CLK1/CLK2-driven signalling at the Leishmania kinetochore is captured by spatially referenced proximity phosphoproteomics.
Journal
Communications biology
ISSN: 2399-3642
Titre abrégé: Commun Biol
Pays: England
ID NLM: 101719179
Informations de publication
Date de publication:
28 11 2022
28 11 2022
Historique:
received:
10
08
2022
accepted:
18
11
2022
entrez:
27
11
2022
pubmed:
28
11
2022
medline:
30
11
2022
Statut:
epublish
Résumé
Kinetochores in the parasite Leishmania and related kinetoplastids appear to be unique amongst eukaryotes and contain protein kinases as core components. Using the kinetochore kinases KKT2, KKT3 and CLK2 as baits, we developed a BirA* proximity biotinylation methodology optimised for sensitivity, XL-BioID, to investigate the composition and function of the Leishmania kinetochore. We could detect many of the predicted components and also discovered two novel kinetochore proteins, KKT24 and KKT26. Using KKT3 tagged with a fast-acting promiscuous biotin ligase variant, we took proximity biotinylation snapshots of the kinetochore in synchronised parasites. To quantify proximal phosphosites at the kinetochore as the parasite progressed through the cell cycle, we further developed a spatially referenced proximity phosphoproteomics approach. This revealed a group of phosphosites at the kinetochore that were highly dynamic during kinetochore assembly. We show that the kinase inhibitor AB1 targets CLK1/CLK2 (KKT10/KKT19) in Leishmania leading to defective cytokinesis. Using AB1 to uncover CLK1/CLK2 driven signalling pathways important for kinetochore function at G2/M, we found a set of 16 inhibitor responsive kinetochore-proximal phosphosites. Our results exploit new proximity labelling approaches to provide a direct analysis of the Leishmania kinetochore, which is emerging as a promising drug target.
Identifiants
pubmed: 36437406
doi: 10.1038/s42003-022-04280-1
pii: 10.1038/s42003-022-04280-1
pmc: PMC9701682
doi:
Substances chimiques
Protein Kinase Inhibitors
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1305Subventions
Organisme : Wellcome Trust
ID : 200807/Z/16/Z
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/P027989/1
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 200807/Z/16/Z
Pays : United Kingdom
Informations de copyright
© 2022. The Author(s).
Références
mBio. 2021 Jun 29;12(3):e0068721
pubmed: 34128702
Cell. 2014 Mar 13;156(6):1247-1258
pubmed: 24582333
J Cell Biol. 2017 Feb;216(2):379-391
pubmed: 28034897
Chromosome Res. 2004;12(6):569-83
pubmed: 15289664
J Cell Biol. 2019 Dec 2;218(12):4042-4062
pubmed: 31676716
Nucleic Acids Res. 2015 Apr 20;43(7):e47
pubmed: 25605792
Cell. 2017 Apr 6;169(2):350-360.e12
pubmed: 28388416
PLoS Genet. 2020 Feb 21;16(2):e1008390
pubmed: 32084124
Open Biol. 2016 Mar;6(3):
pubmed: 26984294
Front Oncol. 2015 Oct 16;5:225
pubmed: 26528436
PLoS Pathog. 2014 Jan;10(1):e1003886
pubmed: 24453978
Nat Biotechnol. 2018 Oct;36(9):880-887
pubmed: 30125270
Curr Biol. 2021 Nov 22;31(22):R1491-R1504
pubmed: 34813757
Nat Commun. 2021 Feb 23;12(1):1244
pubmed: 33623024
J Cell Biol. 2021 Aug 2;220(8):
pubmed: 34081090
Biology (Basel). 2017 Jan 24;6(1):
pubmed: 28125021
BMC Biol. 2020 Mar 2;18(1):23
pubmed: 32122335
Open Biol. 2019 Dec;9(12):190236
pubmed: 31795916
Brief Bioinform. 2018 Jan 1;19(1):1-11
pubmed: 27694351
Cell Microbiol. 2020 May;22(5):e13159
pubmed: 31909863
Sci Rep. 2018 May 21;8(1):7960
pubmed: 29785042
Front Cell Dev Biol. 2018 Jun 19;6:62
pubmed: 29971233
PLoS Pathog. 2019 Dec 12;15(12):e1008129
pubmed: 31830130
Elife. 2020 Sep 08;9:
pubmed: 32897188
Proc Natl Acad Sci U S A. 2014 Jun 17;111(24):E2453-61
pubmed: 24927568
Genome Biol. 2015 Oct 19;16:230
pubmed: 26481451
Nat Commun. 2016 Apr 29;7:11451
pubmed: 27126587
Cell. 2008 May 2;133(3):427-39
pubmed: 18455984
Nature. 2004 Jul 29;430(6999):573-8
pubmed: 15229615
Nat Commun. 2022 Jul 13;13(1):4071
pubmed: 35831302
J Cell Sci. 2020 Apr 29;133(8):
pubmed: 32184264
Eukaryot Cell. 2006 Jul;5(7):1026-35
pubmed: 16835447
PLoS Pathog. 2020 Jun 16;16(6):e1008455
pubmed: 32544189
Curr Biol. 2015 Mar 2;25(5):671-7
pubmed: 25660545
J Cell Biol. 2018 Nov 5;217(11):3886-3900
pubmed: 30209069
Cell Mol Life Sci. 2020 Aug;77(15):2981-2995
pubmed: 32052088
Nucleic Acids Res. 2018 Sep 19;46(16):8216-8231
pubmed: 29931198
R Soc Open Sci. 2017 May 3;4(5):170095
pubmed: 28573017
Nat Microbiol. 2020 Oct;5(10):1207-1216
pubmed: 32661312