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Sequential phosphorylation and multisite interactions characterize specific target recognition by the FHA domain of Ki67

Nature Structural & Molecular Biology volume 12, pages 987–993 (2005)Cite this article

Abstract

The forkhead-associated (FHA) domain of human Ki67 interacts with the human nucleolar protein hNIFK, recognizing a 44-residue fragment, hNIFK226–269, phosphorylated at Thr234. Here we show that high-affinity binding requires sequential phosphorylation by two kinases, CDK1 and GSK3, yielding pThr238, pThr234 and pSer230. We have determined the solution structure of Ki67FHA in complex with the triply phosphorylated peptide hNIFK226–2693P, revealing not only local recognition of pThr234 but also the extension of the β-sheet of the FHA domain by the addition of a β-strand of hNIFK. The structure of an FHA domain in complex with a biologically relevant binding partner provides insights into ligand specificity and potentially links the cancer marker protein Ki67 to a signaling pathway associated with cell fate specification.

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Figure 1: 1H-15N HSQC spectra of hNIFK226–2693P free (black) and in complex with Ki67FHA (red).
Figure 2: Intermolecular NOEs observed for the Ki67FHA–hNIFK226–2693P complex from 3D 13C-edited, 12C/14N-filtered NOE spectra.
Figure 3: Overall structure of the Ki67FHA–hNIFK226–2693P complex.
Figure 4: Intermolecular interactions between Ki67FHA and hNIFK226–2693P.

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Acknowledgements

We thank Y. Yoneda (Osaka University, Osaka, Japan) for providing the hNIFK gene. This work was supported by NIH grants CA87031 and CA69472, by the Genomics Research Center, Taiwan (M.-D.T.) and the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases, and in part by the Intramural AIDS Targeted Antiviral Program of the Office of the Director (A.M.G.). We thank D. Garrett and F. Delaglio for software, J. Baber for technical support, the MS and Proteomics Facility of OSU for MS analyses and D. Vandre of OSU for help in growing HeLa cells.

Author information

Author notes

  1. In-Ja L Byeon and Hongyuan Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health (NIH), Bethesda, 20892, Maryland, USA

    In-Ja L Byeon & Angela M Gronenborn

  2. Departments of Chemistry and Biochemistry and Ohio State Biochemistry Program, The Ohio State University (OSU), Columbus, 43210, Ohio, USA

    Hongyuan Li, Haiyan Song & Ming-Daw Tsai

  3. Program in Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, 15261, Pennsylvania, USA

    Angela M Gronenborn

  4. Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan

    Ming-Daw Tsai

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  1. In-Ja L Byeon
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Correspondence to Angela M Gronenborn or Ming-Daw Tsai.

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Supplementary information

Supplementary Fig. 1

Phosphorylation by cell lysate supports biological relevance. (PDF 220 kb)

Supplementary Fig. 2

Binding of hNIFK(226-269) to Ki67FHA probed by 31P NMR spectroscopy. (PDF 57 kb)

Supplementary Fig. 3

Titration of Ki67FHA with the hNIFK(260-266) heptapeptide. (PDF 78 kb)

Supplementary Table 1

Influence of proline residues on the phosphorylation of hNIFK(226-269). (PDF 58 kb)

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Byeon, IJ., Li, H., Song, H. et al. Sequential phosphorylation and multisite interactions characterize specific target recognition by the FHA domain of Ki67. Nat Struct Mol Biol 12, 987–993 (2005). https://doi.org/10.1038/nsmb1008

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