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.
Highlights
Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions
HP1 is widely conserved among eukaryotes and consists of three functional domains, namely the N-terminal chromo domain (CD) that binds H3K9me[31,7,8], the C-terminal chromoshadow domain (CSD) that mediates HP1 homodimerisation and specific interactions with other regulatory proteins[9,10,11], and a variable hinge region located between the CD and CSD domains that has been shown to interact with DNA and/or RNA12–14
By generating conditional P. falciparum HP1 (PfHP1) phosphomutant cell lines using CRISPR/Cas9-mediated genome editing combined with the DiCre/LoxP system[40,41], we found that P. falciparum casein kinase 2 (PfCK2)-dependent phosphorylation of PfHP1, and phosphorylation of the PfHP1 hinge domain in general, is dispensable for proper PfHP1 localisation, gene silencing, parasite growth and sexual conversion
Summary
Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. 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. We demonstrated that PfHP1 depletion leads to a 25-fold increase in sexual conversion rates, with over 50% of parasites in the progeny differentiating into gametocytes (which are required for malaria transmission via the mosquito vector). This striking phenotype was linked to de-repression of the pfap2-g locus in absence of PfHP1. We showed that the remaining asexual parasites in the PfHP1-depleted progeny failed to enter S-phase, revealing a crucial role for PfHP1 in the control of proliferation[31]
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