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. 2012 May;69(9):1523-35.
doi: 10.1007/s00018-011-0888-y. Epub 2011 Nov 25.

Plasmodium falciparum possesses a unique dual-specificity serine/threonine and tyrosine kinase, Pfnek3

Huiyu Low  1 , Chun Song Chua, Tiow-Suan Sim
Affiliations

Plasmodium falciparum possesses a unique dual-specificity serine/threonine and tyrosine kinase, Pfnek3

Huiyu Low et al. Cell Mol Life Sci. 2012 May.

Abstract

Despite the absence of classical tyrosine kinases encrypted in the kinome of Plasmodium falciparum, biochemical analyses have detected significant tyrosine phosphorylation in its cell lysates. Supporting such phosphorylation is critical for parasite development. These observations have thus raised queries regarding the plasmodial enzymes accountable for tyrosine kinase activities in vivo. In the current investigation, immunoblot analysis intriguingly demonstrated that Pfnek3, a plasmodial mitogen-activated protein kinase kinase (MAPKK), displayed both serine/threonine and tyrosine kinase activities in autophosphorylation reactions as well as in phosphorylation of the exogenous myelin basic protein substrate. The results obtained strongly support Pfnek3 as a novel dual-specificity kinase of the malarial parasite, even though it displays a HGDLKSTN motif in the catalytic loop that resembles the consensus HRDLKxxN signature found in the serine/threonine kinases. Notably, its serine/threonine and tyrosine kinase activities were found to be distinctly influenced by Mg(2+) and Mn(2+) cofactors. Further probing into the regulatory mechanism of Pfnek3 also revealed tyrosine phosphorylation to be a crucial factor that stimulates its kinase activity. Through biocomputational analyses and functional assays, tyrosine residues Y117, Y122, Y172, and Y238 were proposed as phosphorylation sites essential for mediating the catalytic activities of Pfnek3. The discovery of Pfnek3's dual role in phosphorylation marks its importance in closing the loop for cellular regulation in P. falciparum, which remains elusive to date.

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Figures

Fig. 1
Fig. 1
Autophosphorylation characteristics of recombinant Pfnek3. a Recombinant Pfnek3 and its kinase-dead mutant (∆Pfnek3) were transferred to PVDF membrane and probed with antibodies specific against phospho-tyrosine, serine, or threonine (denoted by Anti P-Tyr, Anti P-Ser, and Anti P-Thr, respectively). Coomassie staining of duplicated SDS-PAGE gels indicates similar protein loading for each lane. Representative blots/gel from three independent experiments is shown. b Following protein expression and purification, Pfnek3 was incubated in a kinase assay mix comprising ATP and Mn2+/Mg2+ as shown in the top panel. The mixtures were then subjected to immunoblotting using the specified antibodies. Coomassie staining of duplicated SDS-PAGE gels indicates similar protein loading for each lane. The result shown is representative of three independent experiments. c Densitometric analysis of the results obtained in b. Data is expressed as a percentage of Pfnek3 autophosphorylation in the absence of ATP/Mn2+/Mg2+, with each bar representing the mean autophosphorylation level ± standard deviation from the three independent assays
Fig. 2
Fig. 2
Recombinant Pfnek3 exhibits dual-specificity kinase activity on the exogenous MBP substrate but not Pfmap2. In vitro Pfnek3 kinase assays were performed in the presence of ATP and Mn2+/Mg2+ using a MBP or c ∆Pfmap2 as substrates. The mixtures were resolved by SDS-PAGE and phosphorylation status of the respective substrates was analyzed by Western blot using the indicated phospho-specific antibodies. Coomassie staining of duplicated SDS-PAGE gels indicates similar loading of the respective proteins. Results depicted are representative of three independent experiments. b, d Densitometric quantification of the results obtained in a and c, respectively. For referencing purposes, phosphorylation levels are expressed as percentages of MBP/∆Pfmap2 phosphorylation in the presence of Pfnek3/ATP/Mn2+. Each bar depicts the mean phosphorylation level ± standard deviation from the three independent experiments. e In vitro kinase assays for Pfnek3 were repeated in reaction buffers supplemented with ATP, Mg2+ and either the ∆Pfmap2 or the ∆Pfmap2/T290A substrates. Phosphorylation status of the substrates was subsequently assessed using the Pro-Q® Diamond phosphoprotein gel stain. Coomassie staining of the same gel at the lower panel demonstrates comparable protein loading. A representative result of three independent experiments is illustrated. f Graphical representation of the densitometric analyses for e. Result is expressed as a percentage of ∆Pfmap2 phosphorylation, with each bar representing the mean phosphorylation level ± standard deviation from the three independent assays. ND denotes kinase activity not detected
Fig. 3
Fig. 3
Effects of tyrosine dephosphorylation on the serine/threonine and tyrosine kinase activities of Pfnek3. Recombinant Pfnek3 was treated with PTP1B as described in the Materials and methods section. In control experiments, PTP1B was omitted. a Aliquots of the mixtures were subjected to ELISA-based kinase assays using MBP as substrate to determine the serine/threonine and tyrosine kinase activities of the PTP1B-treated Pfnek3. Results are expressed as percentage of untreated Pfnek3 activity, with each bar representing the mean activity ± standard deviation from two independent assays. b Phosphorylation status of the PTP1B-treated and control Pfnek3 were verified by Western blot using the indicated antibodies. Coomassie staining of duplicated SDS-PAGE gel illustrates similar loading of the proteins. The results illustrated are representative of two independent experiments
Fig. 4
Fig. 4
Positions of predicted phospho-tyrosine sites in the tertiary model of Pfnek3. a The 3D model of Pfnek3 was generated in an earlier study [23] and viewed using the Swiss-PdbViewer. Location of the predicted phospho-tyrosines are shown and highlighted in red. b Neighboring residues that fall within 4 Å of the predicted phospho-tyrosines were identified and are shown in white. Only residues that form hydrogen bonds (indicated by dotted green lines) with the respective tyrosines are labeled
Fig. 5
Fig. 5
Tyrosine phosphorylation levels and catalytic activities of Pfnek3 phenylalanine-substituted mutants. a Purified wild-type Pfnek3 and the indicated site-directed mutants were subjected to Western blot using the phospho-tyrosine antibody. Coomassie staining of duplicated SDS-PAGE gel demonstrates similar protein loading for each lane. The result illustrated is representative of three independent experiments. b Densitometric analysis of the result obtained in a. Data is expressed as a percentage of wild-type Pfnek3 tyrosine autophosphorylation, with each bar representing the mean autophosphorylation level ± standard deviation from the three independent assays. c Via the ELISA-based kinase assay, the in vitro serine/threonine (upper panel) and tyrosine (lower panel) kinase activities of the respective Pfnek3 mutants were determined by their ability to phosphorylate the MBP substrates. Results are presented as a percentage of wild-type Pfnek3 activity, with each bar representing the mean activity ± standard deviation from two independent assays. ND denotes kinase activity not detected
Fig. 6
Fig. 6
Catalytic activities of Pfnek3 tyrosine phospho-mimetic mutants. Residues Y117, Y122, Y172, and Y238 were each replaced with either an aspartate or a glutamate to mimic phosphorylation at these residues. Serine/threonine (upper panel) and tyrosine (lower panel) kinase activities of these mutants were analyzed using the ELISA-based kinase assay. Results are presented as a percentage of wild-type Pfnek3 activity, with each bar representing the mean activity ± standard deviation from two independent assays. ND denotes kinase activity not detected
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