Abstract
SCF ubiquitin ligases target phosphorylated substrates for ubiquitin-dependent proteolysis by means of adapter subunits called F-box proteins. The F-box protein Cdc4 captures phosphorylated forms of the cyclin-dependent kinase inhibitor Sic1 for ubiquitination in late G1 phase, an event necessary for the onset of DNA replication. The WD40 repeat domain of Cdc4 binds with high affinity to a consensus phosphopeptide motif (the Cdc4 phospho-degron, CPD), yet Sic1 itself has many sub-optimal CPD motifs that act in concert to mediate Cdc4 binding. The weak CPD sites in Sic1 establish a phosphorylation threshold that delays degradation in vivo, and thereby establishes a minimal G1 phase period needed to ensure proper DNA replication. Multisite phosphorylation may be a more general mechanism to set thresholds in regulated protein–protein interactions.
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Acknowledgements
We thank G. Gish for helpful discussions and initial peptide synthesis; A. Breitkreutz for assistance with phosphorylation reactions; A. Willems for assistance with modelling; and D. Durocher, A. Amon and L. Harrington for critical comments on the manuscript. This work was supported by grants from the Canadian Institutes of Health Research (CIHR), the National Cancer Institute of Canada (NCIC), the Protein Engineering Network of Centers of Excellence (PENCE) and MDS/Sciex (F.S., T.P. and M.T.), the Human Frontiers Science Program (M.T.), and from the National Institutes of Health (F.G. and M.D.M.). P.N. and X.T. are recipients of CIHR postdoctoral fellowship awards, T.P. is a Distinguished Scientist of the CIHR, M.T. is a Canada Research Chair in Biochemistry and F.S. is an NCIC Scientist.
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Supplementary Methods
Yeast strains and culture
Yeast strain construction, culture growth, mutagenesis and plasmid shuffle experiments was performed as described. Strains, plasmids and oligonucleotides used are listed in Tables S1, S2 and S3. All mutated regions were sequenced in their entirety. For Sic1 half-life experiments, cells bearing wild type and mutant alleles of SIC1HA under control of the GAL1 promoter integrated at the URA3 locus were arrested in G1 phase with a-factor, and induced with galactose for 4 h. After shift to repressive glucose medium, timepoints were processed for immunoblot analysis with an anti-HA antibody as described, except that detection was carried out by quantitative fluorescence on a Storm PhosphorImager. Half-life values for Sic1 mutants were determined by modeling the exponential decay of Sic1HA levels after 20 min to allow for the lag in Cln-Cdc28 kinase accumulation following release from a-factor. The values presented therefore estimate the half-life for Sic1 in the presence of active Cln-Cdc28 at the G1-S boundary. For expression of mutant SIC1 alleles at wild type levels, mutations were introduced into a plasmid based on MDM143(ref. 3), in which the URA3 gene was inserted at a BglII site 769 nucleotides downstream from the SIC1 stop codon to create pMT2702. Mutated regions of interest were cloned into pMT2702 as a SpeI to HpaI fragment encompassing nucleotides +65 to 792 of the SIC1 reading frame and integrated by direct transplacement into the chromosomal SIC1 locus. Note that the endogenous T2/5 phosphorylation sites encoded by the extreme 5' end of the SIC1 coding region were not eliminated due to cloning constraints. The presence of all integrated mutant sequences was confirmed by synthetic restriction sites co-introduced with each mutation. A colony colour sectoring assay was used to monitor rates of chromosome loss of wild type and SIC1CPD strains, as described. Integrated SIC1 and SIC1CPD alleles were tagged at the 3' end of the SIC1 reading frame with green fluorescent protein (GFP) by oligonucleotide directed recombination of a GFP-kan cassette. a-factor arrest-release, centrifugal elutriation and analysis of synchronous fractions were carried out as described previously. GFP positive nuclei were scored for at least 200 cells in unbudded and budded categories.
Recombinant proteins, binding reactions and kinase assays
SCF complexes were purified from Sf9 insect cells infected with recombinant baculoviruses and used in binding assays and ubiquitination reactions essentially as described. GST-Skp1 was expressed in BL21 Codon Plus cells (Stratagene), purified on glutathione resin and used to capture full-length Cdc4 from insect cell lysates. Truncated forms of Cdc4 expressed in in BL21 Codon Plus cells were enriched prior to GST-Skp1 capture by affinity purification of hexahistidine fusion proteins on a metal chelate column (Pharmacia). The Skp1-Cdc4 complexes were released from the GST moiety by cleavage with TEV protease and further purified by size exclusion chromatography on a Superdex S75 or S200 column. Biotin labeled ASPLPSGLLpTPPQSGKKQS, ASPLPSGLLTPPQSGKKQS, and APPLSQEpTFSDLWK were synthesized by addition of d-biotin (Sigma-Aldrich) with an Fmoc-e-aminocaproic acid (Bachem) spacer to carboxy-terminal peptides. Biotinylated peptides were purified by reverse-phase HPLC and confirmed by mass spectroscopy. Streptavidin-agarose beads (Sigma-Aldrich) were incubated in the presence of biotinylated peptide for 90 min at 4˚C and washed 3 times before incubation with lysates from Cdc4 expressing baculovirus-infected Sf9 cells. Captured complexes were washed 4 times and bound proteins resolved by SDS-PAGE then visualized by silver staining. Peptide out-competition of phosphorylated substrates was carried out with immunopurified Skp1FLAG-Cdc4 complexes on anti-FLAG resin (Sigma-Aldrich), which was incubated with phosphopeptides prior to addition of phospho-Sic1 or phospho-Cyclin E-Cdk2. In some experiments, high levels of Cln2-Cdc28 kinase generated a mixture of phosphorylated and hyperphosphorylated forms of Sic1, but each of these isoforms bound equivalently to Cdc4. Kinase inhibition assays were performed with purified recombinant Clb5-Cdc28 complex (30 ng), which was pre-incubated with mutant forms of Sic1 (40 ng) and histone H1 (2 µg) for 60 min at 0°C. Reactions were initiated by addition of 1 µCi [32P]-g-ATP and 50µM cold ATP in a reaction volume of 20 µl and incubated at 25°C for 30 min. Samples were separated by SDS-PAGE and visualized by autoradiography. Concentrations of Sic1 and Clb5-Cdc28 in the reactions were approximately 50 nM, well below that of the estimated 0.5-1 µM concentration of Sic1 in the yeast nucleus. For binding of phospho-Sic1 to Cdc4 and various mutants of Cdc4, Sic1 was purified as a His6-fusion protein, phosphorylated by Cln2-Cdc28 kinase and captured onto Skp1GST-Cdc4 glutathione resin.
Peptide Synthesis
The peptides ASPLPSGLLpTPPQSGKKQS, ASPLPSGLLpTPPQSGK, GLLpTPPQSG, LLpTPP, GLLpTPPQSG, GLLpSPPQSG, GLLpYPPQSG, GLLTPPQSG, GKLpTPPQSG, GLKpTPPQSG, GLLpTAPQSG, GLLpTPKQSG, GLLpTPPKSG, GLLpTPPQKG, GLLpTPPQSK, GLLpTPPK(Ac)SG, FLPpTPVLED, PKPLNLSKPIpSPPPSLKKTA, PPVpTPPMSP, VPVpTPSTTK, TGEFPQFpTPQEQLI, and VEQpTPKKPG were synthesized as described previously. Concatamer peptide sequences were produced from ligation of oligonucleotides listed in Table S3 to the 3’ end of the GST ORF to yield the following sequences:T45-3P, HMVTPSKPVTPSKPVTPSR; T45-6P, TPSKPVTPSKPVTPSRSPVTPSKPVTPSKPVTPSKL; T45-9P TPSKPVTPSKPVTPSRSPVTPSKPVTPSKPVTPSKLDPVTPSKPVTPSKPVTPSKT; T45–12P, TPSKPVTPSKPVTPSRSPVTPSKPVTPSKPVTPSKLDPVTPSKPVTPSKPVTPSKTRPVTPSKPVTPSKPVTPSK; S76-3P, HMGLTSPQRSPFPKSSPPRS;S76-6P, HMGLTSPQRSPFPKSSPPRSGLTSPQRSPFPKSSPPRLD ; S76-9P, HMGLTSPQRSPFPKSSPPRSGLTSPQRSPFPKSSPPRLDGLTSPQRSPFPKSSPPRTR; S76-12P, HMGLTSPQRSPFPKSSPPRSGLTSPQRSPFPKSSPPRLDGLTSPQRSPFPKSSPPRTRGLTSPQRSPFPKSSPPR. Soluble GST-(CPD)n fusion proteins (0.1 µg) were first incubated with Cln2-Cdc28 kinase in the presence of 10 µCi [32P]-g-ATP for 45 min at 30°C, followed by incubation with 2 mM cold ATP for a further 60 min. 32P-labeled GST fusion proteins were captured onto Skp1-Cdc4FLAG resin, washed and resolved by SDS-PAGE. Bound radioactive species were quantitated by PhosphoImager and normalized to 32P-labeled Sic1 (0.1 µg) captured under the same conditions.
Spectroscopic Techniques
Equilibrium binding constant determination was carried out using fluorescence polarization on a Beacon 2000 Fluorescence Polarization System (Pan Vera, WI) equipped with a 100µL sample chamber. Fluorescein-labeled probes were prepared through the reaction of carboxyterminal-peptides with 5-(and-6)-carboxyfluorescein succinimidyl ester (Molecular Probes), purified by reverse-phase HPLC, and confirmed by mass spectrometry. Binding studies were conducted with 5nM fluorescein-labeled probe dissolved in PBS containing 100µg/ml BSA and 1 mM dithiothreitol. Reaction mixtures were allowed to equilibrate for 10 minutes at room temperature prior to each measurement. All fluorescence polarization measurements were conducted at 22˚C. For competition binding studies, calculated IC50 values were obtained from the average of at least three independent experiments and are expressed as a percentage relative to the IC50 of the CycE9pT380 peptide.
Spots array synthesis
Peptide arrays were constructed according to the Spots-synthesis method. Acid-hardened cellulose membranes pre-derivatized with polyethylene glycol (AbiMed – Langfield, Germany) were spotted with a grid of Fmoc b-alanine (Bachem) prior to peptide synthesis. Standard Fmoc chemistry was used throughout. Fmoc protected and activated amino acids were spotted in high density 24 x 18 spot arrays on 130 x 90 mm membranes using an AbiMed ASP422 robot. All washing, Fmoc and side chain deprotection steps were done manually in polypropylene containers. The amino acids were at a concentration of 0.25 M and were twice spotted at a volume of 0.2 µl for each coupling reaction. Following peptide synthesis and side chain deprotection, membranes were blocked overnight in 5% skim milk. Purified Skp1-Cdc4 complex was added at 1µM in TBS and incubated for 1 hour at 4˚C. Membranes were washed three times in TBS and incubated with anti-Skp1 polyclonal antiserum for 30 min, followed by anti-rabbit HRP secondary antibody (Sigma) in TBS. Detection was by SuperSignal enhanced chemiluminescence (Pierce).
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Supplementary Figures
Supplementary Figure S1
(JPG 27. KB)
Cdc4 targets cyclin E1 in vitro and in vivo. a, Capture of recombinant Cdc4 from insect cell lysates, by phosphorylated and non-phosphorylated cyclin E1 derived peptides b, Phosphorylation-dependent ubiquitination of cyclin E1 by SCFCdc4 in vitro. Recombinant cyclin E1MYC6-Cdk2 complexes purified from transfected COS7 cells were incubated with 2 mM ATP for 60 min at 37ËšC to allow autophosphorylation prior to in vitro ubiquitination reactions. CycET380A indicates a mutant cyclin E1 that lacks the T380 phosphorylation site and Cdk2DN indicates a catalytically inactive version of Cdk2 c, Cyclin E1 degradation in yeast depends on Cdc4 function and on phosphorylation of T380. GAL1-cyclin E1 constructs were expressed in the indicated strains by growth in galactose medium then by repressed by addition of glucose and cycloheximide. Decay of cyclin E1 abundance was followed by anti-cyclin E1 immunoblot.
Supplementary Figure S2
(JPG 50.7 KB)
Absence of detectable secondary structure in Sic1. Circular dichroism analysis (inset) of either Sic1 or two N-terminal fragments of Sic1 did not reveal any cooperative folding units. Far -UV CD spectra (200 to 250 nm) were collected on an Aviv model 202 spectropolarimeter utilizing 1.0 mm path-length cells with a 5.0 s averaging time and a 1.0 nm step size (insets). Thermal unfolding experiments were carried out from 4ËšC to 90ËšC in PBS buffer, and monitored at a wavelength of 220nm.
Supplementary Figure S3
(JPG 105 KB)
Alignment of Cdc4 with homologs in C. albicans and S. pombe. Conserved basic amino acid residues are highlighted in blue and the triad of invariant arginine residues essential for Cdc4 binding to CycE9pT380 and phospho-Sic1 are circled in red. WD repeat units and amino acid residue numbers are noted.
Supplementary Figure S4
(JPG 8.84 KB)
Accumulation of Sic1 and Sic1CPD in cdc28-13 and cdc4-1 temperature sensitive strains. Two independent strains of the indicated genotype were incubated at the permissive or restrictive temperature for 3 h and immunoblotted with anti-Sic1 antibody.
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Nash, P., Tang, X., Orlicky, S. et al. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414, 514–521 (2001). https://doi.org/10.1038/35107009
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DOI: https://doi.org/10.1038/35107009
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