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Vol. 17, Issue 4, 2035-2045, April 2006

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Interacting Protein Kinases Involved in the Regulation of Flagellar Length

Bernhard Nocht Institute for Tropical Medicine, Parasitology Section, D-20359 Hamburg, Germany

Submitted October 24, 2005; Revised December 29, 2005; Accepted January 26, 2006
Monitoring Editor: J. Silvio Gutkind

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Protein kinases are central molecules in differentiation, proliferation, stress response, and apoptosis in all eukaryotic cells. A commonly used signaling pathway is that of the mitogen-activated protein (MAP) kinases. Its core module is comprised of a MAP kinase kinase kinase (MKKK), a MAP kinase kinase (MKK), and a MAP kinase. An external signal is sensed by a cell surface receptor, which subsequently undergoes autophosphorylation and recruits first intracellular signaling molecules. These can activate MKKK, which phosphorylates and activates its substrate MKK. MKKs are dual specificity kinases that activate their substrates, the MAP kinases, by phosphorylation on a threonine and tyrosine residue of the TXY motif in the activation lip region. Finally, MAP kinases can phosphorylate transcription factors, directly influencing the expression of certain genes, or they phosphorylate other soluble kinases or structural proteins of the cell. This is a very simplistic view of the processes occurring in a cell after stimulation, because there are among other proteins, a number of scaffold proteins involved that are responsible for the specificity of the phosphorelay. Moreover, protein phosphatases counterbalance the activity of the protein kinases by dephosphorylation.

One aspect of differentiation is the regulation of organelle and overall cell size. Flagellar length regulation is a simple one-dimensional example for maintenance of a defined size of an organelle, the flagellum. A flagellum is a dynamic structure that is built by the assembly of its components at its tip, which is also the site for the disassembly of the structural elements. The building blocks are delivered and removed by a process called intraflagellar transport (IFT). The balance between anterograde (base to tip) and retrograde IFT has been found to determine the length of the flagellum in the green alga Chlamydomonas reinhardtii (Marshall and Rosenbaum, 2001). It is likely, however, that protein phosphorylation adds an additional layer to the regulation of flagellar maintenance because >80 flagellar components have been found to be phosphorylated in Chlamydomonas (Piperno and Luck, 1976; Piperno et al., 1981; Harper et al., 1993; Tuxhorn et al., 1998). Our studies of the protozoan parasite Leishmania have shown that this parasite is a suitable model organism for flagellar length regulation because protein kinases of the MAP kinase signal transduction cascades are critically involved in its regulation (Wiese et al., 2003a; Bengs et al., 2005). Likewise, in Chlamydomonas LF4, a protein kinase with homology to MAP kinases and the human male germ cell-associated kinase (MAK), has been found to influence flagellar length as a null mutant displayed elongated flagella (Berman et al., 2003).

Leishmania parasites have a digenetic life cycle with the sandfly as their insect vector and mammals as their host. The insect stage promastigotes are spindle-shaped cells, 11–20 µm in length and 2 µm in diameter, with a long flagellum protruding from the flagellar pocket, an invagination of the cytoplasmic membrane at the anterior end of the cell. They are transmitted to a mammalian host during the bloodmeal of the sandfly. In the skin of the host, the promastigotes are taken up by macrophages and end up in the lysosome of the host cell, forming the parasitophorous vacuole. Instead of being killed and degraded, triggered by the elevated temperature and the low pH (Zilberstein and Shapira, 1994), the parasites differentiate into the spherical amastigote form, which has an overall reduced cellular volume reflected by a length and width of 5–6 µm, a rudimentary flagellum not protruding from the flagellar pocket, and a different cell surface architecture (McConville and Ferguson, 1993). How the signals, a shift in temperature and in pH, are sensed and translated into differentiation is not known yet. However, it is likely that protein kinases and phosphatases play major roles because early investigations on phosphorylation patterns in different life stages of trypanosomatids revealed stage-specific changes in overall protein phosphorylation (Mukhopadhyay et al., 1988; Aboagye-Kwarteng et al., 1991; Parsons et al., 1991, 1993, 1995; Dell and Engel, 1994).

Using deletion analysis, we found that LmxMPK9 is involved in flagellar length regulation (Bengs et al., 2005). A null mutant displayed significantly elongated flagella compared with wild-type promastigotes. Flagellar length was also affected in a deletion mutant for the MAP kinase kinase LmxMKK (Wiese et al., 2003a). These mutants displayed flagella reduced to one-fifth of the length of the wild-type flagellum, preventing the cells from swimming free in the culture medium.

Here, we analyze LmxMPK3, which is encoded by the only MAP kinase gene investigated so far showing mRNA levels significantly down-regulated in the amastigote stage of Leishmania mexicana (Wiese et al., 2003b). We prove the kinase activity of LmxMPK3 by in vitro kinase assays of the recombinant protein expressed in Escherichia coli. Independently obtained homozygous deletion mutants of LmxMPK3 display flagella significantly reduced in length resembling those of the LmxMKK null mutant (Wiese et al., 2003a). Indeed, the constitutively active LmxMKK phosphorylates and activates LmxMPK3 in vitro and in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Parasites
Promastigotes of L. mexicana MNYC/BZ/62/M379 were grown as described previously (Menz et al., 1991). Female BALB/c mice were infected into the left hind foot pad at the age of 6–8 wk with 1 x 10⁷ promastigote parasites in 30 µl of phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 1.4 mM KH₂PO₄). Using a caliper gauge, the course of infection was followed by measuring lesion size relative to the uninfected right hind foot pad at 4- to 5-wk intervals. For axenic amastigotes, cultures were seeded with stationary phase promastigotes at 4 x 10⁶ cells/ml in Schneider's Drosophila medium (PAN Biotech, Aidenbach, Germany) supplemented with 20% inactivated fetal calf serum and 2 mM glutamine, adjusted to pH 5.5 by the addition of morpholinoethane sulfonic acid, and incubated at 34°C, 5% CO₂. Samples of 1 x 10⁸ cells were taken at defined intervals after initiation of differentiation. For amastigote-to-promastigote differentiation, parasites were isolated from lesions of infected BALB/c mice as described previously (Wiese, 1998). The whole cell suspension was centrifuged for 10 min at 150 x g and 4°C. Then, the supernatant was centrifuged for 10 min at 1500 x g at 4°C. The pellet was resuspended in 10 ml of cold 1x PBS, and the cells were counted. One hundred milliliters of prewarmed (27°C) SDM-79 medium (Brun and Schoenenberger, 1979) was inoculated with 10⁹ amastigotes. Every 5 h, cell pellets of 1 x 10⁸ cells were collected, washed with cold 1x PBS, frozen in liquid nitrogen, and stored at-70°C.

Molecular Cloning Methods
Expand High-Fidelity Polymerase (Roche Diagnostics, Mannheim, Germany) was used for all PCR applications. Plasmid isolation, DNA/RNA isolation and blotting, and hybridizations were performed as described previously (Benzel et al., 2000). Both DNA strands of all constructs derived from PCR were verified by DNA sequencing. LmxMPK3 sequence data have been submitted to DDBJ/EMBL/GenBank databases under accession no. AJ293281 [GenBank] . Splice-addition sites were determined as described previously (Wiese, 1998) using the mini-exon–derived oligonucleotide 5'-CTAACGCTATATAAGTATCAGTTT-3' and two oligonucleotides derived from LmxMPK3, 5'-CTTCGAAGCTGTGGTATT-3' and 5'-GTCGCGACACTTCTTGAT-3', as primers in the reverse transcriptase and polymerase chain reactions.

Antibody Production and Immunoblotting
A rabbit antiserum was produced against the peptide CTAGGSSSKNGSGHHH corresponding to the 15-COOH-terminal amino acids of LmxMPK3 (Eurogentec, Seraing, Belgium) and purified on the peptide. Lysates of 1 x 10⁹ cells ml^-1 in 1x lysis buffer (1x PBS, 0.1% SDS, 50 mM dithiothreitol, 50 µM leupeptin, 25 µM N--p-tosyllysyl-chloromethylketone, 1 mM phenylmethyl-sulfonyl fluoride, 10 mM 1,10-phenanthroline, and 1x SDS sample buffer [0.4% SDS, 4% glycerol, 0.0002% bromphenol blue, 50 mM dithiothreitol, and 12.5 mM Tris-HCl, pH 6.8]) were boiled for 10 min. Then, 20 µl was subjected to SDS-PAGE and blotted to polyvinylidene difluoride membranes. Immunodetection was carried out as described previously (Wiese, 1998) with different rabbit or mouse antisera and goat-anti-rabbit or goat-anti-mouse secondary antibodies coupled to horseradish peroxidase (Dianova, Hamburg, Germany) followed by chemiluminescence detection using the Supersignal system (Pierce Chemical, Rockford, IL).

LmxMPK3 Deletion Constructs
To generate the LmxMPK3 null mutants LmxMPK3::HYG/LmxMPK3::NEO and LmxMPK3::PHLEO/LmxMPK3::NEO abbreviated 1 and 2, respectively, LmxMPK3 and its flanking regions were amplified from a plasmid carrying a DNA-fragment isolated from a genomic DNA library of L. mexicana (Wiese et al., 1995, 2003b). A PCR was performed on this plasmid (5 min at 94°C, 25 x [30 s at 94°C, 30 s at 50°C, 1 min 45 s at 72°C], 7 min at 72°C, 4°C) using the oligomers 5'-GAGAGGGGGAGGACACTT-3' and 5'-TGTGATATCCTCTTCTCGGCTG-3', the latter introducing an EcoRV restriction site into the 5'-untranslated region (UTR) of LmxMPK3. Because a second EcoRV site is present in the 3'-UTR the amplified fragment was cut using EcoRV and ligated into pBluescriptII SK(+) (Stratagene, La Jolla, CA) previously modified to lack a BspLU11I site and linearized at EcoRV, resulting in pBE5upLmxMPK3ds. To replace LmxMPK3 by different resistance marker genes BspLU11I and NheI sites were introduced into pBE5upLmxMPK3ds using the oligomers 5'-CTGTGACTGGTGAGTACTCAACCAAG-3' (covering the ScaI site in the -lactamase gene of the plasmid) and 5'-CTGGCCTAGGACATGTTGGCTACTCTGTGTGC-3' containing an AvrII and a BspLU11I site in one PCR to amplify the 5'-flanking region, and the oligomers 5'-ATGACTTGGTTGAGTACTCACCAGTC-3' (largely complementary to the ScaI-primer mentioned before) and 5'-CAGGCCTAGGCTAGCTAGCGCGCATCTTCTC-3' containing an AvrII and a NheI site in a second PCR to amplify the 3'-flanking region. The resulting DNA-fragments were trimmed using AvrII and ScaI and ligated to each other, resulting in a plasmid with LmxMPK3 replaced by BspLU11I, AvrII, and NheI restriction sites. This plasmid was linearized using BspLU11I and NheI and ligated to DNA-fragments carrying the resistance marker genes for the hygromycin B phosphotransferase HYG, the neomycin phosphotransferase NEO, and the phleomycin binding protein PHLEO, prepared as described previously (Benzel et al., 2000). The constructs were liberated by EcoRV, gel-purified, and used for electroporation of L. mexicana promastigotes in two consecutive rounds as described previously (Bengs et al., 2005).

Expression of LmxMPK3 and LmxMKK(D) in Leishmania
For episomal expression of LmxMPK3 in L. mexicana, the open reading frame (ORF) of LmxMPK3 was amplified using the oligonucleotides 5'-CCAACATGTACAAGAGCAACCAGGAGC-3' and 5'-GTGAAGCTTCTAGTGATGGTGACCGCT-3' to introduce BspLU11I and HindIII restriction sites in a PCR (5 min at 94°C, 25 x [30 s at 94°C, 30 s at 45°C, 1 min at 72°C], 7 min 72°C, 4°C) and cloned into pCR2.1TOPO (Invitrogen, Carlsbad, CA) to generate pCR2.1-23MPK3. LmxMPK3 was released from pCR2.1-23MPK3 using EcoRI and subcloned into MunI of pX63polPHLEO (Wiese, 1998), resulting in pX3ELmxMPK3. This construct was used for transfection of L. mexicana promastigotes as described above. Transformants were selected in SDM-79 medium on 96-well tissue culture plates using 5 µg ml^-1 bleocin.

A 1112-base pair EcoRV/XbaI DNA-fragment was liberated from the pGEX-KG derivative for recombinant expression of LmxMKK(D) generated previously (Wiese et al., 2003a) and used for the construction of a plasmid allowing for integration into the ribosomal DNA gene locus as has been described for LmxMKK (Wiese et al., 2003a). LmxMKK null mutant cells were transfected with 5 µg of a 5.9-kb PacI/PmeI fragment purified from the final construct and recombinants were selected on SDM-79 agar plates containing 20 µM puromycin.

Electron and Light Microscopy and Flagellar Length Determination
For scanning electron microscopy (SEM), Leishmania cells were washed twice in PBS, fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, and postfixed with 1% OsO₄. Samples were dehydrated at increasing ethanol concentrations (30–100%). After critical point drying, samples were treated with gold and analyzed on a Philips SEM 500 electron microscope. For transmission electron microscopy (TEM), the cells were treated as described for SEM, dehydrated with graded ethanol solutions and propylene oxide. The cells were embedded in an epoxy resin (Epon), and 70-nm ultrathin sections were cut (Ultra Cut E; Reichert/Leica, NuBlock, Germany) and counter-stained with uranyl acetate and lead citrate. Sections were examined with a Philips CM 10 transmission electron microscope at an acceleration voltage of 80 kV. Phase contrast and differential interference contrast (DIC) microscopy and flagellar length determination (the length was measured from the cell surface to the tip of the flagellum using the Openlab software; Improvision, Heidelberg, Germany) were performed on a Zeiss Axioskop 2 Plus and a Leica Leitz DMRB microscope as described previously (Bengs et al., 2005).

Recombinant Expression
For recombinant expression of a glutathione S-transferase (GST) fusion protein of LmxMPK3, the ORF was liberated from pCR2.1-23MPK3 (see above) with BspLU11I and HindIII, and the DNA-fragment was gel-purified and ligated into the NcoI/HindIII-cleaved pGEX-KG (Guan and Dixon, 1991), resulting in pGEX-KG5aBHLmxMPK3. To generate an enzymatically inactive version of LmxMPK3, lysine 62 was mutated to methionine by site-directed mutagenesis, resulting in LmxMPK3KM. A PCR reaction was performed on pGEX-KG5aBHLmxMPK3 (5 min at 94°C, 25 x [30 s at 94°C, 30 s at 50°C, 45 s at 72°C], 7 min at 72°C, 4°C) using the oligonucleotides 5'-CTATCCCACAAATTGATAA-3' and 5'-TCGCGACACTTCATGATGGACACCTTC-3'. The amplified fragment was cleaved using NruI and XbaI, a 203-base pair fragment isolated and ligated with the 5952-base pair fragment obtained from pGEX-KG5aBHLmxMPK3 cleaved with NruI and XbaI, resulting in pGEX-KG9BHLmxMPK3KM. Both constructs were transformed into E. coli XL1-Blue (Stratagene). For expression of the fusion proteins, the bacteria were grown in Luria-Bertani medium, induced with 100 µM isopropyl--D-thiogalactopyranoside at an optical density at 600 nm (OD₆₀₀) of 0.9, further incubated overnight at 18°C, and harvested by centrifugation at 4°C and 4500 x g for 15 min. The cells were washed once in cold PBS, resuspended in 50 µl of cold PBS per milliliter of the original culture volume, and the suspension was subjected to sonication on ice with a Branson Sonifier 250 apparatus and a 6-mm tip. The lysate was adjusted to 1% Triton X-100 and end-over-end rotated at 4°C for 1 h. After centrifugation at 4°C and 12,000 x g for 10 min, the recombinant proteins were purified on glutathione-Uniflow resin following the instructions of the manufacturer (BD Biosciences, Heidelberg, Germany). For expression of LmxMKK(D) and LmxMKK(KM) in E. coli XL1-Blue, the constructs described previously (Wiese et al., 2003a) were used under the same conditions as described for LmxMPK3. To cleave the GST-moiety from the fusion protein, 250 µg of protein was incubated with 0.12 U of thrombin (Amersham, Freiburg, Germany) overnight at 20°C.

Kinase Assay
To determine the interaction of LmxMKK with LmxMPK3, 2 µg of LmxMKK(D) or LmxMKK(K91M) was incubated at 27°C with 2 µg of GSTLmxMPK3 or GSTLmxMPK3(KM) and 0.1 mM [-³²P]ATP (1000 cpm/pmol) in a volume of 50 µl containing 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.0, 10 mM MnCl₂, and 0.1 M NaCl. Reactions were terminated after 1 h by the addition of 12.5 µl of 5x SDS sample buffer containing 200 mM dithiothreitol and heating for 10 min at 95°C. Then, 25 µl of the solution was separated on a 12% SDS-PAGE, silver-stained, dried, and exposed to x-ray film at-70°C. For quantification, unlabeled ATP replaced [-³²P]ATP during the phosphorylation of LmxMPK3 by LmxMKK(D) in a reaction of 20 µl, essentially as described previously (Lawler et al., 1997). Then, 350 ng of LmxMKK(D) was incubated with 10 µg of GSTLmxMPK3 at 27°C as described above. After 1 h, 5 µl of this reaction was assayed for GSTLmxMPK3 kinase activity toward MBP at 27°C in a 50-µl reaction containing 50 mM MOPS, pH 7.0, 10 mM MnCl₂, 0.1 M NaCl, 1 mM EGTA, 1 mM Na-orthovanadate, 0.3 µg/µl myelin basic protein (MBP), and 0.1 mM [-³²P]ATP, resulting in an overall molecular ratio of LmxMKK(D)/GSTLmxMPK3/MBP of 1:17:1000. After incubation for 1 h, 40 µl was spotted on phosphocellulose P81 paper (Whatman, Dassel, Germany) and washed four times with 75 mM H₃PO₄ and once with acetone. The papers were air-dried, and the incorporation of phosphate into MBP quantified by Cerenkov counting in a Beckman LS 5000CE liquid scintillation counter.

Phosphoprotein Analysis
For phosphoprotein purification, 2 x 10⁹ promastigotes of L. mexicana wild-type, LmxMKK-/-, and LmxMKK-/- + LmxMKK(D) were harvested. Before lysis, the cells were washed with 20 mM HEPES, pH 7.5, subsequently lysed in 5 ml of phosphoprotein lysis buffer, and subjected to affinity purification as described by the manufacturer (QIAGEN, Hilden, Germany). The phosphoproteins were collected in five eluates from the affinity column, each with a volume of 500 µl, and the protein concentration determined in a Bradford assay. After elution, the single eluates were incubated at 95°C for 10 min in 1x SDS sample buffer. The samples were subjected to immunoblot analysis with the anti-LmxMPK3-antiserum.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Molecular Characterization
LmxMPK3 is one of nine MAP kinase homologues we identified from L. mexicana in a screen using degenerate oligonucleotides encoding highly conserved amino acid regions in subdomains VIb and VIII of MAP kinases (Wiese et al., 2003b). The gene is comprised of an open reading frame of 1164 base pairs coding for a protein of 388 amino acids with a calculated molecular mass of 43.7 kDa. Homologues are present in other kinetoplastids, such as Leishmania major (98% identical amino acids 382/388; our unpublished data), Leishmania infantum (98% identical amino acids 382/388; our unpublished data), Trypanosoma brucei (70% identical amino acids 262/370), and Trypanosoma cruzi (69% identical amino acids 259/373). Less homologous, but still significant, are the proteins from Chlamydomonas reinhardtii (44% identical amino acids 157/350), from Solanum tuberosum (46% identical amino acids 165/356), and the ERK1 kinase from Dictyostelium discoideum (45% identical amino acids 158/351; Gaskins et al., 1994). The sequence of LmxMPK3 displays the typical 12 kinase subdomains and amino acid residues known to be highly conserved in MAP kinases (Figure 1). There are the subdomain I residues G40–G45 forming the phosphate anchor ribbon for ATP binding with the consensus GxGxxG and the p + 1 specificity pocket in subdomain VIII (I198-R203). In addition, there are the catalytic site residues K62, R76, R79, E80, R160, D161, K163, N166, D179 (Mg²⁺ ligand), R184, T194, D195, and Y196, the latter three forming the TXY motif, which is characteristic for MAP kinases and known to be phosphorylated by the activating MAP kinase kinase on threonine and tyrosine to generate the active enzyme. Lysine 62 in subdomain II is a conserved residue found in all protein kinase family members (Hanks et al., 1988) and is essential for catalytic activity (Zoller and Taylor, 1979; Zoller et al., 1981; Kamps and Sefton, 1986; Buechler and Taylor, 1989; Gibbs and Zoller, 1991). The potential common docking (CD)-domain (DEEDE), an accumulation of negatively charged residues that is likely to be involved in protein–protein interactions is depicted in Figure 1 (Tanoue and Nishida, 2003). Splice-acceptor sites for LmxMPK3 were determined using total RNA in a reverse transcriptase-PCR with gene internal and mini-exon primers (see Materials and Methods). Amplified fragments were cloned and sequenced. Three AG splice-acceptor sites were found located 58, 61, and 90 base pairs upstream of the ATG translation initiation codon. Interestingly, all three amplification products were found in RNA obtained from lesion-derived amastigotes despite the fact that the mRNA of LmxMPK3 is down-regulated in this life stage (Wiese et al., 2003b). Southern blot analysis of genomic DNA from L. mexicana proved that LmxMPK3 is a single copy gene in the haploid genome (our unpublished data).

View larger version (65K): [in this window] [in a new window]   Figure 1. Alignment of LmxMPK3 from L. mexicana with MAP kinase amino acid sequences from different species. LmxMPK3, L. mexicana MAP kinase 3 (accession no. AJ293281 [GenBank] ); TbMPK3, Trypanosoma brucei MAP kinase 3 (accession no. GeneDB Tb927.8.3550); TcMPK3, Trypanosoma cruzi MAP kinase 3 (accession no. GeneDB Tc00.1047053509553.60); CrMPK, Chlamydomonas reinhardtii MAP kinase (accession no. AB035141 [GenBank] ); StMPK2, Solanum tuberosum MAP kinase (accession no. AB062138 [GenBank] ); and DdMPK3, Dictyostelium discoideum MAP kinase 3 (accession no. A56042 [GenBank] ). Roman numerals I to XI indicate kinase subdomains. The kinase domain is located between the two inverted triangles. Arrows mark the potential regulatory phosphorylation sites in the TXY motif at Thr194 and Tyr196, filled circles indicate conserved amino acid residues, and open circles depict the residues of the phosphate anchor ribbon for ATP binding. The asterisks show the residues of the p + 1 specificity pocket. The open triangle marks the invariant lysine residue (Lys62), which is essential for the phosphate transfer reaction. The putative CD-domain is indicated. Dashes indicate gaps introduced to optimize the alignment; dots represent identical residues. Numbering corresponds to LmxMPK3.

View larger version (63K): [in this window] [in a new window]   Figure 2. Immunoblot of LmxMPK3 from L. mexicana pro- and amastigote total cell lysates. Lanes 1 and 3, 2 x 107 promastigotes; lanes 2 and 4, 2 x 107 lesion-derived amastigotes. Lanes 1 and 2 were probed with antiserum against a COOH-terminal peptide of LmxMPK3; lanes 3 and 4 were probed with antiserum against recombinant myo-inositol-1-phosphate synthase. The molecular masses of standard proteins are indicated in kilodaltons.

View larger version (66K): [in this window] [in a new window]   Figure 3. Differentiation of amastigotes to promastigotes (A–C) and promastigotes to amastigotes (D–E). Immunoblots from L. mexicana total cell lysates were probed with LmxMPK3 (A and D) and LmxMKK (B and E) antisera. Hours after differentiation initiation are indicated. The molecular masses of standard proteins are given in kilodaltons. The asterisk depicts the bands corresponding to LmxMPK3; the triangle depicts the bands for LmxMKK. C and F, pictures were taken from fixed cells at the indicated time points using DIC microscopy. For better visualization of flagellar lengths, lines were drawn along the flagella. Bar, 10 µm.

View larger version (39K): [in this window] [in a new window]   Figure 4. Immunoblot of LmxMPK3 from L. mexicana wild type and various mutants. Lane 1, L. mexicana wild type; lanes 2 and 3, two independent single-allele knockout mutants; lanes 4 and 5, two null mutants derived from the different single-allele knockouts; and lanes 6 and 7, episomal complementation of the two null mutants. The molecular masses of standard proteins are indicated in kilodaltons.

The Phenotype of the Null Mutant
The overall appearance of the null mutant promastigotes resembled the appearance of those promastigotes generated by the deletion of LmxMKK, the gene for a MAP kinase kinase homologue from L. mexicana (SEM; Figure 5) (Wiese et al., 2003a). If at all, the cells displayed a flagellum rarely reaching one-half of the length of the wild-type flagellum (Figure 5, A and B). However, as in the LmxMKK null mutant, they were able to wiggle slowly with their flagellum, leading to a tumbling locomotion, but keeping the cells at the bottom of the culture flask. The average flagellar length as determined from 400 individual cells depicted randomly from two independent null mutants by phase-contrast microscopy was 2 µm with a size range from 0 to 6.7 µm, whereas the wild type displayed flagella with an average length of 11.8 ± 2.6 µm (Figure 6A). Indeed, the maximal flagellar length differed in the two mutants being 4.6 µm for mutant 1 and 6.7 µm for mutant 2. The null mutants were complemented by introducing the wild-type LmxMPK3 cloned into the plasmid pX63polPAC (Bengs et al., 2005) (Figure 6B). These cells generated long flagella again; however, the length varied from 1 to 18 µm (average flagellar length of 8.2 ± 3.7 µm), which is likely due to different expression levels generally observed in Leishmania harboring an episome (Benzel et al., 2000). The ultrastructure of the mutants was analyzed using transmission electron microscopy on chemically fixed cells (Figures 7 and 9). Wild-type flagella showed the typical (9 + 2) pattern of microtubule doublets in the axoneme and the typical lattice-like structure of the paraflagellar rod (PFR) adjacent to the axoneme (Figure 7A). The deletion mutant clones also revealed the typical axoneme; however, the PFR could never be visualized as clear as in the wild type. Instead, different transverse sections could be found either lacking the PFR entirely (Figure 7B), displaying remnants of the PFR (Figure 7C), or various amounts of undefined material around the axoneme (Figures 7, D–F, and 9C), sometimes also displaying vesicles of different size (Figures 7F and 9B). Again, the two null mutants differed from each other. Whereas in the mutant that revealed the longer flagella (mutant 2), 17% (34/200) of the transverse sections revealed a PFR at least resembling that of the wild type, no such sections could be observed for the mutant with the shorter flagella (mutant 1). Twenty-four percent (48/200) of the sections of mutant 2 and 8.5% (17/200) of the mutant 1 revealed a rudimentary PFR. No PFR was present in 34% (68/200) of the sections of mutant 2 and 29% (58/200) of mutant 1. However, mutant 1 (61/200; 30.5%) revealed twice as many flagella with material present all around the axoneme as mutant 2 (30/200; 15%). Finally, vesicles were present in 10% (20/200) and 32% (64/200) of the flagellar transverse sections for mutant 2 and mutant 1, respectively. In immunofluorescence analysis, 85% of the mutant promastigotes revealed a fluorescence of varying intensity, 15% showed no reaction with the anti-PFR-2 antibody (our unpublished data). The morphology of the flagella was reflected by a quantification of PFR-2, one of the major protein components of the PFR, by immunoblot analysis (Figure 8). The mutants contain on average roughly 20 times less PFR-2 than the wild type, significantly less than expected, because a mere reduction of flagellar length would only lead to 5 times less PFR-2. In the add-back, small numbers of flagellar sections showed no PFR (15/200), material all around the axoneme (2/200), or vesicles (12/200). A rudimentary PFR was found in 15% (30/200) and a relatively normal architecture of the flagellum in 70.5% (141/200) of the sections, indicating that reexpression of LmxMPK3 is able to complement the null mutant phenotype. It is interesting to note that in flagellar transverse sections of the add-backs, the axoneme seemed more condensed (Figure 7, G–I) showing a significantly reduced diameter (160 nm) compared with the wild type (180 nm), but the normal cross-hatched structure of the PFR in the longitudinal section of the flagellum (Figure 7I). Indeed, the null mutant showed the most relaxed structure with an axonemal diameter of 210 nm. As found in the null mutant for LmxMKK, we also observed vesicles and membrane fragments in the flagellar pocket of the LmxMPK3 knockout (Figure 9, A–H). The vesicles revealed single membrane layers (Figure 9, A and H), double layers (Figure 9A), or multiple layers (Figure 9, C–E and G).

View larger version (72K): [in this window] [in a new window]   Figure 5. Scanning electron micrograph of L. mexicana. Wild type (A) and LmxMPK3 null mutant (B) promastigotes at the same magnification. Bar, 10 µm.

View larger version (22K): [in this window] [in a new window]   Figure 6. The LmxMPK3-/-phenotype. Histograms of flagellar lengths from wild-type and mutant L. mexicana promastigotes. The abscissa show groups of flagellar lengths in micrometers; the ordinates indicate the numbers of cells counted. (A) White bars, L. mexicana wild type; black bars, LmxMPK3-/-clone 1 or 1; gray bars, LmxMPK3-/-clone 2 or 2. (B) White bars, LmxMPK3-/-clone 1 + LmxMPK3 or A1; black bars, LmxMPK3-/-clone 2 + LmxMPK3 or A2.

View larger version (138K): [in this window] [in a new window]   Figure 7. Ultrastructure of L. mexicana wild-type and LmxMPK3 mutant (LmxMPK3-/-) flagella. Cross-sections of flagella of chemically fixed L. mexicana wild-type promastigote (A), LmxMPK3-/-promastigotes (B–F), and episomal complementation (G–I) at the same magnification. a, axoneme; p, paraflagellar rod. Bar, 0.25 µm.

View larger version (144K): [in this window] [in a new window]   Figure 9. Ultrastructure of LmxMPK3 null mutant (LmxMPK3-/-) cells and flagella. Longitudinal sections of flagellar pockets and flagella of chemically fixed promastigotes. (A) Vesicles with single and double membranes in flagellar pockets. (B) Vesicles at the flagellar tip. (C) Bloated flagellum. (D, E, and G) Multilayered vesicles in flagellar pockets. (E, F, and H) Membrane fragments (mf) in flagellar pockets. a, axoneme; f, flagellum; fp, flagellar pocket; g, Golgi; k, kinetoplast; m, mitochondrium; mf, membrane fragments; n, nucleus; tj, tight junction; tz, transition zone. Bars, 0.5 µm (A–C), 0.25 µm (D–H).

View larger version (31K): [in this window] [in a new window]   Figure 8. Immunoblot of PFR-2 (mAb L8C4) from L. mexicana wild-type and null mutants. Lane 1, LmxMPK3-/-1 (2 x 107 promastigotes); lane 2, LmxMPK3-/-2 (2 x 107 promastigotes); and lanes 3–8, L. mexicana wild-type promastigotes. Lane 3, 5 x 104; lane 4, 1 x 105; lane 5, 5 x 105; lane 6, 1 x 106; lane 7, 5 x 106; and lane 8, 2 x 107. The molecular masses of standard proteins are indicated in kilodaltons.

L. mexicana wild type, LmxMPK3 single-allele, null mutants, and episomal add-backs were used to infect female BALB/c mice. No significant differences in the progression of lesion development could be observed between the mutant cell lines and the wild-type (our unpublished data), indicating that LmxMPK3 is neither required for the differentiation from promastigotes to amastigotes nor for the proliferation of the amastigotes.

Recombinant Expression and Kinase Assay
LmxMPK3 and its enzymatically inactive version LmxMPK3(K62M) (Carrera et al., 1993) were expressed as glutathione S-transferase fusion proteins and found as 71-kDa proteins on SDS-PAGE (Figure 10A, left, lanes 3 and 4). Both proteins were subjected to kinase assays using MBP as an artificial substrate. As expected, only the wild-type protein displayed auto- and substrate-phosphorylation (Figure 10A, right, lanes 3 and 4). Using recombinant GSTLmxMPK3 and MBP in kinase assays under varying conditions revealed that an increase in temperature from 25 to 40°C at 10 mM Mn²⁺, pH 6.5, led to an increase in auto- and substrate-phosphorylation. Moreover, manganese is preferred over magnesium at promastigote growth temperature of 27°C and pH 6.5. The optimal pH was found to be a pH range from 6.0 to 7.0 at 2 mM Mn²⁺, 10 mM Mg²⁺, and 27°C (our unpublished data).

View larger version (43K): [in this window] [in a new window]   Figure 10. Phosphorylation of LmxMPK3 and LmxMKK. (A) LmxMKK activates LmxMPK3 in vitro. Recombinant kinases were subjected to kinase assays with MBP using 10 mM Mn2+, 50 mM MOPS, pH 7.0, at 27°C either in combination or alone as indicated and resolved by SDS-PAGE followed by autoradiography. Left, silver-stained gel. Middle, autoradiograph of the gel exposed for 6 h. Right, autoradiograph exposed for 5 d. MKK(D), constitutively active aspartate mutant of LmxMKK; MKK(KM), enzymatically inactive mutant of LmxMKK; MPK3 WT, GST-fusion protein of LmxMPK3; MPK3(KM), GST-fusion protein of enzymatically inactive mutant of LmxMPK3. (B) In vivo phosphorylation of LmxMPK3 by LmxMKK. Phosphorylated proteins were enriched from total promastigote cell lysates, subjected to SDS-PAGE, blotted, and LmxMPK3 detected using the polyclonal antiserum against a COOH-terminal peptide. Lane 1, null mutant for LmxMKK (LmxMKK-/-); lane 2, L. mexicana wild type; lane 3, promastigotes expressing LmxMKK(D) from the ribosomal DNA gene locus in LmxMKK (Wiese et al., 2003a). (C) Feedback phosphorylation of LmxMKK by LmxMPK3. Phosphorylation of an enzymatically inactive version of the aspartate mutant of LmxMKK, designated LmxMKK(KM)(D). Recombinant kinases were subjected to kinase assays either in combination or alone and with or without preincubation with LmxMKK(D) to activate LmxMPK3 as indicated, and resolved by SDS-PAGE. Left, Coomassie-stained gel. Right, autoradiograph of the gel exposed for 6 h. The molecular masses of standard proteins are indicated in kilodaltons.

Feedback Phosphorylation
Using 1-h preincubation of GSTLmxMPK3 and GSTLmxMKK(D) as single kinases or together in the presence of ATP followed by an incubation with LmxMKK(K91M)(D), an enzymatically inactive version of the aspartate mutant of LmxMKK, led to the phosphorylation of LmxMKK(K91M)(D) by GSTLmxMPK3 (Figures 10C, lanes 5 and 7). Without activation of LmxMPK3 by LmxMKK(D), the phosphorylation of LmxMKK(K91M)(D) is significantly weaker, indicating that activation of LmxMPK3 by LmxMKK(D) is a prerequisite for full feedback phosphorylation. LmxMKK(K91M)(D) shows no autophosphorylation activity (Figure 10C, lane 1). Likewise, the small amounts of GSTLmxMKK(D) used for the preincubation do not incorporate any label in the radioactive assay (Figure 10C, lane 2). It is interesting to note that GSTLmxMPK3 still reveals high autophosphorylating activity after 1 h of preincubation in the presence of unlabeled ATP (Figure 10C, lane 3). GSTLmxMKK(D) is not able to transphosphorylate LmxMKK(K91M)(D) significantly (Figure 10C, lane 4). The interaction of GSTLmxMKK(D) with GSTLmxMPK3 is reflected by the appearance of a smear in the gel, which is phosphorylated (Figures 10C, lanes 6 and 7). Whether the feedback phosphorylation decreases or augments the activity of LmxMKK has not been investigated yet.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We characterized LmxMPK3, a mitogen-activated protein kinase homologue from L. mexicana. The protein is exclusively expressed in promastigotes, the insect stage of the parasite, as is LmxMKK, a MAP kinase kinase homologue (Wiese et al., 2003a). The stage-specific expression is also reflected by a significant decrease of steady-state mRNA for both genes in Leishmania amastigotes (Duncan et al., 2001; Wiese et al., 2003b). A null mutant for LmxMKK displayed short flagella and shorter promastigote cell bodies that revealed membrane fragments and vesicles in their flagellar pockets (Wiese et al., 2003a). Indeed, independently generated homozygous deletion mutants of LmxMPK3 showed a similar phenotype, indicating that the two kinases are components of a common MAP kinase signal transduction pathway involved in the regulation of flagellar length. In wild-type amastigotes, a rudimentary flagellum that lacks the PFR is maintained in the flagellar pocket not protruding from the cell body. As expected PFR-2, a major protein component of the PFR, is not detectable by immunoblot analysis in the amastigote stage of the parasite (Santrich et al., 1997). During differentiation from amastigotes to promastigotes, the flagellum is extended and the PFR is assembled next to the axoneme but is restricted to the part of the flagellum outside the flagellar pocket. Although LmxMKK appears within 10 h after initiation of differentiation, LmxMPK3 is first detectable after 16 h. At this time, short flagella are detectable by light microscopy. In contrast, in cells differentiating from promastigotes to amastigotes LmxMPK3 almost disappears within 24 h. LmxMKK is still detectable after 72 h after initiation of differentiation despite the complete reduction of the flagellum within 24 h, indicating that LmxMKK might have additional functions beside the regulation of LmxMPK3 activity. Most importantly, we found that LmxMKK is involved in the phosphorylation of LmxMPK3 in vivo. No phosphorylated LmxMPK3 could be detected in promastigotes lacking LmxMKK, whereas promastigotes expressing the constitutively active LmxMKK showed phosphorylated LmxMPK3 like the wild type. Autophosphorylation of LmxMPK3 in vivo seems to play a minor role, if at all. Regulatory molecules such as the two kinases obviously have to be present before effector molecules such as PFR-2 and other structural components of the flagellum are expressed and assembled. Indeed, PFR-2 is first detectable after at least 25 h after promastigote differentiation initiation (our unpublished data). At this time, most of the cells already displayed short flagella resembling those of the two kinase null mutants. Interestingly, in the LmxMKK null mutant most of the cells showed no PFR-2 in immunofluorescence analysis, whereas up to 85% of the LmxMPK3 null mutant promastigotes revealed variable amounts of PFR-2 in their flagella (our unpublished data). However, as revealed by electron microscopy PFR-2 was not properly assembled into the lattice-like structure as in the wild type. This indicates that LmxMKK might be a branch point in the regulation of flagellar morphogenesis and has other substrates in addition to LmxMPK3, which are responsible for the expression of PFR-2, whereas LmxMPK3 might affect the assembly of the PFR. MAP kinase kinases exhibit high levels of substrate specificity (Chang and Karin, 2001); however, yeast Ste7 phosphorylates Kss1 and Fus3, MKK7 phosphorylates different isoforms of SAPK1/JNK1 and p38 (Fleming et al., 2000), and mitogen-activated protein kinase kinase 1 phosphorylates extracellular signal-regulated kinase (ERK)1, ERK2, and a potential Golgi-associated ERK (Acharya et al., 1998). A regulatory motif of 10 nucleotides responsible for a 10-fold down-regulation of the amount of mRNA in the amastigote has been identified in the 3'-UTR of all PFR genes in L. mexicana (Mishra et al., 2003). This element could not be found in the 3'-UTRs of LmxMKK and LmxMPK3. The proposed additional substrate of LmxMKK might be a MAP kinase that phosphorylates an RNA-binding protein that then stabilizes PFR mRNA, leading to the expression of PFR proteins. LmxMPK3, in contrast, might be involved in the regulation of the IFT, supporting the outgrowth of the flagellum and the proper assembly of the PFR because the null mutants never display its cross-hatched structure. Whether the activity of kinesin II, the molecular motor driving anterograde IFT, is regulated by phosphorylation has not been investigated yet; however, potential MAP kinase phosphorylation sites have been identified in both motor proteins forming at least part of the kinesin II complex in Leishmania (Wiese, unpublished data).

Using recombinant proteins, we could demonstrate that LmxMPK3 is phosphorylated by a constitutively active LmxMKK, leading to an increase in phosphorylation of the artificial substrate MBP by the activated LmxMPK3. Moreover, the activated LmxMPK3 was able to phosphorylate its activator, revealing a potential feedback mechanism that might either lead to an increase in kinase activity, inactivate the kinase, or mark it for degradation. Higher eukaryotic Cdc2 kinase and its regulating molecules are examples for different roles of phosphorylation. Negative phosphorylation by Wee1 and Myt1 during interphase inactivates Cdc2. At the onset of mitosis Cdc2 is dephosphorylated by Cdc25 phosphatase which in turn is phosphorylated by Cdc2, increasing its phosphatase activity. Active Cdc2 also phosphorylates its inhibitory kinase Wee1, generating a phospho-degron (signal for degradation). This is recognized by an E3 ubiquitin-ligase, thereby targeting Wee1 for proteasome-dependent degradation, thus preventing inactivation of Cdc2 (Watanabe et al., 2004). Provided that LmxMPK3 behaves like a typical MAP kinase and displays a substrate specificity for serine or threonine followed by a proline residue, there are two potential phosphorylation sites in LmxMKK, serine 117 and threonine 279. It has yet to be tested whether they are indeed phosphorylated to influence the activity or half-life of LmxMKK.

The LmxMKK-LmxMPK3 cascade is not restricted to kinetoplastids because a homologue for LmxMPK3 was found in the flagellated green alga Chlamydomonas reinhardtii. Preceding mitosis the two flagella of Chlamydomonas are resorbed and grow again once the cell has completed division (Cavalier-Smith, 1974). The LmxMPK3 homologue might be involved in the regulation of the outgrowth of the new flagellum in the daughter cells. Although flagellar resorption is coupled to the differentiation to amastigotes in Leishmania, it is likely to be coupled to cytokinesis in Chlamydomonas. Leishmania might have lost this feature because the promastigotes are attached to the wall of the gut of the sandfly by inserting their flagellum between the microvilli of the gut. Resorption of the flagellum before cytokinesis would detach the cell and bear the risk of being excreted with the feces. Nevertheless, Leishmania needs to have a mechanism to initiate the formation of a new flagellum next to the old flagellum before cytokinesis. This cannot be the LmxMKK-LmxMPK3 signaling cascade because amastigotes without expressing the two kinases form a new flagellum before cell division with the difference that it never extends beyond the cell surface. In promastigotes, this mechanism allows for the growth of a short flagellum, lacking an appropriately assembled PFR. A full-length flagellum can only be formed in the presence of both kinases. Because Chlamydomonas lacks a PFR, the regulation of PFR assembly in Leishmania is likely to be an additional function beside the elongation of the flagellum. Indeed, PFR null mutants display full-length flagella (Maga et al., 1999).

The possibility to activate LmxMPK3 will facilitate the identification of the substrate(s) of this kinase, which could either be a gene regulatory molecule affecting components involved in the elongation and maintenance of the flagellum or components of the IFT itself. Now, we have reached a level very close to the elucidation of the regulation of gene expression in an organism lacking RNA polymerase II promoters and the usual eukaryotic transcription factors.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank Andrea MacDonald for expert technical assistance, Keith Gull (University of Oxford, Oxford, United Kingdom) for providing monoclonal antibody L8C4, and Thomas Ilg (Max-Planck-Institute for Biology, Tuebingen, Germany) for the anti-myo-inositol-1-phosphate synthase antiserum.

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-10-0976) on February 8, 2006.

Abbreviations used: IFT, intraflagellar transport; MAP, mitogen-activated protein; PFR, paraflagellar rod.

^* These authors contributed equally to this work.

Address correspondence to: Martin Wiese (martin.wiese{at}bnihamburg.de).

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