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Sci Rep. 2019 Nov 13;9(1):16720. doi: 10.1038/s41598-019-53325-9.

Mapping and functional analysis of heterochromatin protein 1 phosphorylation in the malaria parasite Plasmodium falciparum.

Author information

1
Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, 4051, Basel, Switzerland.
2
University of Basel, 4003, Basel, Switzerland.
3
Department of Microbiology, Monash University, Clayton, Victoria, 3800, Australia.
4
The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia.
5
Department of Medical Biology, University of Melbourne, Parkville, Victoria, 3052, Australia.
6
Biozentrum, University of Basel, 4056, Basel, Switzerland.
7
Centre for Chronic Inflammatory and Infectious Diseases, School for Health and Biomedical Sciences, RMIT University, Bundoora, Victoria, 3083, Australia.
8
Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, 4051, Basel, Switzerland. till.voss@swisstph.ch.
9
University of Basel, 4003, Basel, Switzerland. till.voss@swisstph.ch.

Abstract

Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. However, in the malaria parasite Plasmodium falciparum both the function of HP1 phosphorylation and the identity of the protein kinases targeting HP1 are still elusive. In order to functionally analyze phosphorylation of P. falciparum HP1 (PfHP1), we first mapped PfHP1 phosphorylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of native PfHP1, which identified motifs from which potential kinases could be predicted; in particular, several phosphorylated residues were embedded in motifs rich in acidic residues, reminiscent of targets for P. falciparum casein kinase 2 (PfCK2). Secondly, we tested recombinant PfCK2 and a number of additional protein kinases for their ability to phosphorylate PfHP1 in in vitro kinase assays. These experiments validated our prediction that PfHP1 acts as a substrate for PfCK2. Furthermore, LC-MS/MS analysis showed that PfCK2 phosphorylates three clustered serine residues in an acidic motif within the central hinge region of PfHP1. To study the role of PfHP1 phosphorylation in live parasites we used CRISPR/Cas9-mediated genome editing to generate a number of conditional PfHP1 phosphomutants based on the DiCre/LoxP system. Our studies revealed that neither PfCK2-dependent phosphorylation of PfHP1, nor phosphorylation of the hinge domain in general, affect PfHP1's ability to localize to heterochromatin, and that PfHP1 phosphorylation in this region is dispensable for the proliferation of P. falciparum blood stage parasites.

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