Protein kinase signalling at the Leishmania kinetochore captured by XL-BioID

Elucidating protein kinase signaling pathways is an important but challenging problem in cell biology. Phosphoproteomics has been used to identify many phosphorylation sites, however the spatial context of these sites within the cell is mostly unknown, making it difficult to reconstruct signalling pathways. To address this problem an in vivo proximity capturing workflow was developed, consisting of proximity biotinylation followed by protein cross-linking (XL-BioID). This was applied to protein kinases of the Leishmania kinetochore, leading to the discovery of a novel essential kinetochore protein, KKT26. XL-BioID enabled the quantification of proximal phosphosites at the kinetochore through the cell cycle, allowing the phosphorylation state of the kinetochore to be followed during assembly. A specific inhibitor of kinetochore protein kinases KKT10/KKT19 was used to show that XL-BioID provides a spatially focussed view of protein kinase inhibition, identifying 16 inhibitor-responsive proximal phosphosites, including 3 on KKT2, demonstrating the potential of this approach for discovery of in vivo kinase signalling pathways.

Ctf3/CENP-I provides a docking site for the desumoylase Ulp2 at the kinetochore

The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle–regulated processes.

Over-expression of ZW10 interacting kinetochore protein in human endometrial cancer.

Gynecologic cancers including cancers of the endometrium are a clinical problem (1-4). We mined published microarray data (5, 6) to discover genes associated with endometrial cancers by comparing transcriptomes of the normal endometrium and endometrial tumors from humans. We identified ZW10 interacting kinetochore protein, encoded by ZWINT, as among the most differentially expressed genes, transcriptome-wide, in cancers of the endometrium. ZWINT was expressed at significantly higher levels in endometrial tumor tissues as compared to the endometrium. Importantly, primary tumor expression of ZWINT was correlated with overall survival in patients with endometrial cancer. ZWINT may be a molecule of interest in understanding the etiology and/or progression of human endometrial cancer.

Selfish chromosomal drive shapes recent centromeric histone evolution in monkeyflowers

Centromeres are essential mediators of chromosomal segregation, but both centromeric DNA sequences and associated kinetochore proteins are paradoxically diverse across species. The selfish centromere model explains rapid evolution by both components via an arms-race scenario: centromeric DNA variants drive by distorting chromosomal transmission in female meiosis and attendant fitness costs select on interacting proteins to restore Mendelian inheritance. Although it is clear than centromeres can drive and that drive often carries costs, female meiotic drive has not been directly linked to selection on kinetochore proteins in any natural system. Here, we test the selfish model of centromere evolution in a yellow monkeyflower (Mimulus guttatus) population polymorphic for a costly driving centromere (D). We show that theDhaplotype is structurally and genetically distinct and swept to a high stable frequency within the past 1500 years. We use quantitative genetic mapping to demonstrate that context-dependence in the strength of drive (from near-100%Dtransmission in interspecific hybrids to near-Mendelian in within-population crosses) primarily reflects variable vulnerability of the non-driving competitor chromosomes, but also map an unlinked modifier of drive coincident with kinetochore protein Centromere-specific Histone 3 A (CenH3A). Finally, CenH3A exhibits a recent (<1000 years) selective sweep in our focal population, implicating local interactions withDin ongoing adaptive evolution of this kinetochore protein. Together, our results demonstrate an active co-evolutionary arms race between DNA and protein components of the meiotic machinery inMimulus, with important consequences for individual fitness and molecular divergence.

Differential expression of ZW10 interacting kinetochore protein in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding the ZW10 interacting kinetochore protein, ZWINT, when comparing primary tumors of the breast to the tissue of origin, the normal breast. ZWINT was also differentially expressed in the tumor cells of patients with triple negative breast cancer. ZWINT mRNA was present at significantly higher quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of ZWINT in primary tumors of the breast was correlated with overall survival in patients with basal and luminal A subtype cancer, but in a contrary manner, demonstrating a complex relationship between correlation of primary tumor expression with overall survival based on molecular subtype. ZWINT may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Chromosome segregation fidelity is controlled by small changes in phospho-occupancy at the kinetochore-microtubule interface

SummaryKinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e. all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ∼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ∼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained by ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. Thus, networks of kinases and phosphatases maintain low inherent phospho-occupancy to promote microtubule attachment to kinetochores while providing for high sensitivity of kinetochore-microtubule attachments to very small changes in phospho-occupancy to ensure high mitotic fidelity.