Cellular and viral Fos proteins are degraded by different proteolytic systems

Abstract

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-Fos FBJ and v-Fos FBR 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-Fos FBJ and v-Fos FBR 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 virallyintroduced 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 di erent proteolytic systems in living cells. Oncogene (2001) 20, 942 ± 950.

Identification of a C-terminal tripeptide motif involved in the control of rapid proteasomal degradation of c-Fos proto-oncoprotein during the G0-to-S phase transition

“…Pulsechase experiments, with a chase starting 1.5 h postserum addition, indicated a twofold stabilization of the c-FosDPEST3 protein with a half-life shift from 21+1 to 44+7 minutes ( Figure 2C). Because we have recently shown that the mutations carried by Fos proteins expressed from FBR-MSV and FBJ-MSV oncogenic retroviruses confer resistance to proteasomal degradation which is partially compensated by acquisition of sensitivity to another still-to-be-identi®ed protease (Acquaviva et al, 2001), it was important to investigate whether the three PEST deletion mutants were still subjected to proteasomal degradation. To this aim, synchronization experiments were conducted in the presence of the cell-permeant inhibitors chloroquine, which inhibits lysosomal proteases, E64D, which inhibits a variety of cysteine proteases and MG132, which is a potent inhibitor of the proteasome.…”

“…Balb/c 3T3 cell c-fos mRNA was detected with a mouse-speci®c probe (Plasmid PM381 in ref. (Acquaviva et al, 2001) whereas the ectopic RNA was detected using the c-Fos DNA sequence constructed by Abate et al (1990).…”

“…Understanding the mechanisms whereby c-Fos protein is degraded is thus important since accurate destruction of the protein it encodes participates to keep down the potential oncogenic activity of the c-fos gene. In this respect, it is interesting to note that mutated viral proteins expressed from FBR-MSV and FBJ-MSV are signi®cantly more stable than c-Fos, which permits their accumulation to higher levels (Acquaviva et al, 2001;.…”

“…Interestingly, viral proteins expressed by FBR-MSV and FBJ-MSV are no longer sensitive to proteasomal degradation because of the mutations they have accumulated. Their stabilization is however limited because the same mutations that confer resistance to the proteasome also render them sensitive to other, still to be identi®ed, protease(s) (Acquaviva et al, 2001). Lysosomes and calpains have also been suggested to contribute to cFos breakdown (Aniento et al, 1996;Carillo et al, 1994;Hirai et al, 1991;Salvat et al, 1999;Watt and Molloy, 1993).…”

“…Moreover, ubiquitinylated proteins can sometimes be recognized and degraded independently of their ubiquitin moiety (Shea et al, 2000;Verma and Deshaies, 2000). The proteasome has been shown to play a crucial role in c-Fos destruction in a variety of cell lines during the G 0 -to-S phase transition (Salvat et al, 1998) and asynchronous growth (Acquaviva et al, 2001) as well as in a lymphoma cell line undergoing apoptosis (He et al, 1998). Interestingly, viral proteins expressed by FBR-MSV and FBJ-MSV are no longer sensitive to proteasomal degradation because of the mutations they have accumulated.…”