Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase Xiaojing Tanga, Stephen Orlickya, Tanja Mittagb,1, Veronika Csizmokb, Tony Pawsona,c, Julie D. Forman-Kayb,d,2, Frank Sicheria,c,d,2, and Mike Tyersa,e,2 aCenter for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, ON, Canada M5G 1X5; bProgram in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8; cDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; dDepartment of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; and eInstitute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC, Canada H3C 3J7 Edited by* Angelika Amon, Massachusetts Institute of Technology, Cambridge, MA, and approved November 21, 2011 (received for review October 7, 2011) The ubiquitin ligase SCFCdc4 (Skp1/Cul1/F-box protein) recognizes diversity may facilitate ubiquitination at multiple sites (18) and/ its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multi- or enable long-range electrostatic interactions with the positively site phosphorylation-dependent manner. Although short dipho- charged binding pocket on Cdc4 (7). sphorylated peptides derived from Sic1 can bind to Cdc4 with The human Cdc4 ortholog, called Fbw7, targets phosphory- high affinity, through systematic mutagenesis and quantitative lated forms of cyclin E, Myc, Jun, Notch, SREBP, and other pro- biophysical analysis we show that individually weak, dispersed teins (19). The phosphodegron described for Fbw7 is similar to Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The that of Cdc4, except that an additional phosphorylated residue is affinities of individual phosphoepitopes serve to tune the overall preferred at the P þ 4 position with respect to the primary (P0) phosphorylation site threshold needed for efficient recognition. phosphorylation site (20). The structure of Fbw7 bound to a Notably, phosphoepitope affinity for Cdc4 is dramatically wea- diphospho-CPD peptide from cyclin E revealed a secondary bind- kened in the context of full-length Sic1, demonstrating the impor- P þ 4 ing surface that engages the phosphate and thereby confers BIOCHEMISTRY tance of regional environment on binding interactions. The an overall high affinity for isolated diphosphorylated peptides multisite nature of the Sic1-Cdc4 interaction confers cooperative (21). As Cdc4 also favors a phosphorylated residue at the P þ 4 dependence on kinase activity for Sic1 recognition and ubiquitina- position in isolated peptides, it has been suggested that Cdc4/ tion under equilibrium reaction conditions. Composite dynamic Fbw7 substrates are recognized solely via diphospho-CPDs (21). interactions of low affinity sites may be a general mechanism However, it is not known if the P þ 4 interaction is required for to establish phosphorylation thresholds in biological responses. Sic1 elimination in vivo, nor how the P þ 4 requirement relates to the apparent multisite site recognition threshold. hosphorylation governs many of the protein interactions that Here we investigate these fundamentally different recognition Pdictate cellular responses to growth factors, stress conditions, models and show that the secondary phosphorylation sites in Sic1 and internal regulatory circuits. This control can be exerted are dispensable for multisite recognition, that regional context in through the generation of specific phosphoepitopes that are Sic1 can dramatically reduce the interaction affinity of a short recognized by cognate modular binding domains, the paradig- triphosphopeptide, and that Sic1 recognition and ubiquitination matic example of which is the recognition of pTyr epitopes by the exhibits cooperative dependence on targeting kinase activity SH2 domain (1). Multisite phosphorylation can serve to integrate under equilibrium reaction conditions. The Sic1-Cdc4 multisite signals and often confers nonlinear responses to signal inputs, recognition mechanism illustrates how a dynamic protein inter- referred to as cooperativity or ultrasensitivity (2, 3). The basis for action mode can engender complex system-level behavior. ultrasensitivity lies in the requirement for a threshold level of phosphorylation, which, in the presence of a countervailing phos- Results phatase, confers a nonlinear dependence on kinase activity (3). An Abrupt Phosphorylation-Dependent Transition in the Affinity of Multisite phosphorylation thresholds can derive from enzyme Sic1 for Cdc4. The nine weak CPD sequences in Sic1 all contain activation mechanisms (4), decoy phosphorylation sites (5), bulk mismatches to an optimal CPD motif (14, 15). Consistently, each electrostatic effects (6, 7), and zero-order enzyme saturation (8). of the seven natural CPD phosphopeptides derived from the Phosphorylation of the yeast cyclin-dependent kinase (CDK) NTR bound to a monomeric Skp1-Cdc4 complex with a Ki value inhibitor Sic1 in late G1 phase by G1 CDK (Cln-Cdc28) activity of about 100 μM or greater (Fig. 1A). Pairwise analysis of diphos- drives its recognition by the yeast F-box protein Cdc4, which phopeptides derived from the pS69/pS76/pS80 serine cluster serves to recruit Sic1 for ubiquitination by the core Skp1/Cul1/ F-box protein (SCF) ubiquitin ligase machinery (9–12). Versions of Sic1 that lack multiple CDK phosphorylation sites fail to bind Author contributions: X.T., S.O., T.M., V.C., J.D.F.-K., F.S., and M.T. designed research; X.T., Cdc4, are stabilized in vivo, and cause cell cycle arrest (13). Sic1 S.O., T.M., and V.C. performed research; X.T., S.O., T.M., and V.C. contributed new reagents/ analytic tools; X.T., S.O., T.M., V.C., J.D.F.-K., F.S., and M.T. analyzed data; and X.T., T.P., binds to the WD40 domain of Cdc4 only if a nominal set of six J.D.F.-K., F.S., and M.T. wrote the paper. of the nine CDK consensus sites are phosphorylated, termed The authors declare no conflict of interest. Cdc4 phosphodegron (CPDs) (14). This multisite threshold de- *This Direct Submission article had a prearranged editor. rives from hydrophobic and electrostatic conflicts of the natural Data deposition: Crystallography, atomic coordinates, and structure factors have been CPD sites with the Cdc4 binding pocket, which weakens the in- deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3V7D). dividual site affinities (15). Seven of the nine CPD sites lie in the 1Present address: Department of Structural Biology, St. Jude Children's Research Hospital, intrinsically disordered 90 residue N-terminal targeting region Memphis, TN 38105. (NTR) of Sic1; at least six of the CPD sites in the NTR interact 2To whom correspondence should be addressed. E-mail: [email protected], sicheri@ with Cdc4 in rapid equilibrium (16). An ensemble model of the lunenfeld.ca, or [email protected]. Sic1-Cdc4 complex suggests that the NTR interconverts between This article contains supporting information online at www.pnas.org/lookup/suppl/ dramatically different conformers (17). This conformational doi:10.1073/pnas.1116455109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1116455109 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 29, 2021 K μ region, which binds Cdc4 with a d of 2.4 M in a direct Trp fluor- escence binding assay (7), revealed that a pS76/pS80 peptide bound with similar affinity as the pS69/pS76pS80 peptide, whereas the pS69/pS76 and pS69/pS80 peptides bound with four to fivefold weaker affinities (Table S1). Separation of the pS76 and pS80 sites by an additional residue also reduced affinity by about fourfold. Notably, substitution of the P þ 1 proline adja- cent to either of the pS69 and pS80 sites with alanine decreased binding affinity, indicating that these sites likely contribute to binding by accessing the main P0 binding pocket, which requires a proline at the P þ 1 position (14, 15). Removal of the P þ 1 proline at the pS76 site reduced affinity by 30-fold, suggesting that pS76 is the dominant anchor site in the cluster. Although di- and triphosphorylated peptides exhibited much greater affinity than single CPD peptides, none of the isolated phosphopeptides matched the submicromolar affinity of the full-length NTR (Table S1). These data indicate that multiple sites and effects contribute to both local CPD affinities and the overall high affi- nity interaction of Sic1 with Cdc4. We used surface plasmon resonance (SPR) to assess the quan- titative changes in affinity of Sic1 for Cdc4 as progressively more CPD sites are added (14). A series of Sic1 mutants that contained either three, five, six, or all nine CPD sites were phosphorylated in vitro (called 3p, 5p1, 6p1, 6p2, WT) (see Fig. S1 for all Sic1 variants used in this study) and confirmed by phosphorylation- dependent alterations in isoelectric point (Fig. 1 A and B). This validated series of phosphoisoforms recapitulated the qualitative step-like transition for Sic1 capture onto Cdc4 resin and effects on cell viability (Fig. S2 A and B), as reported previously (14). Equal amounts of each Sic1 phosphoisoform were coupled to low density Biacore SPR sensor arrays (Fig. 1C, Inset) and probed with monomeric Skp1-Cdc4263–744 complex in solution. This con- figuration eliminated potential avidity effects that might arise from high ligand density or Skp1–Cdc4 dimerization (18). As shown by the raw association and dissociation curves, phosphor- ylation on the three or five most genetically important CPD sites on Sic1 (3p, T33/T45/S76; 5p1, T2/T5/T33/T45/S76) conferred little or no interaction signal, whereas phosphorylation on two different combinations of six CPD sites (6p1, T2/T5/T33/T45/ S69/S76; 6p2, T2/T5/T33/T45/S76/S80) or on all nine CPD sites yielded a robust interaction (Fig. 1C). The association rates for the 6p1, 6p2, or WT versions were at least 10-fold higher than that of 5p1, whereas the dissociation rates were similar amongst all phosphoisoforms. Quantitative determinations for each phos- phoisoform revealed that the 6p1, 6p2, and WT versions all K μ bound with a d of 0.4 M or lower (Table S2).