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Comprehensive Substrate Specificity Profiling of the Human bioRxiv preprint doi: https://doi.org/10.1101/515221; this version posted January 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 4 5 Comprehensive Substrate Specificity Profiling of the 6 Human Nek Kinome Reveals Unexpected Signaling Outputs 7 8 9 Bert van de Kooij1, Pau Creixell1, Anne van Vlimmeren1, Brian A. Joughin1, 10 Chad J. Miller2,*, Nasir Haider3, Rune Linding4, Vuk Stambolic3, 11 Benjamin E. Turk2, Michael B. Yaffe1,5,6 12 13 14 15 16 17 18 19 20 21 22 23 24 1. Koch Institute for Integrative Cancer Research, MIT Center for Precision Cancer Medicine, 25 Departments of Biology and Bioengineering, Massachusetts Institute of Technology, 26 Cambridge, MA, USA. 27 2. Department of Pharmacology, Yale School of Medicine, New Haven, CT, USA 28 3. Department of Medical Biophysics, University of Toronto, Toronto, Canada 29 4. Biotech Research and Innovation Center, Faculty of Health and Medical Sciences, University 30 of Copenhagen, Copenhagen, Denmark 31 5. Department of Surgery, Beth Israel Deaconess Medical Center, Divisions of Acute Care 32 Surgery, Trauma, and Critical Care and Surgical Oncology, Harvard Medical School, Boston, 33 USA. 34 6. To whom correspondence should be addressed. E-mail [email protected] 35 * Current address: Department of Biochemistry, University of Washington, Seattle, WA, USA bioRxiv preprint doi: https://doi.org/10.1101/515221; this version posted January 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 36 Abstract 37 Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few 38 substrates are known. We systematically determined the phosphorylation-site motifs for the entire 39 Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic 40 residues in the -3 position, the family separates into four distinct groups based on specificity for a 41 serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other 42 positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates 43 itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto- 44 phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions 45 that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the 46 phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical 47 to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif 48 amplifiers of Plk1 signaling. 2 bioRxiv preprint doi: https://doi.org/10.1101/515221; this version posted January 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 49 Introduction 50 Protein phosphorylation by kinases plays an essential role in nearly all signaling events 51 and physiological processes within the cell. Protein serine/threonine kinases, together with 52 ubiquitin ligases, play a particularly important role in mitosis, during which thousands of sites are 53 phosphorylated (Dephoure et al., 2008). These mitotic phosphorylation events are predominantly 54 mediated by the kinases Cdk1, Plk1, Aurora A and Aurora B, together with several members of 55 the nimA-related kinase (Nek) family (Fry et al., 2017; Joukov et al., 2018). For Cdk1, Plk1 and 56 the Aurora kinases, multiple substrates and detailed downstream signaling functions have been 57 identified (Joukov et al., 2018), though much remains to be learned. In contrast, for the Nek family 58 kinases only a few substrates have been identified, and many of their functions within the complex 59 network of mitotic signaling are not well understood. 60 The Nek kinase family consists of eleven serine/threonine kinases that together form an 61 independent evolutionary branch of the human kinome (Fig. 1A). The first human mitotic Nek 62 kinase to be identified was Nek2, which is required for centrosomal disjunction following their 63 duplication during S-phase, in order to assemble a bipolar mitotic spindle (Fig. 1B; Fry et al., 2017) 64 A similar function was assigned to Nek5, which, in addition, also regulates centrosome 65 composition in interphase (Prosser et al., 2015). Furthermore, Nek6, Nek7 and Nek9 have been 66 shown to cooperate as a signaling module during various stages of mitosis (Belham et al., 2003). 67 In this process, Nek9 is first phosphorylated in early mitosis by Cdk1, creating a binding site for 68 the Plk1 polo-box domain. Plk1-mediated phosphorylation and activation of Nek9 then leads to 69 Nek-9-dependent phosphorylation and interaction-dependent activation of Nek6 and Nek7, 70 allowing them to perform their downstream functions (Roig et al., 2002; Belham et al., 2003; Yin 71 et al., 2003; Richards et al., 2009; Bertran et al., 2011). As a signaling module, Nek6/7/9 have 72 been reported to play a role in centrosome separation and maturation, nuclear envelope 73 breakdown, metaphase and anaphase progression, mitotic spindle formation and cytokinesis 3 bioRxiv preprint doi: https://doi.org/10.1101/515221; this version posted January 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 74 (Fig. 1B; Roig et al., 2002; Yin et al., 2003; Yissachar et al., 2006; O’Regan et al., 2009; Salem 75 et al., 2010; Bertran et al., 2011; Kim et al., 2011; Laurell et al., 2011; Sdelci et al., 2012; Cullati 76 et al., 2017). Notably, the Nek kinase family is not exclusively involved in mitosis, but functionally 77 diverse. Nek kinases have also been reported to play a role in meiosis, ciliary biology, and the 78 response of cells to replication stress and DNA-damage (Fig. 1B; Quarmby et al., 2005; Melixetian 79 et al., 2009; Larissa S Moniz et al., 2011; Choi et al., 2013; Spies et al., 2016; Brieño-Enríquez et 80 al., 2017). The functions of Nek3 and Nek4 are not clear but the current literature suggests that 81 Nek3 may play a role in cell migration (Harrington et al., 2016), and Nek4 might be involved in 82 regulating microtubule stability (Fig. 1B; Doles et al., 2010). 83 We have previously determined the optimal substrate phosphorylation motifs, i.e. the 84 substrate amino acid sequence preferentially phosphorylated by a given kinase, for the mitotic 85 kinases Cdk1, Plk1, Aurora A, Aurora B and Nek2 (Alexander et al., 2011). This motif-information 86 strongly facilitates identification of kinase substrates, and can help to understand the organization 87 of complex phosphorylation networks like those in mitotic cells (Linding et al., 2007; Alexander et 88 al., 2011; Kang et al., 2013). Therefore, to further catalog the optimal substrate motif-atlas for 89 mitotic kinases, and to characterize substrate-specificity of a complete and functionally diverse 90 branch of the human kinome, we sought to determine the optimal phosphorylation-site motif for 91 the Nek-kinases using Oriented Peptide Library Screening (OPLS) (Hutti et al., 2004), followed 92 by direct experimental validation together with molecular modeling. This revealed a surprisingly 93 large amount of diversity in substrate-specificity among the Nek-kinases, and identified Nek10 as 94 a dual-specificity kinase, that can phosphorylate substrates on tyrosine in addition to serine. 95 Furthermore, these studies demonstrated that the mitotic kinases Nek6, Nek7 and Nek9, have a 96 phosphorylation-site motif that is almost identical to the motif of Plk1, suggesting that the Nek6/7/9 97 module functions as a Plk1-motif amplifier. 98 4 bioRxiv preprint doi: https://doi.org/10.1101/515221; this version posted January 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 99 Results 100 Determination of the phosphorylation-site motif for all members of the Nek kinase family 101 To obtain the consensus phosphorylation-site motif for each individual Nek kinase, we 102 performed Oriented Peptide Library Screening (OPLS) as described previously (Hutti et al., 2004; 103 Alexander et al., 2011). In these experiments, each kinase is tested in 198 individual in vitro kinase 104 reactions, each with a different peptide library as substrate (Fig. S1). Each peptide library is 105 composed of a pool of 10-mer peptides containing an equimolar mix of serine and threonine at 106 the phospho-acceptor site, and one other fixed amino acid on a position ranging from five residues 107 upstream (-5 position) to four residues downstream (+4 position) of the phospho-acceptor residue. 108 The fixed amino acid is one of the 20 naturally occurring amino acids, or either phospho-threonine 109 (pT) or phospho-tyrosine (pY). The remaining eight unfixed positions in the library contain an 110 equimolar mix of all natural amino acids except cysteine, serine or threonine. The preference of 111 the protein kinase for each residue at each position is then determined by directly comparing its 112 activity against each individual peptide library.
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