Composite Low Affinity Interactions Dictate Recognition of the Cyclin-Dependent Kinase Inhibitor Sic1 by the Scfcdc4 Ubiquitin Ligase

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 fluorescence 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 adjacent 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 affinity interaction of Sic1 with Cdc4. We used surface plasmon resonance (SPR) to assess the quantitative 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, phosphorylation 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 phosphoisoform revealed that the 6p1, 6p2, and WT versions all K μ bound with a d of 0.4 M or lower (Table S2). This analysis suggested an increase of at least 10-fold in binding affinity for the five-to-six site transition in this particular series of mutants.

Diphospho-CPD Sites are Neither Necessary Nor Sufficient for Sic1 Recognition. Three natural Sic1 CPD sites at T5/S9, T45/T48/T49, and S76/S80 are followed by a serine or threonine residue at the P þ 3 or P þ 4 position (Fig. 1A). Each of the diphosphorylated peptides exhibits a five to tenfold increase in affinity for Cdc4 compared to peptides phosphorylated only on the P0 site (21) Fig. 1. Quantitative analysis of the phosphoSic1-Cdc4 interaction. (A) Sche- (Table S1). These in vitro binding data raise the possibility that matic of Sic1 phosphorylation sites and mutant alleles used in this study. K high affinity diphospho-CPD sites may be necessary or even i values (micromolar) for singly phosphorylated peptides as determined sufficient for the interaction of Sic1 with Cdc4 (21). To test this by fluorescence polarization are shown for each CPD site in the NTR (see hypothesis, we first assessed combinations of Sic1 phosphoryla- Fig. S2C for inhibition curves). Values were not measured for T173 and S191. Effect of overexpression on cell viability is indicated by "+++" (viable) to "−" tion site mutations that altered the T5/S9, T45/T48/T49, and (inviable). Diphospho-CPD degrons are marked by orange bars with P þ 3 or S76/S80 sites. Sic1 that lacked all P þ 3 or P þ 4 sites P þ 4 residues in blue. (B) Indicated versions of Sic1 were phosphorylated 9 ∕ 48 ∕ 49 ∕ 80 (Sic1S A T A T A S A, termed no diphos) was recognized and with Cln2-Cdc28-Cks1, resolved by two-dimensional isoelectric focusing gel ubiquitinated by SCFCdc4 in vitro as effectively as WT Sic1 electrophoresis and detected by anti-Sic1 immunoblot. (C) Phosphorylated (Fig. 2 A and B, Fig. S3A). In contrast, versions that contained versions of Sic1 shown in B were coupled to CM4 low-density Biacore chips only a single pair of adjacent phosphorylated sites in the absence and analyzed for interactions with monomeric Skp1-Cdc4 complex. Levels of phospho-Sic1 immobilization are shown in the Inset. Where measurable, cal- 1S76∕S80 1S69∕S76 of all other sites, termed Sic or Sic , were neither culated K values are indicated next to each trace. On and off rate data is 4 d bound nor ubiquitinated by SCFCdc (Fig. 2 A and B), despite provided in Table S2.

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1116455109 Tang et al. Downloaded by guest on September 29, 2021 A B we determined the structure of a pS69/pS76/pS80 20-mer CPD T 263–744 W no diphosS76/S80S69/S76 1 4 -+No. sites phosphorylated + IEF triphosphopeptide bound to Skp -Cdc at 2.3-Å resolution

01 234562345 7 (Table S3). The pS76 and pS80 sites and adjacent residues were 73 ladder visible in the structure (Fig. S4A). The core CPD sequence G -L- Sic1- (Ub)n 77 S76/S80 T-pS-P engaged the primary P0 pocket in a similar manner as S69/S76 in the Cdc4-CPDcyclinE structure (15), but with weaker interac- unphos S76/S80 tions of the nonoptimal T75 residue in the important hydrophobic P-1 pocket. Notably, pS80 engaged the positively charged Sic1 22345 3 4 5 6 7 8 9 10 ladder surface adjacent to the primary P0 binding pocket, with hydrogen

bound no diphos bond interactions between the phosphate and residues R443, S464, T465, and R485 (Fig. S4 A and B). The Cdc4/Fbw7 residues input WT that form the P þ 4 engagement surface are highly conserved, WT no diphos WT 4p3 although the fission yeast homologue Pop2 contains isoleucine C diphos D instead of threonine at position 465, and may exhibit less favor- 5p2 3 Ala able interactions with the P þ 4 phosphate. The Sic1 diphospho- T48A 3 diphos CPD interaction with Cdc4 thus closely resembles the human cyclin E diphospho-CPD interaction with Fbw7. 4p7 To evaluate the role of the P þ 4 site in Sic1 recognition, we WT S69/S76/S80 6p2 5p3 mutated the three nonessential residues in Cdc4 that engage the P þ 4 phosphate (R443A, S464A, T465I), either alone or in com- 0p0p 3 diphos bination. Each Cdc4 mutant bound to a singly phosphorylated 4p4 WT CPD peptide as well as WT Cdc4 but had reduced affinity for a diphospho-CPD peptide (Fig. 3A), as shown previously for similar mutations in Fbw7 (21). Despite this defect in CPD recogni- Cdc4 P þ 4 Fig. 2. Interaction of Sic1 with SCF does not depend on phosphor- tion in vitro, each CDC4 allele, including the CDC4AAI triple ylation sites. (A) The indicated Sic1 proteins were phosphorylated and either CDC4 B 4FLAG Cdc4 mutant, complemented function in vivo (Fig. 3 ). The captured onto Cdc resin or incubated with complete SCF reaction CDC4AAI mixture and detected by anti-Sic1 immunoblot. (B) Input Sic1 phosphoiso- strain had normal cell morphology, but exhibited a slight

C BIOCHEMISTRY forms were detected by two-dimensional isoelectric focusing gel electrophor- accumulation of cells with 1N DNA content (Fig. 3 ). These re- esis followed by Coomassie blue stain. Partial Sic1 phosphorylation reactions sults demonstrate that the P þ 4 binding surface is not essential were used to generate a marker ladder. (C, D) Strains bearing the indicated for Cdc4 activity in vivo. GAL1-HASIC1 (C)orGAL1-SIC1 (D) alleles were grown for 2 d on 2% We then assessed the performance of the CDC4AAI allele in the galactose medium at 30 °C. face of excess SIC1 dosage in vivo. To titrate CDC4 capacity, a series of SIC1 phosphorylation site mutants expressed from the the moderately high affinity of the isolated diphosphorylated GAL1 promoter were evaluated for effects on viability. Whereas peptides for Cdc4 (21) (Table S1). WT CDC4 allowed cells to cope with overexpression of versions We then determined if the diphospho-CPD sites influenced of SIC1 that contained six or more phosphorylation sites, the elimination of Sic1 in vivo. Overexpression of partially stabilized CDC4AAI strain was unable to cope even with the loss of the weak forms of Sic1 causes a lethal G1 phase arrest, which is a sensitive CPD sites at T173 and S191 (Fig. 3D). As a control, a version of readout for Sic1 elimination in vivo (14). Strains that over- SIC1 S9A T49A S9A/S80A that was targeted solely by a high affinity singly phosphory- SIC1 SIC1 SIC1 CPD expressed the , ,or alleles were in- lated CPD derived from cyclin E, termed SIC1 (14), did not B AAI distinguishable from WT (Fig. S3 ), whereas overexpression of compromise growth of the CDC4 strain. From these genetic SIC1T48A SIC1S9A/T48A/S80A either or (no diphos) caused a mild titration experiments, we conclude that the P þ 4 pocket serves C B SIC1 growth defect (Fig. 2 , Fig. S3 ). Conversely, alleles that to tune the number of CPD sites needed for efficient Sic1 elim- encoded only the three diphospho-CPD sites, the high affinity ination, and that the weak CPD sites at T173 and S191 contribute serine cluster (S69/S76/S80) alone, or that lacked only the serine to Sic1 recognition in vivo. cluster (3 Ala) caused inviability when overexpressed, similar to a C D control that lacked all single CPD sites (Fig. 2 and ). These Effect of Regional Sequence Context on CPD Interaction Affinities. We effects on viability were paralleled by effects on Sic1 stability in used NMR to examine interactions of the isolated WD40 domain vivo (Fig. S3C). Addition of the T45 site to the serine cluster of Cdc4 (Cdc4WD40) with each CPD site in N15-labeled phos- (4p3) did not rescue viability, whereas further addition of the phorylated full-length Sic1. Resonances of each phosphorylated low affinity T33 site (5p2) restored growth (Fig. 2D). Surprisingly, residue have a pronounced downfield chemical shift due to the removal of the T2/T5 diphospho-CPD from the 6p1 and 6p2 electronegative influence of the phosphate (Fig. 4A). The titra- alleles (T33/T45/S69/S76 and T33/T45/S76/S80, termed 4p7 and tion of Cdc4WD40 against full-length Sic1 caused a similar degree 4p4, respectively) actually improved viability, whereas the mini- of resonance broadening for each CPD site in the NTR (Fig. 4B, mal T33/T45/S76 allele (3p) caused lethality (Fig. 2D, Fig. S2A). Fig. S5), as previously observed (16). Additional resonances cor- These data demonstrate that the originally inferred six site re- responding to T173 and S191, which lie outside of the NTR and quirement for Sic1 recognition is not absolute, but depends on immediately proximal to the CDK inhibitory domain of Sic1, the particular combination of four or more sites. Although the were also broadened by Cdc4WD40 (Fig. 4B), consistent with the pT2/pT5 diphospho-CPD site binds to Cdc4 with moderate genetic effects of T173 and S191 (Fig. 3D). This NMR analysis affinity in vitro (Table S1), these sites may antagonize Sic1 degra- demonstrates that the dynamic equilibrium engagement of multi- dation in vivo. Overall, we conclude that diphospho-CPD motifs ple CPD sites occurs in the context of full-length Sic1. are not required for the recognition and degradation of Sic1 in Despite the relatively high affinity interaction of the isolated vivo, despite the moderately high affinity interactions between pS69/pS76/pS80 triphosphopeptide with Cdc4 (7), this extended diphospho-CPD peptides and Cdc4 in vitro. degron was insufficient for Sic1 elimination in vivo (Fig. 2C). We speculated that the inherent affinity of the serine cluster de- The P þ 4 Binding Surface is Dispensable but Adjusts the Phosphoryla- gron might be reduced in its natural regional context. We used an tion Threshold. To ascertain that the diphospho-CPD binding intrinsic Trp fluorescence binding assay to directly compare the mode of yeast Cdc4 is analogous to that of human Fbw7 (21), affinity of the pS69/pS76/pS80 triphosphopeptide to that of

Tang et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 29, 2021 Fig. 3. The P þ 4 binding site on Cdc4 is dispensable in vivo but helps establish the precise phosphorylation threshold for Sic1 recognition. (A) Binding affinities of singly and doubly phosphorylated cyclin E-derived peptides with wild type and P þ 4 binding surface mutants of Cdc4. Values are the mean of three independent experiments SEM. (B) The indicated P þ 4 binding surface alleles on a hCDC4 TRP1 CENi plasmid were introduced into a cdc4ΔHIS3 hCDC4 URA3 CENi strain, streaked on either 5-fluorooro- tic acid medium to select against the WT allele or, as a control, on -Trp or -Ura medium and grown at 30 °C for 3 d. (C) WT and CDC4S464AT465IR443A strains were assessed for DNA content by FACS analysis (Upper) and microscopy (Lower) at early log phase in rich glucose medium. Approximately 1 and 4% of the cell population had an elongated- bud phenotype in WT and CDC4S464AT465IR443A strains, respectively. (D) Serial dilutions of WT or Cdc4S464AT465IR443A strains bearing the indicated GAL1-SIC1 alleles were spotted on glucose or 0.5% galactose medium and incubated at 30 °C for 3 d. Schematic shows phosphorylation sites in each allele; Inset shows sequence surrounding the high affinity CPD site.

full-length Sic1 that was phosphorylated only on the same three experiment with Sic1CPD, which requires only a single phosphor- 69∕ 76∕ 80 residues (Sic1S S S ). This comparison revealed that although ylation event for efficient capture by Cdc4 (14). Despite some K μ the isolated peptide had a d of 2.4 M for Cdc4, the affinity of nonspecific binding in the absence of phosphorylation in these the serine cluster degron was reduced by at least 25-fold, to an experiments, the Sic1CPD-Cdc4 interaction exhibited a more K > 50 μ estimated d M, when placed in the context of full-length graded dependence on CDK activity with an estimated nH of 1.8 Sic1 (Fig. 4C). The constraint on local degron affinity imposed by (Fig. 5 B and C). We then subjected a series of Sic1 phosphor- the NTR reveals the important role of regional context in disor- ylation reactions to a simultaneous SCFCdc4 ubiquitination reac- dered protein interactions. tion over a prolonged incubation period. Conversion of Sic1 to highly ubiquitinated species also exhibited a strongly cooperative Ultrasensitive Dependence of Sic1 Recognition on CDK Activity. The dependence on input CDK concentration (Fig. 5D). This step multisite phosphorylation threshold should render Sic1 recogni- transition in ubiquitination was manifest despite the opportu- tion and ubiquitination ultrasensitive with respect to CDK kinase nity for transiently phosphorylated forms of Sic1 to become ubi- activity, provided that phosphorylation is distributive and that a quitinated by SCFCdc4 and trapped as ubiquitinated species at competing phosphatase back-reaction establishes an equilibrium lower kinase concentrations. These data demonstrate that Sic1 distribution of phosphorylation states (3). We developed an in 4 recognition and ubiquitination by SCFCdc exhibits ultrasensitive vitro recombinant system for equilibrium phosphorylation of Sic1 dependence on the targeting kinase under the equilibrium reac- by titrating input Cln2-Cdc28-Cks1 against a fixed level of Cdc14, tion conditions expected to exist in vivo. which is a physiological phosphatase for Sic1 (22). Phosphoryla- tion of full-length untagged Sic1 by Cln2-Cdc28-Cks1 in these Discussion reactions was distributive (Fig. S6A) and equilibrium levels of Our biochemical, genetic and structural data suggest that the phosphorylation were obtained over prolonged incubation peri- multiple weak CPD sites in Sic1 engage Cdc4 in rapid dynamic ods in the presence of Cdc14 (Fig. S6 B and C), provided a con- equilibrium. This binding mode depends on manifold variables taminating insect phosphatase was inactivated with okadaic acid including the number, spacing, and individual affinities of differ- (Fig. S6D). The dependence of Sic1 phosphoisoform distribu- ent CPD sites, decoy sites, and regional sequence context effects. tions on input kinase concentration was confirmed by isoelectric These variables allow the recognition threshold to be tuned in a focusing, although the distribution balance varied in different substrate-specific fashion, such that interactions can be dictated kinase preparations (compare Fig. 5A to Fig. S6C). Sic1 from primarily by high affinity diphospho-CPD sites (19–21, 23), or by each phosphorylation reaction was captured on monomeric a combination of individually weak CPD sites, such as in Sic1 and SCF-Cdc4Flag resin and quantitated by immunoblot. This analysis other proteins (24, 25). This type of dynamic interaction contrasts revealed a step transition in the Sic1-Cdc4 interaction from neg- starkly with static engagement models that are typically invoked ligible to near maximal levels over a mere twofold increase in to explain regulated protein interactions (26). In this dynamic CDK concentration, with a calculated Hill coefficient (nH ) of 4.8 view, the X-ray structure of Cdc4 in complex with the pS76 and (Fig. 5C, Table S4). As a control, we carried out an analogous pS80 sites represents only a snapshot of one of many possible

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1116455109 Tang et al. Downloaded by guest on September 29, 2021 A - No. sites phosphorylated + IEF 0 1 2 3 4 5 6 7 8 9

0 kinase units

kinase units B 1 2 4 8 160 1 2 4 816

bound

unbound

Sic1 WT Sic1CPD C D 00.251 2 4 8 16kinase 35 units

30 Sic1-(Ub)n 25

15 Signal intensity 10 Sic1 WT, nH = 4.8 5 CPD, nH = 1.8 Cdc14Flag

0 2 4 6 8 10 12 14 16 18 Cdc4Flag Kinase units (SCF)

Cdc4

Fig. 5. Dependence of Sic1 recruitment and ubiquitination by SCF on BIOCHEMISTRY CDK activity under equilibrium reaction conditions. (A) Sic1 or Sic1CPD were incubated with the indicated arbitrary units of Cln2-Cdc28-Cks1 kinase in the presence of fixed Cdc14 phosphatase and SCFCdc4 resin at 30 °C for 60 min. Phospho-Sic1 species for the indicated reactions were resolved by two- dimensional isoelectric focusing gel electrophoresis and Coomassie blue stain. Minor Sic1 species detected between phosphoisoforms are due to partial carbamoylation during sample preparation. (B) Capture of Sic1 or Sic1CPD from reactions in A onto Cdc4 resin and detection by anti-Sic1 immunoblot (Upper) or Coomassie blue stain (Lower). (C) Quantitation of Sic1 or Sic1CPD captured onto Cdc4 and estimation of Hill coefficients (nH). Plots are average 40 of duplicate experiments. (D) Ubiquitination of Sic1 under equilibrium phos- Fig. 4. Dynamics of full-length Sic1-Cdc4WD interaction and regional con- 1 15 phorylation conditions. A similar but independent experiment as in A was text effects on CPD interaction affinity. (A) NMR HN- N correlation spectra performed except that components for SCFCdc4-dependent ubiquitination of phosphorylated residues of full-length Sic1 in the absence (black) and pre- 40 were included in the reaction and incubated at 30 °C for 60 min. sence (red) of Cdc4WD . Residues with multiple resonances from cis- and trans-prolyl isomers are denoted with a prime. (B) Intensity ratios of amide resonances of full-length Sic1 upon titration with unlabeled Cdc4WD40 at ylation of more than one site generate a cooperative response pSic1∶Cdc4WD40 molar ratios of 1∶0, 1∶0.1, 1∶0.25, 1∶0.5, 1∶0.75, and curve (3). Multisite phosphorylation can also lead to ultrasen- 1∶1.2, represented by lines of increasing darkness. Red stars indicate phos- sitive responses through preferred decoy sites, which serve to phorylation sites; red bar indicates serine cluster degron. Intensity ratios of increase the concentration of kinase needed to phosphorylate amide resonances of residues T173 and S191 are reported separately. (C)Ex- critical effector sites of lower affinity (5). In a variation on this perimental binding curves for an isolated pS69/pS76/pS80 triphosphopeptide (blue) and phosphorylated full-length Sic1S69∕S76∕S80 (red) compared to theo- phenomenon, the T2/T5 sites in Sic1 may act as decoys that con- retical fluorescence binding curves at 500 nM protein concentration for the tribute to binding but adversely affect a downstream step in the indicated affinity constants. As the titration of Skp1-Cdc4263–744 with phos- elimination of Sic1. Multiple phosphorylation events can also 1S69∕S76∕S80 K pho-Sic did not reach saturation, a numerical d could not be cal- engender switch-like effects by causing transitions in electrostatic culated. Data represent two independent experiments. potential. Charge neutralization of the highly basic NTR by the binding modes of pSic1. We speculate that the rapidly fluctuating addition of multiple phosphates has been posited to help estab- structure of Sic1 weakens the interactions of individual sites, such lish the threshold for Sic1 recognition (7). Similarly, multisite as the triphosphorylated serine cluster, in part through net repul- phosphorylation of the Ste5 scaffold protein cooperatively dis- sive electrostatic interactions (7). Two additional points are rupts its membrane localization through electrostatic repulsion worthy of emphasis. First, although Sic1 is a highly disordered between the multiple phosphates and the negatively charged protein, it nevertheless exhibits a surprisingly compact structure phospholipid membrane surface (6). Finally, the biochemical (17), which may contribute to its dynamic interactions with Cdc4 phenomenon of zero-order ultrasensitivity, first described for me- through electrostatic averaging effects (7). Second, although the tabolic enzymes (2), can also operate in signaling complexes SCFCdc4 holocomplex is a dimer in solution, the affinity of Cdc4 where the local concentration of phosphorylation sites exceeds for Sic1 does not depend on dimerization, which precludes a the catalytic capacity of the kinase (8). These different types model based on simultaneous engagement of two spatially sepa- of multisite dependencies all serve to enable cellular decision rated binding pockets (18). making processes. Although models based on entropic, electrostatic, and/or kinetic considerations have been proposed for the Multisite Phosphorylation and Switch-Like Biological Responses. En- Sic1-Cdc4 interaction (7, 27–29), the physicochemical basis for zyme activation or protein interactions that depend on phosphor- the precise multisite threshold remains to be determined.

Tang et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 29, 2021 Intrinsically Disordered Regions and Protein Recognition. Proteins plex was equilibrated in running buffer [10 mM Hepes (pH 7.3), 150 mM NaCl, with disordered regions often lie at the nexus of cellular regula- 3 mM EDTA, 0.005% Surfactant P2] and injected at between 29 and 1,860 nM tory networks (26, 30). Disordered regions can interact via at 100 μL∕ min for 2 min. Following 4 min of dissociation, two 1 min pulses of coupled folding and binding, via simple modular recognition of 2 M MgCl2 were applied to regenerate the surface. Sensorgrams were re- short linear sequence motifs or, as shown here, via low affinity corded on a Biacore 3000 and kinetic parameters extracted using BIAevalua- 1∶1 linear motifs that interact in rapid equilibrium. The spectrum tion Software v3.1. Binding curves were fit with a Langmuir interaction 2 ¼ 2 of potential binding modes afforded by disordered regions is par- model with Chi or better. For fluorescence polarization, unlabeled pep- ticularly suited to control by phosphorylation, which has a strong tides were competed against an N-terminal fluorescein-labeled cyclin E peptide (GLLpTPPQSG) in 50 mM Hepes (pH 7.5), 50 mM NaCl, 1 mM DTT, propensity to occur in such regions (31). The rapid equilibrium and 0.1 mg∕mL BSA (14). Intrinsic tryptophan fluorescence changes upon mode for multisite engagement is readily tuned to different bio- peptide addition to 50–500 nM Skp1-Cdc4263–744 were measured as described logical thresholds and also inherently able to integrate different (7). Immunoblot signal intensity was quantitated on a Fluor-S MultiImager signals through phosphorylation of different sites. Many disor- and curve fitting performed with GraphPad Prism software. dered regions harbor multiple short linear motifs that mediate moderate to low affinity interactions (30, 32, 33). The elucidation X-ray Structure and NMR Analysis. Skp1-Cdc4263–744 was purified and crystal- of the biophysical mechanisms that underlie the combinatorial lized as previously reported (15). Crystals of the apo form of Skp1-Cdc4263–744 weak interactions of linear motifs in disordered proteins with were grown and transferred to a solution containing 100 mM Tris (pH 8.5), their receptors will be critical for understanding how the cell con- 1.5 M ammonium sulfate, 15% glycerol, and 1 mM of Ac-GMTpSPFNGLT- structs precise thresholds in various biological responses (3, 26). pSPQRpSPFPKS-NH2 peptide for 10 min prior to freezing. Data were collected at BioCars 14-MBC at 0.9 Å and the structure solved by molecular replace- Materials and Methods ment with 1NEX. For NMR experiments, the WD40 domain of Cdc4 was Yeast Culture. Strains and plasmids used in this study are listed in Tables S5 and expressed from pET-Sumo (Invitrogen), isolated by Ni-nitrilotriacetate (NTA) S6. Methods for cell culture, genetic manipulation, protein extraction, immu- affinity chromatography, digested with Ulp protease, and further purified by noblot, and immunoprecipitation were carried out as described (15, 18). Ni-NTA and Superdex 200 chromatography. NMR titrations of the phosphorylated NTR and full-length Sic1 with Cdc4WD40 were performed on a Varian Binding and Ubiquitination Assays. Recombinant proteins were produced, Inova 500 MHz spectrometer at 5 °C. Assignments for NTR resonances were phosphorylated, and dephosphorylated as described (15, 18, 22). Sic1 was transferred to full-length Sic1. T173 and S191 were assigned by mutagenesis, phosphorylated by Cln2-Cdc28-Cks1 for 1 h at 30 °C in kinase buffer [50 mM and peak intensities in the heteronuclear single quantum correlation spectra 40 Hepes (pH 7.2), 55 mM NaCl, 1 mM ATP, 10 mM MgCl2, 1 mM DTT]. For equi- in the presence and absence of Cdc4WD were analyzed by FuDA (16). librium binding, Sic1, FLAGCdc4, and Cdc14 were mixed before addition of kinase. For ubiquitination, reactions were initiated by adding Cln2-Cdc28- Cdc4 ACKNOWLEDGMENTS. We thank Lewis Kay, Mikael Borg, and Hue-Sun Chan Cks1 to premixed Sic1, Cdc14, human E1, Cdc34, SCF , and ubiquitin in for discussions. Supported by grants from the Canadian Cancer Society kinase buffer. For SPR assays, purified Sic1 isoforms bearing a C-terminal Research Institute (J.D.F.-K.) and the Canadian Institutes of Health Research cysteine were phosphorylated then diluted approximately 10-fold with im- (CIHR) (T.P., J.D.F.-K., F.S., and M.T.). Support was provided by the Terry Fox mobilization buffer (10 mM NaOAc, pH 5.0) to 25 μg∕mL and immobilized Foundation (T.M.), the CIHR Strategic Training Program in Protein Folding to a CM4 sensor chip (GE Healthcare) at 5 μL∕ min using thiol coupling to and Interaction Dynamics (V.C.), and by Canada Research Chairs in Structural a level of approximately 100 RU. Analyte Skp1-Cdc4263–744 monomeric com- Biology (F.S.) and in Systems and Synthetic Biology (M.T.).

1. Pawson T (1995) Protein modules and signaling networks. Nature 373:573–580. 18. Tang X, et al. (2007) Suprafacial orientation of the SCFCdc4 dimer accommodates 2. Goldbeter A, Koshland DE, Jr (1984) Ultrasensitivity in biochemical systems controlled multiple geometries for substrate ubiquitination. Cell 129:1165–1176. by covalent modification. Interplay between zero-order and multistep effects. J Biol 19. Welcker M, Clurman BE (2008) FBW7 ubiquitin ligase: A tumor suppressor at the Chem 259:14441–14447. crossroads of cell division, growth and differentiation. Nat Rev Cancer 8:83–93. 3. Ferrell JE, Jr (1996) Tripping the switch fantastic: How a protein kinase cascade can 20. Welcker M, et al. (2003) Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E convert graded inputs into switch-like outputs. Trends Biochem Sci 21:460–466. degradation. Mol Cell 12:381–392. 4. Ferrell JE, Jr, Machleder EM (1998) The biochemical basis of an all-or-none cell fate 21. Hao B, et al. (2007) Structure of a Fbw7-Skp1-cyclin E complex: Multisite-phosphory- switch in Xenopus oocytes. Science 280:895–898. lated substrate recognition by SCF ubiquitin ligases. Mol Cell 26:131–143. 5. Kim SY, Ferrell JE, Jr (2007) Substrate competition as a source of ultrasensitivity in the 22. Visintin R, et al. (1998) The phosphatase Cdc14 triggers mitotic exit by reversal of inactivation of Wee1. Cell 128:1133–1145. Cdk-dependent phosphorylation. Mol Cell 2:709–718. 6. Strickfaden SC, et al. (2007) A mechanism for cell-cycle regulation of MAP kinase 23. Bao MZ, et al. (2010) Multisite phosphorylation of the Saccharomyces cerevisiae fila- signaling in a yeast differentiation pathway. Cell 128:519–531. mentous growth regulator Tec1 is required for its recognition by the E3 ubiquitin li- 7. Borg M, et al. (2007) Polyelectrostatic interactions of disordered ligands suggest a gase adaptor Cdc4 and its subsequent destruction in vivo. Eukaryotic Cell 9:31–36. physical basis for ultrasensitivity. Proc Natl Acad Sci USA 104:9650–9655. 24. Moberg KH, et al. (2004) The Drosophila F box protein archipelago regulates dMyc 8. Malleshaiah MK, Shahrezaei V, Swain PS, Michnick SW (2010) The scaffold protein Ste5 protein levels in vivo. Curr Biol 14:965–974. directly controls a switch-like mating decision in yeast. Nature 465:101–105. 25. Liu Q, et al. (2011) SCFCdc4 enables mating type switching in yeast by cyclin-dependent 9. Bai C, et al. (1996) SKP1 connects cell cycle regulators to the ubiquitin proteolysis kinase-mediated elimination of the Ash1 transcriptional repressor. Mol Cell Biol machinery through a novel motif, the F-box. Cell 86:263–274. 31:584–598. 10. Feldman RM, Correll CC, Kaplan KB, Deshaies RJ (1997) A complex of Cdc4p, Skp1p, 26. Boehr DD, Nussinov R, Wright PE (2009) The role of dynamic conformational ensem- and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p. bles in biomolecular recognition. Nat Chem Biol 5:789–796. Cell 91:221–230. 27. Varedi KS, Ventura AC, Merajver SD, Lin XN (2010) Multisite phosphorylation provides 11. Skowyra D, et al. (1997) F-box proteins are receptors that recruit phosphorylated an effective and flexible mechanism for switch-like protein degradation. PLoS One 5: substrates to the SCF ubiquitin-ligase complex. Cell 91:209–219. e14029. 12. Petroski MD, Deshaies RJ (2005) Function and regulation of cullin-RING ubiquitin 28. Lenz P, Swain PS (2006) An entropic mechanism to generate highly cooperative and ligases. Nat Rev Mol Cell Biol 6:9–20. specific binding from protein phosphorylations. Curr Biol 16:2150–2155. 13. Verma R, et al. (1997) Phosphorylation of Sic1p by G1 Cdk required for its degradation 29. Klein P, Pawson T, Tyers M (2003) Mathematical modeling suggests cooperative and entry into S phase. Science 278:455–460. interactions between a disordered polyvalent ligand and a single receptor site. Curr 14. Nash P, et al. (2001) Multisite phosphorylation of a CDK inhibitor sets a threshold for Biol 13:1669–1678. the onset of DNA replication. Nature 414:514–521. 30. Gibson TJ (2009) Cell regulation: Determined to signal discrete cooperation. Trends 15. Orlicky S, et al. (2003) Structural basis for phosphodependent substrate selection and Biochem Sci 34:471–482. orientation by the SCFCdc4 ubiquitin ligase. Cell 112:243–256. 31. Iakoucheva LM, et al. (2004) The importance of intrinsic disorder for protein phosphor- 16. Mittag T, et al. (2008) Dynamic equilibrium engagement of a polyvalent ligand with a ylation. Nucleic Acids Res 32:1037–1049. single-site receptor. Proc Natl Acad Sci USA 105:17772–17777. 32. Mittag T, Kay LE, Forman-Kay JD (2010) Protein dynamics and conformational disorder 17. Mittag T, et al. (2010) Structure/function implications in a dynamic complex of the in molecular recognition. J Mol Recognit 23:105–116. intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase. Structure 33. Hazy E, Tompa P (2009) Limitations of induced folding in molecular recognition by 18:494–506. intrinsically disordered proteins. ChemPhysChem 10:1415–1419.

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