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

Abstract

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.

ULK4 and Fused/STK36 interact to mediate assembly of a motile flagellum

“…Clonal cell lines were selected by limiting dilution and the modification of both alleles was confirmed via endpoint PCR (using primers that spanned the inserted BirA* tag encoding region; F: 5’-CGGGGCACTGAGCAACTTTGT-3’, R: 5’-CACCAGTGGGCAGTGTGAGC-3’). Biotinylated proteins were captured using the XL-BioID protocol, which includes a cross-linking step prior to affinity capture (Geoghegan et al, 2021): Log phase promastigote cultures (seeded at 2×10 6 cell/ml) were allowed to grow in complete M199 supplemented with 150 μM biotin (B-4639, Sigma-Aldrich) for 18-24 hours. 4×10 8 parasites were harvested via centrifugation (800 g for 5 minutes), washed twice with PBS, and cross-linked with 1 mM dithiobis(succinimidyl propionate) (DSP) in 10 ml PBS plus 5% dimethyl sulfoxide (DMSO) for 10 minutes at 28°C.…”

Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome

“…Magenta hexagons indicate members of the BDF5, BDF3, HAT2 complex reported in T. brucei. To identify the functional properties of the environment proximal to BDF we applied an insitu proximity labelling technique, cross-linking BioID (XL-BioID) 51 . The promiscuous biotin ligase BirA*, which generates a locally reactive (~10 nm) biotinoyl-5'-AMP 52 , was fused to the N-terminus of BDF5 by endogenous tagging.…”

“…However, we were again unable to detect any acetylated peptides derived from histones. The capacity of XL-BioID to enrich large amounts of proximal material allows it to be combined with other methods, such as phosphoproteomics 51 . We engineered a cell line to carry BDF5::miniTurboID for faster labelling kinetics and higher temporal resolution, allowing us to explore BDF5-proximal phosphorylation events across the cell cycle of hydroxyurea synchronised cultures.…”

“…Samples were processed using the XL-BioID workflow, then proximal phosphopeptides were enriched using Ti-IMAC resin prior to LC-MS/MS analysis. The resulting dataset was compared to a reference phosphopeptide dataset derived from the kinetochore protein KKT3 51 . Two BDF5-proximal phosphopeptides were identified in early-S phase which then rose to 19 and 13 as the cells progressed through the S and G2/M phases respectively (Fig.…”

“…Peptides were then desalted and prepared for mass spectrometry. For a full protocol see Geoghegan et al 51 Hypothetical proteins were screened for remote structural homology using HHPRED 31 and Phyre2 75 to identify putative domains in these proteins.…”