Abstract
ABSTRACTDuring mitosis, eukaryotic cells must duplicate and separate their chromosomes in a precise and timely manner. The apparatus responsible for this is the kinetochore, which is a large protein structure that links chromosomal DNA and spindle microtubules to facilitate chromosome alignment and segregation. The proteins that comprise the kinetochore in the protozoan parasite Trypanosoma brucei are divergent from yeast and mammals and comprise an inner kinetochore complex composed of 24 distinct proteins (KKT1 to KKT23, KKT25) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2, and KKT3. We recently reported the identification of a specific trypanocidal inhibitor of T. brucei CLK1, an amidobenzimidazole, AB1. We now show that chemical inhibition of CLK1 with AB1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. Here, we show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 by CLK1 to be essential for KKT2 function and for kinetochore assembly. Additionally, KKT2 protein kinase activity is required for parasite proliferation but not for assembly of the inner kinetochore complex. We also show that chemical inhibition of the aurora kinase AUK1 does not affect CLK1 phosphorylation of KKT2, indicating that AUK1 and CLK1 are in separate regulatory pathways. We propose that CLK1 is part of a divergent signaling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2.
Highlights
From yeast to humans, the majority of the Centromere-Associated Network (CCAN) assembly can be subdivided into four discrete units, and their stability depends critically on reciprocal interactions [6].the recruitment of components of the CCAN in these species depends on a specialized centromeric histone H3 variant, the centromere protein A (CENP-A) [9]
Two proteins with protein kinase domains (KKT2-3) are constitutively localized to centromeres throughout the cell cycle, most likely acting as functional orthologues of the eukaryotic CCAN proteins [15, 16]. This parasite has a set of KKT-interacting proteins (KKIP1-12), which are related to outer kinetochore proteins Ndc80 and Nuf2 [17] and a cohort of proteins localized to the nucleus during interphase and to the spindle during mitosis (NuSAPs) involved in regulating spindle dynamics and chromosome segregation [18]
Given the clinical importance of T. brucei bloodstream forms for drug intervention and the advantage of using a chemical tool to study the kinetochore regulation, here we demonstrate that CLK1 phosphorylates KKT2 at S508 during early metaphase, and its inhibition affects the posterior recruitment of inner kinetochore components affecting chromosome segregation, in a pathway that is independent to Aurora Kinase B
Summary
The majority of the CCAN assembly can be subdivided into four discrete units, and their stability depends critically on reciprocal interactions [6]. Two proteins with protein kinase domains (KKT2-3) are constitutively localized to centromeres throughout the cell cycle, most likely acting as functional orthologues of the eukaryotic CCAN proteins [15, 16] This parasite has a set of KKT-interacting proteins (KKIP1-12), which are related to outer kinetochore proteins Ndc and Nuf2 [17] and a cohort of proteins localized to the nucleus during interphase and to the spindle during mitosis (NuSAPs) involved in regulating spindle dynamics and chromosome segregation [18]. Given the clinical importance of T. brucei bloodstream forms for drug intervention and the advantage of using a chemical tool to study the kinetochore regulation, here we demonstrate that CLK1 phosphorylates KKT2 at S508 during early metaphase, and its inhibition affects the posterior recruitment of inner kinetochore components affecting chromosome segregation, in a pathway that is independent to Aurora Kinase B. These results suggest that KKT2 and KKT3 are centromere-anchored proteins [15], they respond differently to CLK1 inhibition and that TbCLK1 is a critical regulator of inner kinetochore component dynamics
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