Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs

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Abstract

Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the −3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif amplifiers of Plk1 signaling.

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Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates.

Andrew Elia, Lewis Cantley, Michael Yaffe (2003)

We have developed a proteomic approach for identifying phosphopeptide binding domains that modulate kinase-dependent signaling pathways. An immobilized library of partially degenerate phosphopeptides biased toward a particular protein kinase phosphorylation motif is used to isolate phospho-binding domains that bind to proteins phosphorylated by that kinase. Applying this approach to cyclin-dependent kinases (Cdks), we identified the polo-box domain (PBD) of the mitotic kinase polo-like kinase 1 (Plk1) as a specific phosphoserine (pSer) or phosphothreonine (pThr) binding domain and determined its optimal binding motif. This motif is present in known Plk1 substrates such as Cdc25, and an optimal phosphopeptide containing the motif disrupted PBD-substrate binding and localization of the PBD to centrosomes. This finding reveals how Plk1 can localize to specific sites within cells in response to Cdk phosphorylation at those sites and provides a structural mechanism for targeting the Plk1 kinase domain to its substrates.

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Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion

Martin Thomsen, Morten A. Nielsen (2012)

Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed).

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mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin.

Benjamin E. Turk, Gail Kang, Mary Pacold … (2013)

The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs life span in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin, as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals.

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Author and article information

Contributors

Michael B Yaffe:

ORCID: https://orcid.org/0000-0002-9547-3251

Margaret C Frame: Role: Reviewing Editor

Jonathan A Cooper: Role: Senior Editor

Journal

Journal ID (nlm-ta): eLife

Journal ID (iso-abbrev): Elife

Journal ID (publisher-id): eLife

Title: eLife

Publisher: eLife Sciences Publications, Ltd

ISSN (Electronic): 2050-084X

Publication date (Electronic, pub): 24 May 2019

Publication date Collection: 2019

Volume: 8

Electronic Location Identifier: e44635

Affiliations

[1 ]deptDepartment of Biology Massachusetts Institute of Technology CambridgeUnited States

[2 ]deptDepartment of Biological Engineering Massachusetts Institute of Technology CambridgeUnited States

[3 ]deptKoch Institute for Integrative Cancer Research Massachusetts Institute of Technology CambridgeUnited States

[4 ]deptMIT Center for Precision Cancer Medicine Massachusetts Institute of Technology CambridgeUnited States

[5 ]deptDepartment of Pharmacology Yale School of Medicine New HavenUnited States

[6 ]deptDepartment of Medical Biophysics University of Toronto TorontoCanada

[7 ]deptBiotech Research and Innovation Center, Faculty of Health and Medical Sciences University of Copenhagen CopenhagenDenmark

[8 ]deptDepartment of Surgery, Beth Israel Deaconess Medical Center, Divisions of Acute Care Surgery, Trauma, and Critical Care and Surgical Oncology Harvard Medical School BostonUnited States

[9 ]deptPrincess Margaret Cancer Center University Health Network TorontoCanada

University of Edinburgh United Kingdom

Fred Hutchinson Cancer Research Center United States

University of Edinburgh United Kingdom

Author notes

[†]

Department of Biochemistry, University of Washington, Seattle, United States.

Author information

Bert van de Kooij https://orcid.org/0000-0003-1042-8409

Brian A Joughin https://orcid.org/0000-0003-1022-9450

Vuk Stambolic http://orcid.org/0000-0001-8853-3239

Michael B Yaffe https://orcid.org/0000-0002-9547-3251

Article

Publisher ID: 44635

DOI: 10.7554/eLife.44635

PMC ID: 6570481

PubMed ID: 31124786

SO-VID: 688db408-22f6-4078-91a5-2cf3527eddcb

License:

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

History

Date received : 21 December 2018

Date accepted : 13 May 2019

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100009729, Ludwig Institute for Cancer Research;

Award Recipient : Bert van de Kooij

Funded by: FundRef http://dx.doi.org/10.13039/501100004622, Dutch Cancer Society;

Award ID: BUIT 2015-7546

Award Recipient : Bert van de Kooij

Funded by: FundRef http://dx.doi.org/10.13039/100000054, National Cancer Institute;

Award ID: K99CA226396

Award Recipient : Pau Creixell

Funded by: FundRef http://dx.doi.org/10.13039/100011102, European Union Seventh Framework Programme;

Award ID: FP/2007-2013 ERC grant (KINOMEDRIFT)

Award Recipient : Rune Linding

Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;

Award ID: R01-GM104047

Award Recipient : Benjamin E Turk Michael B Yaffe

Funded by: The Charles and Marjorie Holloway Foundation;