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Regulation of Plasmodium falciparum Pfnek3 relies on phosphorylation at its activation loop and at threonine 82

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Abstract

A mitogen-activated protein kinase (MAPK), Pfmap2, has been identified in Plasmodium falciparum. However, its bona fide activator remains elusive as no MAPK kinase (MAPKK) homologues have been found so far. Instead, Pfnek3, a NIMA (never in mitosis, Aspergillus)-related kinase, was earlier reported to display a MAPKK-like activity due to its activating effect on Pfmap2. In this study, the regulatory mechanism of Pfnek3 was investigated. Pfnek3 was found to possess a SSEQSS motif within its activation loop that fulfills the consensus SXXXS/T phospho-activating sequence of MAPKKs. Functional analyses of the SSEQSS motif by site-directed mutagenesis revealed that phosphorylation of residues S221 and S226 is essential for mediating Pfnek3 activity. Moreover, via tandem mass-spectrometry, residue T82 was uncovered as an additional phosphorylation site involved in Pfnek3 activation. Collectively, these results provide valuable insights into the potential in vivo regulation of Pfnek3, with residues T82, S221 and S226 functioning as phospho-activating sites.

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Acknowledgments

We are grateful to Professor Christian Doerig (University of Glasgow) for providing the plasmid carrying the Pfmap2 gene. We would also like to express our sincere gratitude to Dr. Manfred Raida, Ms Ee Kim Huey and Mr Li Rong (A*STAR Experimental Therapeutics Centre) for providing technical expertise in the LC-MS/MS analyses.

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  1. Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4A #04-02, 5 Science Drive 2, Singapore, 117597, Singapore

    Huiyu Low, Chun Song Chua & Tiow-Suan Sim

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  1. Huiyu Low
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  2. Chun Song Chua
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  3. Tiow-Suan Sim
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Correspondence to Tiow-Suan Sim.

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Low, H., Chua, C.S. & Sim, TS. Regulation of Plasmodium falciparum Pfnek3 relies on phosphorylation at its activation loop and at threonine 82. Cell. Mol. Life Sci. 66, 3081–3090 (2009). https://doi.org/10.1007/s00018-009-0101-8

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  • DOI: https://doi.org/10.1007/s00018-009-0101-8

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