Transgenic analysis of the Leishmania MAP kinase MPK10 reveals an auto-inhibitory mechanism crucial for stage-regulated activity and parasite viability

Abstract

Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.

Figure 1. Recombinant MPK10 shows no significant substrate-specific kinase activity.

In vitro kinase assays using non-mutated GST-Strep-MPK10 (NM) and the corresponding inactive mutant GST-Strep-MPK10-K51A (K/A). Analysis of the reaction samples was performed by SDS-PAGE, gels were stained with Coomassie (upper panels) and phosphotransfer was visualized by auto-radiography (lower panels). All gels are representative of three independent experiments. (A) Purified GST-Strep-MPK10 NM was incubated with (+) or without (−) the canonical MAPK substrate MBP for 30 min at pH 7.5 and 30°C or 37°C. (B) GST-Strep-MPK10 NM and -K/A were incubated with different canonical substrates, including 12 µg of histone H1, 9 µg of Ets1, 36 µg of casein, and 25 µg of MBP. Recombinant human MEK1 was used as positive control with MBP as substrate. Kinase assays were performed for 30 min at pH 7.5 and 37°C.

Figure 2. Deletion of the C-terminal domain increases auto-phosphorylation activity.

(A) Partial tryptic digestion of recombinant MPK10. 50 µg of Strep3-MPK10 were digested with 0.25 µg trypsin at RT. Aliquots were taken at the indicated time points and the reaction was stopped either by adding Laemmli buffer (for N-terminal sequencing) or by lowering the pH to 5.0 and subsequent freezing (for mass determination by SELDI-TOF). For N-terminal sequencing, samples were separated by SDS-PAGE, transferred on PVDF membrane and stained by amidoblack. N-terminal sequencing was performed at the protein analysis platform at the Institut Pasteur. For mass determination, samples were immobilized on a H4 ProteinChip Array (C16 reversed phase surface) and peptide masses identified by SELDI-TOF. Results of the N-terminal sequencing are represented by the cartoon in (B), and the sequences are indicated in (C). Italic characters represent the Strep3-tag and bold characters represent the sequence of Leishmania major MPK10. White and grey arrowheads indicate respectively lysine or arginine residues recognized by trypsine, including K12, K24, K30 and R392. The white arrow at the position D387 indicates the position of the last cleaved residue resulting in the generation of the form lacking the last 46 amino acids of MPK10. (D) In vitro kinase assay using recombinant His-MPK10 (NM) and the truncated kinase mutants His-MPK10-ΔC (ΔC), and His-MPK10-ΔC_K51A (ΔC_K/A). Results are representative of three independent experiments. Purified proteins were incubated with four different substrates, including 12 µg of histone H1, 9 µg of Ets1, 36 µg of casein, and 25 µg of MBP. Recombinant human MEK1 was used as positive control with MBP as substrate. Kinase assays were performed at the same time for 30 min at pH 7.5 and 37°C and reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (right), and signals were revealed by auto-radiography with the same exposure time between the different gels (left). The brackets in (D) indicate auto-phosphorylation (Auto-P) and substrate phosphorylation (Substrate-P) signals.

Figure 3. MPK10 activity is enhanced during axenic amastigote differentiation.

(A) In vitro kinase assay. GFP-MPK10 NM fusion protein was purified using anti-GFP antibody from logarithmic (Log) and stationary (Sta) growth phase promastigotes (Pro) and after 12 h, 24 h, 36 h, 48 h, 72 h, 96 h and 120 h of axenic amastigote (Ax. ama.) differentiation. Kinase assays were performed for 30 min at 37°C with 25 µg of MBP as substrate. Reaction samples were separated by SDS-PAGE, gels were stained with Coomassie (Coom.), and signals were revealed by auto-radiography (Auto-rad.) using the indicated exposure time. The numbers represent the level of radioactive counts as determined in a scintillation counter. The results were expressed relative to GFP-MPK10 NM from promastigotes of logarithmic culture set to 100%. (B) Signal quantification. The histogram plot represents the level of radioactive counts as determined in a scintillation counter. The results were expressed relative to GFP-MPK10 NM from promastigotes of logarithmic culture set to 100%. Histograms represent mean values and the error bars correspond to the standard deviation of three independent experiments. Statistical significance was calculated by t-test or Mann-Whitney Rank Sum test. (C) Assessment of tyrosine phosphorylation. Non-mutated GFP-MPK10 (NM), and mutated GFP-MPK10-K51A (K/A), -Y192F (Y/F) and -T190A_Y192F (T/A_Y/F) were purified from promastigote (Pro.) and axenic amastigote (Ax. ama.) after 48 h of differentiation, and analyzed by western blotting using anti-phospho-tyrosine (α-pTyr) and anti-MPK10 (α-MPK10). (D) Analysis of tyrosine phosphorylation during axenic amastigote differentiation. Samples used in (A) were analyzed by western blotting using mouse anti-phospho-tyrosine (α-pTyr) and rabbit anti-MPK10 (α-MPK10) antibodies. (E) SRM analysis. Digested proteins from whole cell lysates of L. mexicana promastigotes (Pro) and axenic amastigotes (Ax. Ama), were subjected to TiO₂ enrichment, eluates were desalted on C8 STAGE-tips and vacuum dried. Prior to LC-MS/MS analysis, the eluates were reconstituted in 0.1% formic acid. The acquired data were imported into the Pinpoint method file for analysis. T-H-pY, peptides represent single phosphorylation of Y192; pT-H-pY, peptides represent dual phosphorylation of residues T190 and Y192. This value of T-H-pY represents the sum of the values obtained with two different peptides (THYVTHR and EDTADANKTHYVTHR). All blots and auto-radiograms are representative of three independent experiments.

Figure 4. Both threonine 190 and tyrosine 192 are required for MPK10 activity.

(A) In vitro kinase assay. Purified protein from promastigotes expressing non-mutated GFP-MPK10 (NM), and mutated GFP-MPK10-K51A (K/A), -T190A (T/A), -Y192F (Y/F) and -T190A_Y192F (T/A_Y/F) were incubated for 30 min at 37°C and pH 7.5 with 25 µg MBP as substrate. Reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (Coom.), and signals revealed by auto-radiography (Auto-rad.) using the indicated exposure time. The numbers represent the level of radioactive counts as determined in a scintillation counter. The results were expressed relative to GFP-MPK10 NM from promastigotes of logarithmic culture set to 100%. Results are representative of three independent experiments. (B) Proliferation and viability analysis. 1×10⁶ amastigotes were cultured for 8 days and aliquots were taken every 24 h for analysis. Cell number and percent of cell death were assessed by flow cytometry. Upper panel: Untransfected Control, UC (dotted line, open squares); non-mutated GFP-MPK10, NM (grey line, grey circles); GFP-MPK10-K51A, K/A (black line, open triangles). Lower panel: GFP-MPK10-T190A, T/A (black line, black triangles); GFP-MPK10-Y192F, Y/F (black line, white diamonds); GFP-MPK10-T190A_Y192F, T/A_Y/F (black line, black diamonds). Mean values of the results obtained in two independent experiments in triplicate were plotted, with standard deviations indicated by the bars. (C) In vitro kinase assay. Non-mutated GFP-MPK10 (NM), and mutated GFP-MPK10-K51A (K/A), -T190A (T/A), -Y192F (Y/F) and -T190A_Y192F (T/A_Y/F) obtained from amastigotes at 48 h (left) and 96 h (right) during axenic differentiation were incubated for 30 min at 37°C and pH 7.5 with 25 µg MBP as substrate. Reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (Coom.), and signals revealed by auto-radiography (Auto-rad.) using the indicated exposure time. The numbers represent the level of radioactive counts as determined by a scintillation counter. The results were expressed relative to GFP-MPK10 NM from promastigotes of logarithmic culture set to 100%. Results are representative of three independent experiments.

Figure 5. The C-terminal domain negatively regulates MPK10 activity in situ.

(A), (D) Proliferation and viability analysis. The analyses represent the combined results of two triplicates experiments. 2×10⁵ promastigotes (A) and 1×10⁶ amastigotes (D) were cultured for 8 days and aliquots were taken every 24 h for analysis. Cell number and percent of cell death were assessed by flow cytometry. The cell lines that were tested are GFP-MPK10, NM (grey line, grey circles), GFP-MPK10-ΔC, ΔC (black line, black squares), GFP-MPK10-ΔC-K51A, ΔC-K/A (black line, open triangles), GFP-MPK10-ΔC-Y192F, ΔC-Y/F (black line, black circles). Mean values of the results obtained in two independent experiments in triplicate were plotted, with standard deviations indicated by the bars. (B) In vitro kinase assay. GFP-MPK10-ΔC_K51A (ΔC_K/A) and GFP-MPK10-ΔC_Y192F (ΔC_Y/F) obtained from respective transgenic promastigotes were incubated for 30 min at 37°C and pH 7.5 with 25 µg MBP as substrate. Reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (Coom.), and signals revealed by auto-radiography (Auto-rad.) using the indicated exposure time. (C) Western blot analysis. Proteins were purified from promastigotes and analyzed by western blotting using anti-phospho-tyrosine (α-pTyr) and anti-MPK10 (α-MPK10) antibodies. All blots and auto-radiograms are representative of three independent experiments.

Figure 6. The regulatory Serine 395 residue shows stage-specific phosphorylation.

(A) Multiple sequence alignment of MPK10 orthologs from trypanosomatids generated with Clustal-X and visualized with BioEdit. The phospho-serine residue S395 is marked by the grey arrow. LmjF, L. major Friedlin; LmxM, L. mexicana MHOM/GT2001/U1103; LinJ, L. infantum JPCM5; LdBPK, L. donovani BPK282A1; LbrM, L. braziliensis MHOM/BR/75/M2904; LtaP, L. tarentolae Parrot-TarlI; Tb, T. brucei; Tbg, T. brucei gambiense DAL972; TcIL3000, T. congolense IL3000; TcCLB, T. cruzi CL Brener; TCSYLVIO, T. cruzi Sylvio X10/1; Tc_MARK, T. cruzi marinlellei strain B7; TvY486, T. vivax Y486. Color code: Black, identical residues; Grey, similar residues; White, no conservation. (B) The level of phosphorylated S395 (pS395) isolated from promastigotes (P) and axenic amastigotes (Ax) was determined by SRM analysis as described in legend of Figure 3E. (C), (F) Proliferation and viability analysis. The analyses represent the combined results of two triplicates experiments. 2×10⁵ promastigotes (C) and 1×10⁶ amastigotes (F) were cultured for 8 days and aliquots were taken every 24 h for analyses. Cell number and percent of cell death were assessed by flow cytometry. The cell lines that were tested are GFP-MPK10 NM (grey line, grey circles), GFP-MPK10-S395A, S/A (black line, black crosses). Mean values of the results obtained in three independent triplicate experiments were plotted, with standard deviations indicated by the bars (*: p<0.05; **: p<0.01; ***: p<0.001). (D) In vitro kinase assay. Non-mutated GFP-MPK10 NM and mutated GFP-MPK10-S395A (S/A) obtained from promastigotes (Pro) and axenic amastigotes (Ax. Ama) at 48 h and 96 h during axenic differentiation were incubated for 30 min at 37°C and pH 7.5 with 25 µg MBP as substrate. Reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (Coom.), and signals revealed by auto-radiography (Auto-rad.) using the indicated exposure time. The numbers represent the level of radioactive counts as determined by a scintillation counter. Signals were normalized to the counts obtained with GFP-MPK10 NM on MBP and set at 100%. (E) Histogram plot of the radioactive counts obtained from MBP after kinase assay normalized to the values obtained with GFP-MPK10 NM on MBP and set at 100%. Histograms represent mean values and the standard deviation is denoted by the error bars. Results are representative of three independent experiments. The asterisks represent statistical significance, with *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 7. Model of MPK10 regulation based on our data.

We propose a model in which MPK10 is not inactivated but partially active in promastigotes as judged by tyrosine phosphorylation and structural conformation. At this stage the kinase is kept in a standby configuration by auto-inhibition. During the first 48 h of axenic amastigote differentiation, MPK10 is released from auto-inhibition, which correlates with T190 phosphorylation and S395 dephosphorylation. This activity seems to be controlled by a feedback loop where MPK10 regulates its own tyrosine phosphorylation levels. Thereafter, MPK10 activity is decreased likely due to dephosphorylation of the TxY motif and phosphorylation of S395.