unstable transcription factors that heterodimerize within the AP-1 transcription complex. Their accumulation is transiently induced at the beginning of the G0-to-S phase transition in quiescent cells stimulated for growth. To address the mechanisms responsible for rapid clearance of c-Fos and c-Jun proteins under these experimental conditions, we have used the ts20 mouse embryo fibroblasts which express a thermosensitive mutant of the E1 enzyme of the ubiquitin pathway. The use of cell-permeant protease inhibitors indicates that both proteins are degraded by the proteasome and excludes any major contribution for calpains and lysosomes during the G0-to-S phase transition. Synchronisation of ts20 cells at the non permissive temperature blocks the degradation of c-Jun, indicating that this process is E1-dependent. In contrast, c-Fos is broken down according to an apparently E1-independent pathway in ts20 cells, although a role for ubiquitinylation in this process cannot be formally ruled out. Interestingly, c-Jun is highly unstable in c-Fos-null mouse embryo fibroblasts stimulated for growth. Taken together, these observations show that in vivo during a G0-to-S phase transition (i) the precise mechanisms triggering c-Fos and c-Jun directing to the proteasome are not identical, (ii) the presence of c-Fos is not an absolute prerequisite for the degradation of c-Jun and (iii) the degradation of c-Jun is not required for that of c-Fos.

Identification of a C-terminal tripeptide motif involved in the control of rapid proteasomal degradation of c-Fos proto-oncoprotein during the G 0 -to-S phase transition

c-Fos proto-oncoprotein is rapidly and transiently expressed in cells undergoing the G0-to-S phase transition in response to stimulation for growth by serum. Under these conditions, the rapid decay of the protein occurring after induction is accounted for by efficient recognition and degradation by the proteasome. PEST motifs are sequences rich in Pro, Glu, Asp, Ser and Thr which have been proposed to constitute protein instability determinants. c-Fos contains three such motifs, one of which comprises the C-terminal 20 amino acids and has already been proposed to be the major determinant of c-Fos instability. Using site-directed mutagenesis and an expression system reproducing c-fos gene transient expression in transfected cells, we have analysed the turnover of c-Fos mutants deleted of the various PEST sequences in synchronized mouse embryo fibroblasts. Our data showed no role for the two internal PEST motifs in c-Fos instability. However, deletion of the C-terminal PEST region led to only a twofold stabilization of the protein. Taken together, these data indicate that c-Fos instability during the G0-to-S phase transition is governed by a major non-PEST destabilizer and a C-terminal degradation-accelerating element. Further dissection of c-Fos C-terminal region showed that the degradation-accelerating effect is not contributed by the whole PEST sequence but by a short PTL tripeptide which cannot be considered as a PEST motif and which can act in the absence of any PEST environment. Interestingly, the PTL motif is conserved in other members of the fos multigene family. Nevertheless, its contribution to protein instability is restricted to c-Fos suggesting that the mechanisms whereby the various Fos proteins are broken down are, at least partially, different. MAP kinases-mediated phosphorylation of two serines close to PTL, which are both phosphorylated all over the G0-to-S phase transition, have been proposed by others to stabilize c-Fos protein significantly. We, however, showed that the PTL motif does not exert its effect by counteracting a stabilizing effect of these phosphorylations under our experimental conditions.

Multiple degradation pathways for Fos family proteins.

Abstract: c‐Fos protooncoprotein is a short‐lived transcription factor with oncogenic potential. It is massively degraded by the proteasome in vivo under various experimental conditions. Those include consititutive expression in exponentially growing cells and transient induction in cells undergoing the G0/G1 phase transition upon stimulation by serum. Though there is evidence that c‐Fos can be ubiquitinylated in vitro, the unambigous demonstration that prior ubiquitinylation is necessary for degradation by the proteasome in vivo is still lacking. c‐Jun, one of the main dimerization partners of c‐Fos within the AP‐1 transcription complex, is also an unstable protein. Its degradation is clearly proteasome dependent. However, several lines of evidence indicate that the mechanisms by which it addresses the proteasome are different from those operating on c‐Fos. Moreover, genetic analysis has indicated that c‐Fos is addressed to the proteasome via pathways that differ depending on the conditions of expression. c‐Fos has been transduced by two murine osteosarcomatogenic retroviruses in mutated forms, which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of one of the main c‐Fos destabilizers but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. However, although viral Fos proteins have acquired full resistance to proteasomal degradation, stabilization is limited because the mutations they have accumulated, during or after c‐fos gene transduction, confer sensitivity to an unidentified proteolytic system(s). This observation is consistent with the idea that fos‐expressing viruses have evolved expression machineries to ensure controlled protein levels in order to maintain an optimal balance between prooncogenic and proapoptotic activities of v‐Fos proteins.

JunB Breakdown in Mid-/Late G2 Is Required for Down-Regulation of Cyclin A2 Levels and Proper Mitosis

ABSTRACT JunB, a member of the AP-1 family of dimeric transcription factors, is best known as a cell proliferation inhibitor, a senescence inducer, and a tumor suppressor, although it also has been attributed a cell division-promoting activity. Its effects on the cell cycle have been studied mostly in G1 and S phases, whereas its role in G2 and M phases still is elusive. Using cell synchronization experiments, we show that JunB levels, which are high in S phase, drop during mid- to late G2 phase due to accelerated phosphorylation-dependent degradation by the proteasome. The forced expression of an ectopic JunB protein in late G2 phase indicates that JunB decay is necessary for the subsequent reduction of cyclin A2 levels in prometaphase, the latter event being essential for proper mitosis. Consistently, abnormal JunB expression in late G2 phase entails a variety of mitotic defects. As these aberrations may cause genetic instability, our findings contrast with the acknowledged tumor suppressor activity of JunB and reveal a mechanism by which the deregulation of JunB might contribute to tumorigenesis.

Cellular and viral Fos proteins are degraded by different proteolytic systems

c-Fos proto-oncoprotein is a short-lived transcription factor degraded by the proteasome in vivo. Its mutated forms expressed by the mouse osteosarcomatogenic retroviruses, FBJ-MSV and FBR-MSV, are stabilized two- and threefold, respectively. To elucidate the mechanisms underlying v-FosFBJ and v-FosFBR protein stabilization, we conducted a genetic analysis in which the half-lives and the sensitivities to various cell-permeable protease inhibitors of a variety of cellular and viral protein mutants were measured. Our data showed that the decreased degradation of v-FosFBJ and v-FosFBR is not simply explained by the deletion of a c-Fos destabilizing C-terminal domain. Rather, it involves a complex balance between opposing destabilizing and stabilizing mutations which are distinct and which include virally-introduced peptide motifs in both cases. The mutations in viral Fos proteins conferred both total insensitivity to proteasomal degradation and sensitivity to another proteolytic system not naturally operating on c-Fos, explaining the limited stabilization of the two proteins. This observation is consistent with the idea that FBR-MSV and FBJ-MSV expression machineries have evolved to ensure controlled protein levels. Importantly, our data illustrate that the degradation of unstable proteins does not necessarily involve the proteasome and provide support to the notion that highly related proteins can be broken down by different proteolytic systems in living cells.

Degradation of cellular and viral Fos proteins.

c-Fos proto-oncoprotein is a short-lived transcription factor with oncogenic potential. We have shown that it is massively degraded by the proteasome in vivo under various experimental conditions. Other proteolytic systems including lysosomes and calpains, might, however, also marginally operate on it. Although there is evidence that c-Fos can be ubiquitinylated in vitro, the unambiguous demonstration that ubiquitinylation is necessary for its addressing to the proteasome in vivo is still lacking. c-Jun, one of the main dimerization partners of c-Fos within the AP-1 transcription complex, is also an unstable protein. Its degradation is clearly proteasome- and ubiquitin-dependent in vivo. Interestingly, several lines of evidence indicate that the addressing of c-Fos and c-Jun to the proteasome is, at least in part, governed by different mechanisms. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses under mutated forms which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of c-Fos main destabilizer but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. Though mutations in viral Fos proteins confer full resistance to proteasomal degradation, stabilization is limited because mutations also entail sensitivity to an unidentified proteolytic system. This observation is consistent with the idea that Fos-expressing viruses have evolved to ensure control protein levels to avoid high protein accumulation-linked apoptosis. In conclusion, the unveiling of the complex mechanism network responsible for the degradation of AP-1 family members is still at its beginning and a number of issues regarding the regulation of this process and the addressing to the proteasome are still unresolved.

Evasion from proteasomal degradation by mutated Fos proteins expressed from FBJ-MSV and FBR-MSV osteosarcomatogenic retroviruses.

c-Fos proto-oncoprotein is highly unstable, which is crucial for rapid gene expression shut-off and control of its intrinsic oncogenic potential. It is massively degraded by the proteasome in vivo in various situations. Although there is evidence that c-Fos can be ubiquitinylated in vitro, the unambiguous demonstration that ubiquitinylation is necessary for recognition and subsequent hydrolysis by the proteasome in vivo is still lacking. Moreover, genetic analysis have also indicated that c-Fos can be addressed to the proteasome via different mechanisms depending on the conditions studied. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses under mutated forms which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of a C-terminal c-Fos destabilizer but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. Though mutations in viral Fos proteins confer full resistance to proteasomal degradation, stabilization is limited because mutations also entail sensitivity to (an) unidentified proteolytic system(s). This observation is consistent with the idea that Fos-expressing viruses have evolved gene expression controls that avoid high protein accumulation-linked apoptosis.