Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex
- Author(s)
- Nebl, T; Prieto, JH; Kapp, E; Smith, BJ; Williams, MJ; Yates, JR; Cowman, AF; Tonkin, CJ;
- Details
- Publication Year 2011-09,Volume 7,Issue #9,Page e1002222
- Journal Title
- PLOS PATHOGENS
- Publication Type
- Journal Article
- Abstract
- Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca(2+)-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of (32)[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified similar to 546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca(2+)-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.
- Publisher
- PUBLIC LIBRARY SCIENCE
- Keywords
- PROTEIN-KINASE 1; GONDII MYOSIN-A; SELECTABLE MARKER; MASS-SPECTROMETRY; XIV MYOSIN; EXPRESSION; MEMBRANE; MOTILITY; GENE; IDENTIFICATIONS
- Research Division(s)
- Systems Biology And Personalised Medicine; Infection And Immunity
- Publisher's Version
- https://doi.org/10.1371/journal.ppat.1002222
- Open Access at Publisher's Site
10.1371/journal.ppat.1002222- Terms of Use/Rights Notice
- Copyright: © 2011 Nebl et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Creation Date: 2011-09-01 12:00:00