Control of cell cycle-dependent degradation of c-Ski proto-oncoprotein by Cdc34

Abstract

It is known that excess amounts of Ski, or any member of its proto-oncoprotein family, causes disruption of the transforming growth factor beta signal transduction pathway, thus causing oncogenic transformation of cells. Previous studies indicate that Ski is a relatively unstable protein whose expression levels can be regulated by ubiquitin-mediated proteolysis. Here, we investigate the mechanism by which the stability of Ski is regulated. We show that the steady-state levels of Ski protein are controlled post-translationally by cell cycle-dependent proteolysis, wherein Ski is degraded during the interphase of the cell cycle but is relatively stable during mitosis. Furthermore, we demonstrate that the ubiquitin-conjugating enzyme Cdc34 mediates cell cycle-dependent Ski degradation both in vitro and in vivo. Overexpression of dominant-negative Cdc34 stabilizes Ski and enhances its ability to antagonize TGF-b signaling. Our data suggest that regulated proteolysis of Ski is one of the key mechanisms that control the threshold levels of this proto-oncoprotein, and thus prevents epithelial cells from becoming TGF-b resistant.

The Ski oncoprotein is upregulated and localized at the centrosomes and mitotic spindle during mitosis

“…This degradation seems to occur in G0/G1 phase, as the lowest levels of Ski were observed in those phases of the cell cycle. This result agrees with recently published data (Macdonald et al, 2004).…”

“…Recently, Macdonald et al (2004) showed a higher stability of Ski protein in Xenopus egg extracts prepared from the mitotic phase of the cell cycle. Here we show that the highest levels of Ski and the phosphorylated form of Ski were present in nocodazole-prophase arrested cells, a time when the (Nigg, 2001), and that the Ski protein level correlated directly with the levels of cyclin B.…”

Transforming Growth Factor β Depletion Is the Primary Determinant of Smad Signaling Kinetics

“…First, we established the dose-response relationship between TGF-␤ concentration and luciferase reporter activity in mink lung epithelial cells stably infected with a luciferase reporter gene driven by a Smad-sensitive promoter (hereafter referred to as PE25 cells [30]) (Fig. 1A).…”

Cdh1-Anaphase-Promoting Complex Targets Skp2 for Destruction in Transforming Growth Factor β-Induced Growth Inhibition

“…SnoN and Ski, two corepressors of transcription, are rapidly removed upon stimulation with TGF-␤ in order to initiate TGF-␤-induced transactivation. In particular, degradation of SnoN has been reported to require the involvement of anaphase-promoting complex/cyclosome (APC/C), whereas destruction of Ski is suggested to be through the SCF (Skp1-Cul1-F-box) complex (21,38,43). Our recent studies systematically measuring protein profiles in response to TGF-␤ stimulation show that several proteins are significantly altered in a rapid manner that suggests protein degradation (unpublished data).…”