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Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates

Abstract

Cell-cycle events are controlled by cyclin-dependent kinases (CDKs), whose periodic activation is driven by cyclins. Different cyclins promote distinct cell-cycle events, but the molecular basis for these differences remains unclear1,2. Here we compare the specificity of two budding yeast cyclins, the S-phase cyclin Clb5 and the M-phase cyclin Clb2, in the phosphorylation of 150 Cdk1 (Cdc28) substrates. About 24% of these proteins were phosphorylated more efficiently by Clb5–Cdk1 than Clb2–Cdk1. The Clb5-specific targets include several proteins (Sld2, Cdc6, Orc6, Mcm3 and Cdh1) involved in early S-phase events. Clb5 specificity depended on an interaction between a hydrophobic patch in Clb5 and a short sequence in the substrate (the RXL or Cy motif). Phosphorylation of Clb5-specific targets during S phase was reduced by replacing Clb5 with Clb2 or by mutating the substrate RXL motif, confirming the importance of Clb5 specificity in vivo. Although we did not identify any highly Clb2-specific substrates, we found that Clb2–Cdk1 possessed higher intrinsic kinase activity than Clb5–Cdk1, enabling efficient phosphorylation of a broad range of mitotic Cdk1 targets. Thus, Clb5 and Clb2 use distinct mechanisms to enhance the phosphorylation of S-phase and M-phase substrates.

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Acknowledgements

We thank E. Woodbury for Fin1 reagents and assistance; J. Ubersax for numerous reagents and discussions; K. Shokat for N6-(benzyl)ADP, E. Phizicky for the GST open reading frame library; E. K. O'Shea and J. S. Weissman for TAP-tagged strains; and C. Carroll, L. Holt, M. Matyskiela, D. Randle and other members of the Morgan laboratory for discussions and comments on the manuscript. This work was supported by funding from the National Institute of General Medical Sciences.

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Correspondence to David O. Morgan.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Table 1

Cyclin specificity in the phosphorylation of 150 Cdk1 substrates. (XLS 49 kb)

Supplementary Table 1 Legend (DOC 22 kb)

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Figure 1: Identification of Clb5-specific Cdk1 substrates in budding yeast.
Figure 2: Clb5 specificity depends on an interaction between the Clb5 hydrophobic patch and an RXL motif in the substrate.
Figure 3: Clb5-specific substrate phosphorylation occurs in vivo.

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