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Purification of Cdk-CyclinB-Kinase–Targeted Phosphopeptides from Nuclear Envelope

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The Nuclear Pore Complex

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2502))

Abstract

We describe a method for rapid identification of protein kinase substrates within the nuclear envelope. Open mitosis in higher eukaryotes is characterized by nuclear envelope breakdown (NEBD) concerted with disassembly of the nuclear lamina and dissociation of nuclear pore complexes (NPCs) into individual subcomplexes. Evidence indicates that reversible phosphorylation events largely drive this mitotic NEBD. These posttranslational modifications likely disrupt structurally significant interactions among nucleoporins (Nups), lamina and membrane proteins of the nuclear envelope (NE). It is therefore critical to determine when and where these substrates are phosphorylated. One likely regulator is the mitotic kinase: Cdk1-Cyclin B. We employed an “analog-sensitive” Cdk1 to bio-orthogonally and uniquely label its substrates in the NE with a phosphate analog tag. Subsequently, peptides covalently modified with the phosphate analogs are rapidly purified by a tag-specific covalent capture and release methodology. In this manner, we were able to confirm the identity of known Cdk1 targets in the NE and discover additional candidates for regulation by mitotic phosphorylation.

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Acknowledgments

The corresponding author would first like to thank the late Günter Blobel for his mentorship and encouragement. Also, we would like to Andrew Krutchinsky and Kevan Shokat for their support. We thank Thomas Cattabiani for critical reading of the manuscript. This work was supported in part by the Howard Hughes Medical Institute. J.S.G. was supported by the National Institutes of Health Individual National Research Service Award 5F32GN20520 and NIGMS-7R15GM119118.

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Authors and Affiliations

  1. Pacific Biosciences, Menlo Park, CA, USA

    Justin D. Blethrow

  2. Department of Biochemistry and Molecular Biophysics, University of Chicago, Chicago, IL, USA

    Amanda L. DiGuilio

  3. Department of Pharmaceutical Sciences, Fisch College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA

    Joseph S. Glavy

Authors
  1. Justin D. Blethrow
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  2. Amanda L. DiGuilio
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  3. Joseph S. Glavy
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Corresponding author

Correspondence to Joseph S. Glavy .

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Editors and Affiliations

  1. Department of Biosciences, Durham University, Durham, UK

    Martin W. Goldberg

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Blethrow, J.D., DiGuilio, A.L., Glavy, J.S. (2022). Purification of Cdk-CyclinB-Kinase–Targeted Phosphopeptides from Nuclear Envelope. In: Goldberg, M.W. (eds) The Nuclear Pore Complex. Methods in Molecular Biology, vol 2502. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2337-4_18

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  • DOI: https://doi.org/10.1007/978-1-0716-2337-4_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2336-7

  • Online ISBN: 978-1-0716-2337-4

  • eBook Packages: Springer Protocols

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