Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCF^Cdc4ubiquitin ligase

Abstract

The ubiquitin ligase SCF Cdc4 (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses.

Multisite Light-Induced Phosphorylation of the Transcription Factor PIF3 Is Necessary for Both Its Rapid Degradation and Concomitant Negative Feedback Modulation of Photoreceptor phyB Levels in Arabidopsis

“…Most notable perhaps is Sic1, involved in cell cycle regulation in yeast, where phosphorylation of a minimum of six residues appears to set a threshold for SCF E3-ligase recognition, thereby providing a sensitive switch mechanism for the induction of the G1/S transition (Deshaies and Ferrell, 2001;Nash et al, 2001). The molecular basis for this multisite recognition in this and other systems has not been resolved, but possibilities include the cooperative action of multiple low-affinity sites that bind to multiple basic cavities in the E3-ligase substrate binding subunit (Nash et al, 2001;Orlicky et al, 2003;Hao et al, 2007;Deshaies and Joazeiro, 2009;Bao et al, 2010;Varedi K et al, 2010;Zhao et al, 2010;Kõivomägi et al, 2011;Tang et al, 2012). Although the number of phosphorylation sites apparently functionally involved in light-induced PIF3 degradation, identified here by targeted mutagenesis, appears large compared with these other reported systems (Varedi K et al, 2010), our data suggest that a smaller subset of these residues (e.g., those identified in PIF3-A6: Ser-58, Ser-88, Ser-102, Ser-151, Ser-152, and Ser-153) may contribute more strongly to the collective activity of the sites than other residues ( Figures 5E and 5F).…”

Hst3 is turned over by a replication stress-responsive SCF Cdc4 phospho-degron

“…Recent work on the canonical Cdc4 target Sic1 shows that maximal degradation of Sic1 by SCF Cdc4 also requires multiple phospho-degrons (36,37). We find that in Hst3, two phosphorylation sites-S420 and S421-are required for maximal turnover even though the two sites alone are not sufficient for any turnover.…”

“…Cdc4 often recognizes its substrates through a phosphodegron containing two phosphorylation sites spaced closely together (20,21,23), although data on Sic1 in budding yeast suggests that this spacing may not be strictly required (36). We find that maximal degradation of Hst3 requires two such phosphodegrons, and each phospho-degron contributes to the increased turnover in response to replication stress.…”

CRL4-like Clr4 complex in Schizosaccharomyces pombe depends on an exposed surface of Dos1 for heterochromatin silencing

“…Dos1WD shows the strongest structural similarity to the human F-box protein Fbw7 and its homolog in budding yeast, Cdc4, which function in cell cycle regulation by guiding the ubiquitylation of cyclin E (28,29). The putative substrate-binding surface of Dos1WD, the narrow side of the toroid, shows an electrostatic surface charge that is predominantly negative (Fig.…”