Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism

Abstract

Ubiquitin-dependent degradation of regulatory proteins controls many cellular processes, including cell cycle progression, morphogenesis, and signal transduction. Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin ligases composed of a core complex including Skp1p, Cdc53p, one of multiple F-box proteins that are thought to provide substrate specificity to the complex, and the ubiquitinconjugating enzyme, Cdc34p. It is not understood how SCF complexes are regulated and how physiological conditions alter their levels. Here we show that three F-box proteins, Grr1p, Cdc4p, and Met30p, are unstable components of the SCF, and are themselves degraded in a ubiquitin-and proteasome-dependent manner in vivo. Ubiquitination requires all the core components of the SCF and an intact F-box, suggesting that ubiquitination occurs within the SCF complex by an autocatalytic mechanism. Cdc4p and Grr1p are intrinsically unstable, and their steady-state levels did not f luctuate through the cell cycle. Taken together, our results suggest that ubiquitin-dependent degradation of F-box proteins allows rapid switching among multiple SCF complexes, thereby enabling cells to adapt quickly to changing physiological conditions and progression through different phases of the cell cycle.Post-translational modification by ubiquitin targets many proteins for rapid degradation by the 26S proteasome (1). Covalent attachment of ubiquitin onto lysine residues of the substrate requires the coordinated action of three classes of enzymes: the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin ligases (2). Whereas the E1 and E2 enzymes are primarily involved in activating and transferring ubiquitin through high-energy thioester bonds, E3 enzymes play the critical step in providing the specificity of substrate recognition. At least two multiprotein complexes function as E3-ubiquitin ligases for many cell cycle regulators (3): the anaphase-promoting complex

Mutant B-RAF signaling and cyclin D1 regulate Cks1/S-phase kinase-associated protein 2-mediated degradation of p27Kip1 in human melanoma cells

“…This notion is consistent with studies by showing that overexpression of p107 downregulates Skp2 levels in fibroblast cells (Rodier et al, 2005). F-box proteins are known to be intrinsically unstable (Galan and Peter, 1999;Wirbelauer et al, 2000). We show that Skp2 levels are regulated by Cks1.…”

Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

“…This was described, for example, for the self-ubiquitination of the HECT ligase Rsp5, 12 and the F-box protein Grr1p. 16 Consistent with the concept of intramolecular modification, it appears that the self-ubiquitinating activity of ITCH and other HECT ligases like NEDD4-1, NEDD4-2, SMURF2, and WWP1, is regulated through intramolecular interactions that are modulated by modifications such as phosphorylation, and that involve the HECT domain. [17][18][19] In an attempt to decipher the biological role of self-ubiquitination, it was proposed that it serves to target the ligase for degradation, 11 which has been observed indeed as a means of negative feedback for Mdm2, 6,20 E6-AP, 21 CBL ligases, 22 and the substrate receptor subunits of CRL complexes.…”

“…[34][35][36][37] Interestingly, it was observed that the degradation of several F-box proteins is attenuated by their respective substrates. 16,34,36 It was recently uncovered that a WD40 repeat, which is present in a large subset of substrate receptors, can bind ubiquitin. This ubiquitin-binding property was required for self-ubiquitination and degradation of Cdc4 (cell division cycle 4).…”

Auxin signaling and regulated protein degradation

“…Although post-transcriptional or post-translational regulation of SCF TIR1 and/or the RUB pathway proteins is a possibility, no such activity has been identified. However, it has been found that some F-box proteins are generally unstable owing to autoubiquitination [28,29]. Although there are no data available on the stability of TIR1, it is possible that the TIR1 protein is degraded through autoubiquitination in the absence of auxin (Figure 1).…”