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PLoS Pathog. 2015 Nov 6;11(11):e1005268. doi: 10.1371/journal.ppat.1005268. eCollection 2015.

Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress.

Author information

1
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
2
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
3
Stark Neuroscience Research Center, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
4
The Francis Crick Institute, London, United Kingdom.

Abstract

Members of the family of calcium dependent protein kinases (CDPK's) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii.

PMID:
26544049
PMCID:
PMC4636360
DOI:
10.1371/journal.ppat.1005268
[Indexed for MEDLINE]
Free PMC Article

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