Abstract
Cks is an evolutionarily conserved protein that regulates cyclin-dependent kinase (CDK) activity. Clarifying the underlying mechanisms and cellular contexts of Cks function is critical because Cks is essential for proper cell growth, and its overexpression has been linked to cancer. We observe that budding-yeast Cks associates with select phosphorylated sequences in cell cycle–regulatory proteins. We characterize the molecular interactions responsible for this specificity and demonstrate that Cks enhances CDK activity in response to specific priming phosphosites. Identification of the binding consensus sequence allows us to identify putative Cks-directed CDK substrates and binding partners. We characterize new Cks-binding sites in the mitotic regulator Wee1 and discover a new role for Cks in regulating CDK activity at mitotic entry. Together, our results portray Cks as a multifunctional phosphoadaptor that serves as a specificity factor for CDK activity.
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Acknowledgements
The authors acknowledge R. Cook of the Massachusetts Institute of Technology Biopolymers group for synthesis of peptide arrays and E. van Veen for providing valuable advice regarding peptide arrays. The authors thank E. Chen for assistance with CD experimental design and analysis. This research was supported by funding to S.M.R. from the American Cancer Society (RSG-12-131-01-CCG) and by a targeted financing scheme and an institutional grant IUT2-21 from the Estonian government to M.L.
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D.A.M., E.R.M.B., M.K., R.L., D.R.K., M.L. and S.M.R. designed the study. D.A.M., E.R.M.B., M.K., R.L., M.V.M. and A.H. performed experiments. All authors analyzed data. D.A.M., E.R.M.B. and S.M.R. wrote the manuscript.
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Supplementary Figure 2 Cks1 alkylation control.
Alkylation of Cks1 does not abrogate binding of phosphopeptides.The wild-type phosCdc62-9 peptide was probed with Cks1 and alkylated Cks1 as described for Figure 2a. Alkylation, which was confirmed by mass spectrometry, occurs on a single cysteine that is distal to the phosphate-binding pocket.
Supplementary Figure 3 Solution behavior of fusion crystallization construct.
Domain swapping and binding properties of the Cks1-Cdc6 fusion construct. (a) ITC data demonstrate that the phosCdc63-9-Cks11-112 fusion protein does not bind phosphorylated Cdc6 peptide in trans (left), while the unphosphorylated fusion binds the Cdc6 peptide similar to wild-type Cks1 (right). These measurements demonstrate that the phosCdc6 sequence in the fusion can bind Cks1 in cis at the appropriate site and exclude the added peptide. (b) In the crystal structure, the fusion undergoes domain swapping in which the phosCdc6 sequence at the C-terminus of one fusion molecule extends to bind the cationic pocket site on a different Cks1 molecule, related by crystallographic symmetry. Four molecules (cyan) are observed in the asymmetric unit. Unlike in several other Cks crystals34,35, there is no evidence that Cks11-112-phosCdc63-9 forms domain-swapped dimers by exchanging strand 4, although the electron density for the loop preceding strand 4 is weak. Instead, dimers are formed between a molecule in the asymmetric unit (cyan) and its crystallographic symmetry mate (yellow) by the swapping of appended phosCdc63-9 sequences. (c) Superdex 75 gel filtration analysis demonstrates that the phosphorylated fusion behaves as a monomer in gel filtration. The unphosphorylated fusion (maroon line) elutes from gel filtration as a double peak, consistent with a monomer-dimer equilibrium in solution and suggesting that the construct undergoes domain swapping similar to Cks1. The phosphorylated Cks11-112-phosCdc63-9 (blue dashes) elutes as a single peak and in the same volume as the monomer-promoting mutant Cks11-112/P93A-Cdc63-9 (green dots)55. We conclude that Cks11-112-phosCdc63-9 fusion protein is a monomer in solution. It is likely that the phosphorylated Cdc6 sequence binds to Cks1 in cis, which disrupts the domain-swapped dimerization that occurs through strand 4 in the wild-type protein in vitro at high concentrations.
Supplementary Figure 4 Cks-mutant structure-function controls.
Mutations to Cks that disrupt consensus sequence binding do not disrupt folding or Cdk binding. (a) Circular dichroism spectra of wild-type and mutant Cks1 proteins at a concentration of ~10 μM were obtained as previously described55. The spectra demonstrate that the mutations do not disrupt proper folding of Cks1. (b) Binding of Cks1 consensus site mutants to recombinant human Cdk2. The Cks-Cdk interface is highly conserved, and budding yeast Cks1 binds Cdk2 with micromolar affinity55,56. We used Cdk2 to facilitate the in vitro binding experiment with recombinant proteins. We mixed 12.5 μM purified His6-Cdk2 with 12.5 μM of the indicated GST-tagged Cks1 protein in a buffer containing 150 mM NaCl, 25mM Tris, and 1 mM DTT (pH 8). The 1 mL binding reaction was mixed with 100 μL GS4B-Sepharose beads and the precipitated beads were washed with binding buffer and eluted with SDS-PAGE sample loading buffer. Input (I), flow-through unbound (U), and bound (B) protein fractions were run on SDS-PAGE and stained with Coomassie Blue. Wild-type and consensus site mutants all bind Cdk2 similarly, which is consistent with the structural observation that the phosphopeptide and Cdk binding interfaces are on opposite faces of Cks (Figure 3a).
Supplementary Figure 5 Kinetic priming assay controls.
Comparison in Cdk kinase reaction of unprimed to primed substrate. Kinase reactions are carried out as described in Figure 4 with wild-type Cks. Both the H1 peptide and Sic1ΔC-T5 construct contain single phosphoacceptor sites. In the Sic1ΔC-T5 construct, all the phosphoacceptor sites except T5 are mutated. The Cdc6 peptide contains phosphorylated T7 and a phosphoacceptor site at T23. The kcat for the reaction with the H1 peptide (marked with *) was previously determined9. Absolute kcat values reported here and in Figure 4 were calculated by comparison of the Vmax value in each experiment to the Vmax determined here for the H1 peptide.
Supplementary Figure 6 Cks1-hsCks1 structure comparison.
Comparison of the phosCdc6-Cks1 structure determined here with the structure of phosp27-hsCks1-Skp2 (PDB code: 2AST)37.
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Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Table 1 (PDF 8203 kb)
Supplementary Table 2
Putative Cks-binding sequences (XLSX 32 kb)
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McGrath, D., Balog, E., Kõivomägi, M. et al. Cks confers specificity to phosphorylation-dependent CDK signaling pathways. Nat Struct Mol Biol 20, 1407–1414 (2013). https://doi.org/10.1038/nsmb.2707
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DOI: https://doi.org/10.1038/nsmb.2707
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