Regulation of protein phosphatase type 1 and cell cycle progression by PfLRR1, a novel leucine‐rich repeat protein of the human malaria parasite Plasmodium falciparum

Abstract

SummaryThe protein called 'suppressor of the dis2 mutant ( sds22 + )' is an essential regulator of cell division in fission and budding yeasts, where its deletion causes mitotic arrest. Its role in cell cycle control appears to be mediated through the activation of protein phosphatase type 1 (PP1) in Schizosaccharomyces pombe . We have identified the Plasmodium falciparum Sds22 orthologue, which we designated PfLRR1 as it belongs to the leucine-rich repeat protein family. We showed by glutathione-S-transferase pull-down assay that the PfLRR1 gene product interacts with PfPP1, that the PfLRR1-PfPP1 complex is present in parasite extracts and that PfLRR1 inhibits PfPP1 activity. Functional studies in Xenopus oocytes revealed that PfLRR1 interacted with endogenous PP1 and overcame the G2/M cell cycle checkpoint by promoting progression to germinal vesicle breakdown (GVBD). Confirmatory results showing the appearance of GVBD were observed when oocytes were treated with anti-PP1 antibodies or okadaic acid. Taken together, these observations suggest that PfLRR1 can regulate the cell cycle by binding to PP1 and regulating its activity.

Plasmodium falciparum Inhibitor-3 Homolog Increases Protein Phosphatase Type 1 Activity and Is Essential for Parasitic Survival

“…PP1 belongs to the serine/threonine phosphatase family with representatives in animals (including helminth parasites), plants, and unicellular eukaryotes, including Apicomplexa, that share a high level of identity of amino acid sequences (Ͼ80%) (19,21,44). Several lines of evidence indicate that PP1 contributes to a wide range of physiological processes, including glycogen metabolism, smooth muscle contraction, and sperm motility (45)(46)(47)(48).…”

“…Assays for PfPP1 and Effect of PfI3-The activity of PfPP1 with p-nitrophenyl phosphate (pNPP) as substrate was assayed exactly as described previously (19). To investigate the role of PfI3 on PfPP1 activity, different amounts of PfI3 were added to PfPP1 and preincubated for 30 min at 37°C before testing the PfPP1 phosphatase activity.…”

“…In addition, okadaic acid has been shown to inhibit parasite growth in vitro, mainly by blocking PP1-like activity (18). In this parasite, very little is known about the role of endogenous regulatory subunits of PP1, although we recently reported the first data on an inhibitory subunit of PfPP1, PfLRR1 (19). The gene product of PfLRR1 belongs to the leucine-rich repeat protein family and is the ortholog of Sds22 described in yeast (20).…”

Plasmodium falciparum Dynein Light Chain 1 Interacts with Actin/Myosin during Blood Stage Development

“…To further assess the localization pattern, cytoplasmic, nuclear, and membranes fractions of parasites from asynchronous culture were prepared as previously described (29) and analyzed by immunoblotting. The detergent-resistant pellet (cytoskeletal fraction) was then dissolved in 5% (w/v) SDS in 10 mM sodium phosphate, pH 7.4, and subjected to Western blot analysis.…”

“…The blots were revealed by ECL under autoradiography conditions. B-D, cytoplasm, nuclear, and membranes fractions were prepared as previously described (29). Lanes 1 and 2 correspond to blots incubated with rat and rabbit preimmune sera, respectively.…”