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Not your Mother’s MAPKs: Apicomplexan MAPK function in daughter cell budding

  • William J. O’Shaughnessy ,

    Contributed equally to this work with: William J. O’Shaughnessy, Pravin S. Dewangan

    Affiliation Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas, United States of America

    ⨯
  • Pravin S. Dewangan ,

    Contributed equally to this work with: William J. O’Shaughnessy, Pravin S. Dewangan

    Affiliation Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas, United States of America

    ⨯
  • E. Ariana Paiz,

    Affiliation Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas, United States of America

    ⨯
  • Michael L. Reese

    michael.reese@utsouthwestern.edu

    Affiliations Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas, United States of America, Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas, Texas, United States of America

    ⨯

Abstract

Reversible phosphorylation by protein kinases is one of the core mechanisms by which biological signals are propagated and processed. Mitogen-activated protein kinases, or MAPKs, are conserved throughout eukaryotes where they regulate cell cycle, development, and stress response. Here, we review advances in our understanding of the function and biochemistry of MAPK signaling in apicomplexan parasites. As expected for well-conserved signaling modules, MAPKs have been found to have multiple essential roles regulating both Toxoplasma tachyzoite replication and sexual differentiation in Plasmodium. However, apicomplexan MAPK signaling is notable for the lack of the canonical kinase cascade that normally regulates the networks, and therefore must be regulated by a distinct mechanism. We highlight what few regulatory relationships have been established to date, and discuss the challenges to the field in elucidating the complete MAPK signaling networks in these parasites.

Citation: O’Shaughnessy WJ, Dewangan PS, Paiz EA, Reese ML (2022) Not your Mother’s MAPKs: Apicomplexan MAPK function in daughter cell budding. PLoS Pathog 18(10): e1010849. https://doi.org/10.1371/journal.ppat.1010849

Editor: Bjorn F.C. Kafsack, Joan and Sanford I Weill Medical College of Cornell University, UNITED STATES

Published: October 13, 2022

Copyright: © 2022 O'Shaughnessy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the National Institutes of Health (NIH; R01AI150715 to M.L.R), the National Science Foundation (MCB1553334 to M.L.R.), and the Welch Foundation (I-2075-20210327 to M.L.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The mitogen-activated protein kinases (MAPKs), together with their regulators, comprise a core eukaryotic signaling module. The canonical MAPK module translates extracellular signals across a 3-tiered kinase cascade to provoke cellular responses (Fig 1; [1–4]). Typically, MAPKs respond to cellular stresses as well as growth and developmental cues (“mitogens”). Originally discovered in mammals and yeast, the MAPK cascades of standard model organisms have been largely well characterized. Apicomplexan parasites, however, encode atypical MAPKs whose functional networks are just beginning to be understood. Notably, apicomplexan parasites do not encode for any predicted G protein-coupled receptors or receptor kinases, which serve as the initiators of many MAPK cascades in model organisms. Apicomplexan parasites also lack STE family kinases [5–7] that are integral components of canonical MAPK cascades.

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Fig 1. Overview of MAPK signaling.

(A) In a canonical MAPK signaling cascade, a signal results in activation of the upstream MAP3K (the “MAPK kinase kinase”), which phosphorylates and activates the MAP2K (the “MAPK kinase”), which in turn phosphorylates its target MAPK on both Thr and Tyr. Apicomplexan parasites lack the STE kinase family, to which all MAP2Ks belong. They therefore encode no MAP2Ks or MAP3Ks. (B) Domain architecture of the apicomplexan MAPKs (not to scale). ERK7 and MAPKL1 both have long CTEs that are predicted to be intrinsically disordered by IUPRED [75]. The ERK7 CTE contains sequence repeats (green; Sarcocystidae only). In Toxoplasma, both phosphorylation (yellow diamonds) and fucosylation (orange hexagons) sites have been identified. The Plasmodium ERK7 CTE also has one or more predicted nuclear localization signals (purple rectangles), though no post-translational modifications of the CTE have been identified as of yet. The Toxoplasma MAPKL1 CTE is also phosphorylated. Many MAPKL1 family members have an extended activation loop that reaches a maximum of approximately 100 residues in Sarcocystidae. Most MAPK2 proteins have a disordered N-terminal extension. The activation motifs for each of the apicomplexan MAPK subfamilies are indicated above the kinase domain. CTE, C-terminal extension; MAPK, mitogen-activated protein kinase.

https://doi.org/10.1371/journal.ppat.1010849.g001

Genetic data from Toxoplasma have demonstrated that catalytic activity is critical to each of the 3 apicomplexan MAPK cellular functions [8–10]. These data indicate that the kinases are activated despite the lack of a canonical cascade, which begs the question: What is the mechanism of activation? Moreover, the identities of both the regulators and downstream targets of apicomplexan MAPKs remain largely unknown. Many questions remain in the biology of the apicomplexan MAPKs: What proteins do they phosphorylate to carry out their functions? What upstream signals do they respond to? By what mechanism do they become activated—by a non-standard activating kinase, an allosteric activator, or simply by scaffolding and regulatory inhibition? We also have much to learn about negative regulation of the pathways. Are apicomplexan MAPK signals tuned by phosphatases? By protein homeostasis? We will discuss these ideas as they relate to Apicomplexa in the context of the broader understanding of MAPK signaling in other organisms.

1) Overview of apicomplexan MAPKs

1.1) Clarification of apicomplexan MAPK nomenclature

Three distinct MAPKs have been identified in Apicomplexa: ERK7, MAPKL1, and MAPK2 (Fig 1B, Table 1). Two of the 3 apicomplexan MAPKs are found in Plasmodium. The Plasmodium ortholog of ERK7 was identified concurrently by 2 groups, who named it PfMAP-1/PfMRP [11,12]. The second Plasmodium MAPK was identified as PfMAP-2 [13]. Toxoplasma gondii encodes all 3 of the apicomplexan MAPKs. These are called TgMAPKL1 [8] (originally named TgMAPK1 [14]), TgMAPK2 (the ortholog of PfMAP-2, also called TgMAPK3 [15]), and TgERK7 (also called TgMAPK2 [15,16]). The lack of consistent naming conventions has led to some confusion in the field, and, for the sake of clarity, we will refer to these kinases as ERK7 (e.g., TgERK7, PfMAP-1; found in all Apicomplexa), MAPKL1 (e.g., TgMAPKL1; missing in Plasmodium), and MAPK2 (e.g., TgMAPK2, PfMAP-2; Alveolate-specific; found in all Apicomplexa) from here on.

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Table 1. Apicomplexan MAPK gene models.

https://doi.org/10.1371/journal.ppat.1010849.t001

1.2) Evolution of MAPK signaling in Apicomplexa

Mammalian ERK7 (called “ERK8” or “MAPK15” in humans) has been described as able to robustly autophosphorylate both the Thr/Tyr of its activation loop, and thus autoactivate [17]. Such autoactivation is an unusual property of a MAPK, since the cascades are typically characterized as tightly regulated. Other members of the CMGC kinase family autophosphorylate on their activation loop Tyr [18], and ancestral reconstruction of the MAPK family supports a model where the ancestral MAPK was autoactivating [19], and traded this activity to enable more tightly responsive signaling. ERK7s are early-branching MAPKs and appear to be the most broadly conserved members of the family throughout eukaryotes [10,19]. Thus, an ERK7-like molecule is likely the original MAPK.

While it is possible that ERK7 is the ancestor of all extant apicomplexan MAPKs, such a model is not supported by phylogenetic analysis (Fig 2). The number of available apicomplexan genomes has exploded in recent years and have been supplemented by the genomic sequences of closely related organisms such as those of the phylum Chromerida [20]. Notably, Chromerida, unlike Apicomplexa, appear to encode the upstream activating MAPK kinases (or “MAP2Ks”) and a much larger number of MAPKs (Fig 2), suggesting they possess a functional canonical MAPK cascade. Comparing the MAPKs across Apicomplexa with a diverse set of outgroups reveals that ERK7 from apicomplexan species branches with the orthologs from all other organisms compared (Fig 2). Furthermore, the MAPKL1 and MAPK2 clades clearly branch well outside of the ERK7 family. MAPK2 is well conserved throughout Alveolate phyla (Fig 2; [6,7,9]). There is strong support for MAPKL1 orthologs both in Cryptosporidium and in the Chromerids. It would therefore appear that each of the 3 apicomplexan MAPKs were found in the ancestral organism and that these proteins originally evolved in the context of a fully functional MAPK cascade.

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Fig 2. Phylogenetic analysis of the apicomplexan MAPKs.

Phylogenetic trees of multiple sequence alignments of the MAPKs from the indicated organisms were estimated using IQTREE2 [76]. Subtrees were collapsed for space considerations, indicated by triangles. When present, numbers to the right of the triangles indicate number of kinases in the subtree. Expanded subtrees for the apicomplexan MAPK clades are shown in inset boxes. MAPK activation loop motifs (e.g., TDY, TxH) have been indicated, where appropriate, to demonstrate that the TxH motif is not confined to the MAPK2 clade. Organisms: Metazoan (human, mouse, fruit fly), Apicomplexa (T. gondii, E. falciformis, E. maxima, C. parvum, G. niphandrodes, P. berghei, P. falciparum, P. vivax), Chromerid (C. velia, V. brassicaformis), Dinoflagellate (S. microadriaticum), Ciliate (T. thermophila), green plant (A. thaliana, C. reinhardtii), red algae (C. crispus, P. purpureum, P. yezoensis). Note: The estimated number of MAPKs in V. brassicaformis is approximately 40 and approximately 75 in C. velia; note that analysis is complicated by current low quality of genome build and lack of verification of gene models by transcript sequencing. MAPK, mitogen-activated protein kinase.

https://doi.org/10.1371/journal.ppat.1010849.g002

1.3) Apicomplexan MAPKs are each atypical

While the apicomplexan MAPKs are divergent, phylogenetic analysis robustly supports their identification as bona fide members of the family [5] (Fig 2). Typical MAPK proteins have little more than a kinase domain and have specialized their functions by variation in the MAPK scaffolding domains ([21]; described in detail below). Each of the 3 apicomplexan MAPKs have regions apart from the kinase domain (Fig 1B). Both ERK7 and MAPKL1 have sizeable C-terminal extensions (CTEs) that are predicted to be largely intrinsically disordered, while MAPK2 contains a shorter N-terminal extension. Many MAPKL1 proteins also have an extension in the activation loop region between the DFG and APE motifs, which is most extensive (approximately 100 residues) and potentially structured in Sarcocystidae.

The ERK7 CTE is a hallmark of this MAPK subfamily and has been implicated in regulation of kinase activity and subcellular localization in the mammalian protein [22]. Notably, the TgERK7 CTE is extensively post-translationally modified, including a number of phosphorylation [23] and fucosylation sites [24], though their functional importance has yet to be investigated. Furthermore, the ERK7 CTE in P. berghei is required for its nuclear localization, due to 2 nuclear localization signals (NLSs) in the region [25]. Intriguingly, ERK7 nuclear localization is not conserved in all Apicomplexan, as the NLS are notably absent in the Cryptosporidium and Toxoplasma proteins, and TgERK7 does not localize to the parasite nucleus, at least in tachyzoites [10].

Dual phosphorylation on the activation loop Thr/Tyr is the key to MAPK regulation (Fig 1A). The MAPK2 subfamily is therefore quite unusual in that it replaced the TxY activation motif with a TxH. This indicates that the MAPK2 family must be activated in a totally distinct mechanism from typical MAPKs, even in organisms that encode the full MAPK cascade. While His is not normally considered a phosphorylatable residue in eukaryotes, this may be due to difficulty in detection. Indeed, His acts as a transition phosphoacceptor in bacterial and plant 2-component signaling [26]. Furthermore, recent advances in mass spectrometry [27,28] and development of phospho-His-specific antibodies [29] have revealed potential roles for phospho-His in metazoan biology. Finally, Apicomplexa encode for members of the nucleoside diphosphate kinase family (e.g., TGME49_295350), which has been suggested to moonlight as a His kinase in metazoa [30]. Nevertheless, it is unclear how the conserved activation loop His affects MAPK2 activation or function, and certainly remains untested whether this residue is phosphorylated in parasites. Intriguingly, some (but not all) red algae encode MAPKs with a similar TxH motif to MAPK2 [31]. It is thus tantalizing to hypothesize that the MAPK2 family evolved from horizontal gene transfer from the red algal secondary endosymbiont shared among Alveolates. Phylogenetic analysis of available sequences, however, suggests that a TxH activation motif may have evolved multiple times in the MAPKs of red algal-derived lineages (Fig 2).

2) Regulation of the apicomplexan MAPKs

2.1) Phosphorylation is the first layer of MAPK regulation

As described above, MAPKs are unusual in requiring phosphorylation on both a Thr and Tyr in their activation loops in order to signal, though the MAP2Ks that carry this out are missing in Apicomplexa (Fig 1A). While ERK7 family members autoactivate, and therefore do not require an upstream kinase [17,19], it unclear, however, whether the same is true for MAPKL1 or MAPK2. No interacting partners have yet been identified for a MAPKL1 protein. In Plasmodium, 2 NEK kinases, PfNEK1 and PfNEK3, have been demonstrated to be able to phosphorylate the PfMAPK2 activation loop in vitro, and therefore have been suggested to act as activating kinases for this protein [32,33]. However, such an activating relationship between the kinases has not been rigorously validated. It has yet to be demonstrated that PfNEK1 nor PfNEK3 are required for PfMAPK2 function within the parasites. Importantly, kinases that are bona fide activators of MAPKs typically contain a docking sequence motif that facilitates efficiency and specificity of activation (Fig 3; described below). Thus, identification of apicomplexan MAPKs regulators is likely to come from interactome studies.

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Fig 3. MAPKs use docking sites to recognize substrates and regulators.

(A) Canonical MAPKs use the conserved D-site (orange) to recognize kinase-interaction-motifs such as that of MKP3 bound to ERK2 (blue; PDB: 2FYS). This site lies distal to the active site and substrate recognition region. Some MAPKs use a second docking site, the F-site (yellow) to recognize F-x-F-P motifs such as that found in ORP45 (purple; PDB: 7OPM). (B) The inhibitory scaffold AC9 wraps around the Toxoplasma ERK7 kinase domain (blue; PDB: 6V6A) and occupies both the D-site (orange), active site, and substrate-recognition region. The site where the F-site would be localized on the TgERK7 structure is indicated in yellow, though no F-site binding partners have yet been identified for an apicomplexan MAPK. It is possible that apicomplexan MAPKs do not use F-site recognition. (C) Alignment of the sequences comprising the D-site of the indicated MAPKs. Polar (mostly acidic) sites that are thought to provide specificity to the KIMs recognized are indicated by arrowheads and red text. Sites that typically make backbone or hydrophobic interactions with KIMs are boxed. (Hs–human; Tg–T. gondii; Cp–C. parvum; Pf–P. falciparum) KIM, kinase-interacting motif; MAPK, mitogen-activated protein kinase.

https://doi.org/10.1371/journal.ppat.1010849.g003

As MAPKs require dual phosphorylation to activate, they can also be deactivated by dephosphorylation, which is carried out by specific dual-specificity phosphatases (DUSPs). DUSPs are a critical component of tuning MAPK response [34,35] and recognize their cognate MAPKs through scaffolding interactions on the D-site (described below; Fig 3). Note that kinases that robustly autoactivate, such as ERK7, cannot be primarily regulated by dephosphorylation without some additional inhibitory interaction, as the dephosphorylated kinase would quickly reactivate. MAPKL1 and MAPK2, however, may well be regulated by a more typical mechanism. We note that putative DUSP family members are encoded in all apicomplexan genomes (e.g., 9 in Toxoplasma, 3 in P. falciparum), though these have not yet been phenotypically or biochemically characterized. Importantly, many DUSPs regulate diverse substrates beyond MAPKs [36], so it is entirely possible these proteins regulate processes distinct from MAPK signaling in Apicomplexa.

2.2) Scaffolding interactions define the architecture of a MAPK cascade

Pawson and colleagues spearheaded the understanding of signaling cascades as being assembled by combinations of interactions between modular domains [37,38]. While scaffolding proteins certainly guide the architecture of canonical MAPK cascades [39–42], MAPKs, in general, recognize both their regulators and substrates using conserved docking sites on the kinase domain [43]. Docking site interactions typically have affinities in the high nanomolar to low micromolar range and have been demonstrated to be the main drivers of signaling specificity [21]. Therefore, defining such docking interactions for the apicomplexan MAPKs is potentially the most direct method by which we can identify potential activators and substrates and thus delineate their signaling pathways.

Two distinct docking sites have been identified on the MAPK kinase domain. The first, called the D-site or common docking (CD) domain, is thought to be present in all MAPKs. The D-site lies on the face opposite of the kinase active site and binds so-called “kinase-interacting motifs” or KIMs [43–46] (Fig 3). Most often, KIMs are short linear sequence motifs containing an N-terminal basic patch followed by a hydrophobic patch (e.g., [K/R]-X-[K/R]-X2-4-[I/L]-X-[I/L]) and are unfolded until binding [47,48]. Some interacting proteins, however, such as the dual-specificity phosphatases that down-regulate canonical MAPKs, bind the D-site using a folded surface [49]. The second site, called the F-site or DEF-site, lies on the C-lobe just below the activation loop [47,50] and typically recognizes short linear motifs containing the sequence F-X-F-P [51] (Fig 3). While the D-site appears conserved in all MAPKs, the F-site binding seems to be missing in some MAPKs and has therefore been proposed to provide an additional layer of specificity to substrate recognition [51–53]. Even though the motifs recognized by the 2 docking sites are relatively relaxed, there is a surprising degree of functional specificity in practice, suggesting there is still much to learn about the determinants of recognition. We note that the primary sequence and structural elements of both docking sites appear intact in each of the 3 apicomplexan MAPKs, suggesting that they recognize similar motifs those found in metazoan proteins.

2.3) Regulation by inhibitory interactions

In addition to regulation by phosphorylation, MAPKs, like other kinases, may be inactivated by binding inhibitor proteins. The scaffolding protein PEA-15 sequesters metazoan ERK2 in the cytoplasm [54]. PEA-15 binds near the ERK2 F-site, which results in conformational changes to the activation loop, the Gly-rich loop, and the α-C helix, inactivating the kinase [53]. A number of pathogens, such as KSHV and Yersinia, use proteins with optimized docking site motifs to compete off the endogenous partners of kinases such as mammalian RSK and ERK2 [50,55], thereby blocking kinase function.

Such a mechanism of regulation is likely especially important for kinases such as ERK7 that are autoactivating. Indeed, we have found that that an inhibitory scaffold is essential to ERK7 function in Toxoplasma. We demonstrated that Toxoplasma apical cap protein 9 (AC9) binds tightly to the ERK7 kinase domain, and is required for its recruitment to the parasite apical cap, and thus for its essential function [56]. AC9 interacts with TgERK7 with a surprisingly large interface, wrapping around the kinase to occupy the D-site docking domain, the ATP binding site, and the substrate recognition site of the kinase [56] (Fig 3 –docking domain). While the net binding affinity of AC9 for ERK7 is relatively high (KD approximately 50 nM), the distribution of the binding energy over such a large surface leads to an interaction that is relatively dynamic. This led to a model whereby AC9 acts both to concentrate TgERK7 at its site of action and to block phosphorylation of nonspecific substrates. AC9 can be effectively competed off by substrates containing motifs that recognize the D-site with only moderate affinity (1 to 10 μM) [56]. In fact, we recently found that another apical cap protein, AC10, is one such substrate of ERK7 [57]. Furthermore, it appears that AC9 and AC10 use multivalent interactions to form an amorphous oligomer at the apical cap IMC to concentrate TgERK7 at this site [57] and facilitate its function.

2.4) Regulation by protein homeostasis

Another major mechanism of kinase regulation is, of course, protein homeostasis. Each of the 3 Toxoplasma MAPKs show strong cell cycle dependence of their transcript levels (see EupathDB; [58,59]). All 3 Toxoplasma MAPKs have transcript levels that vary with the tachyzoite cell cycle (ToxoDB v53). While TgERK7 protein is found throughout the cell cycle [10], TgMAPKL1 is found only during S-phase and mitosis [60]. TgMAPK2 protein levels also appear cell cycle regulated, and the protein is undetectable from late budding through cytokinesis [9]. Notably, the TgMAPK2-AID phenotype was not completely rescued by expression of a non-degradable (i.e., not AID-tagged) copy of the protein [9]. Thus, a careful balance of protein expression and degradation may be critical to the tuning of apicomplexan MAPK signaling.

3) Biological functions associated with the apicomplexan MAPKs

Recent work has defined functions for each of the apicomplexan MAPKs (Fig 4). As the majority of work has been conducted in Toxoplasma and Plasmodium, we will focus on the current understanding of MAPK function in those organisms.

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Fig 4. MAPK function in Toxoplasma tachyzoite replication.

ERK7 (red kinase) localizes to the maternal and daughter bud apical caps, just below the conoid (orange rings). MAPKL1 (green kinase) localizes to the pericentrosomal material surrounding the centrosome (green circle). MAPK2 (yellow kinase) is cytosolic. Right panels show phenotypes resulting from knockdown of each of MAPKs. Loss of ERK7 results in destruction of the conoid. Loss of MAPKL1 and MAPK2 has opposing effects on centrosome duplication. MAPK, mitogen-activated protein kinase.

https://doi.org/10.1371/journal.ppat.1010849.g004

3.1) MAPKL1

TgMAPKL1 localizes to the outer core of the centrosome of dividing Toxoplasma tachyzoites, and use of a temperature-sensitive allele demonstrated that its loss-of-function results in over-duplication of centrosomes [60]. This led to the model that nuclear and cellular division are controlled separately in Toxoplasma and related organisms [60]; MAPKL1 and other outer core centrosome components are proposed to control parasite budding, while inner core components control nuclear division. Given its tight association with the centrosome, MAPKL1 likely has multiple tight interacting partners, though no interactome study for this kinase has yet been published.

Notably, MAPKL1 has been demonstrated to be susceptible to inhibition by at least 2 available compounds that are marketed as selective. The ALK4,5,7 inhibitor SB505124 was demonstrated to block parasite replication both by inhibiting MAPKL1 and by altering host HIF1α signaling [8]. MAPKL1 also has a relatively small “gatekeeper” residue (Ser191) that renders it susceptible to inhibition by bumped inhibitors such as 1NM-PP1 [61]. Notably, TgCDPK1 is also potently inhibited by bumped inhibitors, blocking parasite motility and invasion [62]. The block on cell cycle could be rescued, however, by mutating the MAPKL1 gatekeeper to a more typical, bulky Tyr, verifying MAPKL1 as the relevant target [61]. While these studies highlight the potential (and mostly untapped) value of pharmacological inhibition to study biological function of apicomplexan kinases, they also demonstrate the importance of verifying specificity of a given drug when using it a new system (i.e., most parasites), and thus serve as important cautionary tales.

3.2) MAPK2

In Plasmodium, MAPK2 was found to be essential for male gametogenesis and ex-flagellation, a process by which the male gametocytes undergo 3 rounds of replication leading to the production of 4 flagellated gametes [63,64]. While MAPK2 was demonstrated dispensable in P. berghei asexual stages, early attempts to knock out the kinase in P. falciparum in the blood stages [65] led to the hypothesis that MAPK2 may have different functions in the 2 species in the asexual stages. However, a recent, unconditional, knockout of PfMAPK2 demonstrates that MAPK2 is dispensable in the asexual stages of both species [63,64]. Notably, while Plasmodium MAPK2 is primarily nuclear-localized in gametocytes [63], expression of an exogenous copy in the blood stages yields primarily cytosolic localization [65].

Somewhat surprisingly, given its function in Plasmodium and conservation throughout Alveolates, MAPK2 is essential in the asexual tachyzoite stage of Toxoplasma [9], though its role in the sexual stages has yet to be tested. When TgMAPK2 was conditionally depleted using the auxin-inducible degron system, parasite replication was arrested prior to the initiation of centrosome duplication and daughter cell budding [9]. This appeared to be a true arrest, as wash-out of auxin (and restoration of TgMAPK2 protein), rescued the phenotype in the majority of parasites. Remarkably, both the parasite cell and its organelles continued to grow in size without segregation of contents into buds or parasite division [9]. Thus, TgMAPK2 is required for maintaining the coupling of cell growth with division.

Intriguingly, MAPKL1 and MAPK2 loss-of-function in Toxoplasma both manifest in opposing phenotypes associated with centrosome duplication. While the loss of TgMAPKL1 results in over-duplication of the centrosome, loss of TgMAPK2 blocks duplication. Interestingly, while TgMAPKL1 is tightly associated with the centrosome [60], TgMAPK2 is broadly cytosolic and does not co-localize with any known cellular structures, including the centrosome [9]. Thus, TgMAPK2 likely facilitates centrosome duplication more indirectly than does TgMAPKL1. As with MAPKL1, there are, as of yet, no validated interactors for MAPK2 in any organism, so both its upstream regulators and downstream targets are a mystery. Notably, MAPKs often regulate transcription factor localization and activity, including the AP2 transcription factors in plants [66,67], which are also conserved in Apicomplexa [68,69]. One possibility that deserves consideration is that kinases, such as MAPK2, play a similar role in Apicomplexa.

3.3) ERK7

Compared to metazoa, where it is one of the most poorly understood MAPKs, in Apicomplexa, the best understood MAPK pathway is that of Toxoplasma ERK7, which is essential to the tachyzoite lytic cycle [10]. TgERK7 localizes to the apical cap of the parasite inner membrane complex (IMC), both in daughter buds and in mature parasites [10], though its staining becomes weaker in the mature parasites as they approach cytokinesis. When TgERK7 was inducibly knocked-down with the auxin-degron system, Toxoplasma tachyzoites replicated normally within a vacuole, but were immotile and therefore incapable of egress and invasion of new host cells [10]. In parasites without ERK7, these phenotypes are all due to the loss of the parasite conoid, the central organizing hub of the apical complex. As the conoid was preserved in early daughter buds, but missing in mature parasites grown without ERK7, the kinase was posited to play a role late in conoid assembly [10]. Recent findings have called into question this mechanism of action.

We recently completed an interactome study of TgERK7 [70]. In addition to its known regulatory scaffolds AC9 [56] and AC10 [57], we identified a putative E3 ligase called CSAR1 that directly interacted with the ERK7 by yeast-2-hybrid. Remarkably, knockout of CSAR1 suppressed the ERK7 loss-of-function phenotype, allowing parasites to mature with intact conoids. Thus, it appears loss-of-function of ERK7 leads to an aberrant function in CSAR1, causing the premature degradation of daughter conoids [70]. This idea is consistent with recent proteomics that indicated loss of ERK7 results in degradation of components of the apical complex [71]. Intriguingly, ERK7 has been strongly linked both to the biogenesis and maintenance of primary cilia in metazoa [72,73] as well as to regulation of ubiquitin-mediated degradation of specific proteins [74]. It seems likely, therefore, that different apicomplexan parasites have adapted conserved ERK7 functions to their varied life cycles.

In stark contrast to findings in Toxoplasma, the Plasmodium falciparum ERK7 ortholog (PfMAP-1) is apparently dispensable both for the blood stage and mosquito stages of the parasite’s life cycle [65]. What the kinase is doing in these parasites is therefore unknown. Plasmodium ERK7 localization is dynamic as parasites develop: P. berghei ERK7 was found to be enriched in the parasite nuclei in early liver stage schizonts. In the cytomere stage, PbERK7 was instead concentrated to comma- and ring-shaped structures that no longer co-localized with the nucleus, while in merozoites ERK7 displayed uniform cytosolic localization [25]. It is unfortunate that PbERK7 localization has not yet been experimentally compared with any additional marker, such as the IMC. Notably, PbERK7 expression is much higher in parasite stages such as ookinetes and sporozoites, suggesting its localization and function may be distinct in these stages.

Concluding remarks

Much of the fungal and metazoan MAPK signaling networks were elucidated by a combination of genetic screens, early systems biology methods (e.g., yeast-2-hybrid), and painstaking biochemistry. Identifying the full complement of activators, negative regulators, scaffolds, and downstream substrates of the apicomplexan MAPKs will take similar efforts. Importantly, the depth of understanding of human MAPK signaling has led to >10 FDA-approved small molecules to date (with more in clinical trials) and a plethora of tool compounds that target these pathways. Kinase signaling therefore represents an, as yet, untapped bounty of targets to treat apicomplexan infections. Of course, modern proteomics and bioinformatics methods can greatly accelerate discovery. However, to gain the depth of knowledge required to unravel the complexity of parasite kinase signaling will require the same kind of careful, hypothesis-driven cell biological, and biochemical work that enabled our current understanding in well-studied models.

References

  1. 1. Cobb MH, Goldsmith EJ. How MAP kinases are regulated. J Biol Chem. 1995;270:14843–14846. pmid:7797459
  2. 2. Lewis TS, Shapiro PS, Ahn NG. Signal transduction through MAP kinase cascades. Adv Cancer Res. 1998;74:49–139. pmid:9561267
  3. 3. Peti W, Page R. Molecular basis of MAP kinase regulation. Protein Sci Publ Protein Soc. 2013;22:1698–1710. pmid:24115095
  4. 4. Raman M, Chen W, Cobb MH. Differential regulation and properties of MAPKs. Oncogene. 2007;26:3100–3112. pmid:17496909
  5. 5. Talevich E, Mirza A, Kannan N. Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa. BMC Evol Biol. 2011;11:321. pmid:22047078
  6. 6. Talevich E, Tobin AB, Kannan N, Doerig C. An evolutionary perspective on the kinome of malaria parasites. Philos Trans R Soc Lond B Biol Sci. 2012;367:2607–2618. pmid:22889911
  7. 7. Ward P, Equinet L, Packer J, Doerig C. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote. BMC Genom. 2004;5:79. pmid:15479470
  8. 8. Brown KM, Suvorova E, Farrell A, McLain A, Dittmar A, Wiley GB, et al. Forward genetic screening identifies a small molecule that blocks Toxoplasma gondii growth by inhibiting both host- and parasite-encoded kinases. PLoS Pathog. 2014;10:e1004180. pmid:24945800
  9. 9. Hu X, O’Shaughnessy WJ, Beraki TG, Reese ML. Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division. mBio. 2020;11. pmid:33173004
  10. 10. O’Shaughnessy WJ, Hu X, Beraki T, McDougal M, Reese ML. Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure in Toxoplasma gondii. Mol Biol Cell. 2020;31:881–888. pmid:32073987
  11. 11. Doerig CM, Parzy D, Langsley G, Horrocks P, Carter R, Doerig CD. A MAP kinase homologue from the human malaria parasite, Plasmodium falciparum. Gene. 1996;177:1–6. pmid:8921836
  12. 12. Lin DT, Goldman ND, Syin C. Stage-specific expression of a Plasmodium falciparum protein related to the eukaryotic mitogen-activated protein kinases. Mol Biochem Parasitol. 1996;78:67–77. pmid:8813678
  13. 13. Dorin D, Alano P, Boccaccio I, Cicéron L, Doerig C, Sulpice R, et al. An atypical mitogen-activated protein kinase (MAPK) homologue expressed in gametocytes of the human malaria parasite Plasmodium falciparum. Identification of a MAPK signature. J Biol Chem. 1999;274:29912–29920. pmid:10514473
  14. 14. Brumlik MJ, Wei S, Finstad K, Nesbit J, Hyman LE, Lacey M, et al. Identification of a novel mitogen-activated protein kinase in Toxoplasma gondii. Int J Parasitol. 2004;34:1245–1254. pmid:15491587
  15. 15. Lacey MR, Brumlik MJ, Yenni RE, Burow ME, Curiel TJ. Toxoplasma gondii expresses two mitogen-activated protein kinase genes that represent distinct protozoan subfamilies. J Mol Evol. 2007;64:4–14. pmid:17160647
  16. 16. Huang H, Ma YF, Bao Y, Lee H, Lisanti MP, Tanowitz HB, et al. Molecular cloning and characterization of mitogen-activated protein kinase 2 in Toxoplasma gondii. Cell Cycle. 2011;10:3519–3526. pmid:22030559
  17. 17. Abe MK, Kahle KT, Saelzler MP, Orth K, Dixon JE, Rosner MR. ERK7 is an autoactivated member of the MAPK family. J Biol Chem. 2001;276:21272–21279. pmid:11287416
  18. 18. Lochhead PA, Sibbet G, Morrice N, Cleghon V. Activation-loop autophosphorylation is mediated by a novel transitional intermediate form of DYRKs. Cell. 2005;121:925–936. pmid:15960979
  19. 19. Sang D, Pinglay S, Wiewiora RP, Selvan ME, Lou HJ, Chodera JD, et al. Ancestral reconstruction reveals mechanisms of ERK regulatory evolution. eLife. 2019;8. pmid:31407663
  20. 20. Woo YH, Ansari H, Otto TD, Klinger CM, Kolisko M, Michálek J, et al. Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites. eLife. 2015;4:e06974. pmid:26175406
  21. 21. Won AP, Garbarino JE, Lim WA. Recruitment interactions can override catalytic interactions in determining the functional identity of a protein kinase. Proc Natl Acad Sci U S A. 2011;108:9809–9814. pmid:21628578
  22. 22. Abe MK, Kuo WL, Hershenson MB, Rosner MR. Extracellular signal-regulated kinase 7 (ERK7), a novel ERK with a C-terminal domain that regulates its activity, its cellular localization, and cell growth. Mol Cell Biol. 1999;19:1301–1312. pmid:9891064
  23. 23. Treeck M, Sanders JL, Elias JE, Boothroyd JC. The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites’ boundaries. Cell Host Microbe. 2011;10:410–419. pmid:22018241
  24. 24. Bandini G, Haserick JR, Motari E, Ouologuem DT, Lourido S, Roos DS, et al. O-fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii. Proc Natl Acad Sci U S A. 2016;113:11567–11572. pmid:27663739
  25. 25. Wierk JK, Langbehn A, Kamper M, Richter S, Burda P-C, Heussler VT, et al. Plasmodium berghei MAPK1 displays differential and dynamic subcellular localizations during liver stage development. PLoS ONE. 2013;8:e59755. pmid:23544094
  26. 26. Alex LA, Simon MI. Protein histidine kinases and signal transduction in prokaryotes and eukaryotes. Trends Genet TIG. 1994;10:133–138. pmid:8029829
  27. 27. Hardman G, Perkins S, Brownridge PJ, Clarke CJ, Byrne DP, Campbell AE, et al. Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation. EMBO J. 2019;38:e100847. pmid:31433507
  28. 28. Hardman G, Eyers CE. High-Throughput Characterization of Histidine Phosphorylation Sites Using UPAX and Tandem Mass Spectrometry. Methods Mol Biol. 2020;2077:225–235. pmid:31707662
  29. 29. Fuhs SR, Meisenhelder J, Aslanian A, Ma L, Zagorska A, Stankova M, et al. Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation. Cell. 2015;162:198–210. pmid:26140597
  30. 30. Adam K, Lesperance J, Hunter T, Zage PE. The Potential Functional Roles of NME1 Histidine Kinase Activity in Neuroblastoma Pathogenesis. Int J Mol Sci. 2020;21:E3319. pmid:32392889
  31. 31. Li C, Kong F, Sun P, Bi G, Li N, Mao Y, et al. Genome-wide identification and expression pattern analysis under abiotic stress of mitogen-activated protein kinase genes in Pyropia yezoensis. J Appl Phycol. 2018;30:2561–2572.
  32. 32. Dorin D, Le Roch K, Sallicandro P, Alano P, Parzy D, Poullet P, et al. Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum Biochemical properties and possible involvement in MAPK regulation. Eur J Biochem. 2001;268:2600–2608. pmid:11322879
  33. 33. Lye YM, Chan M, Sim T-S. Pfnek3: an atypical activator of a MAP kinase in Plasmodium falciparum. FEBS Lett. 2006;580:6083–6092. pmid:17064692
  34. 34. Caunt CJ, Keyse SM. Dual-specificity MAP kinase phosphatases (MKPs): shaping the outcome of MAP kinase signalling. FEBS J. 2013;280:489–504. pmid:22812510
  35. 35. Owens DM, Keyse SM. Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases. Oncogene. 2007;26:3203–3213. pmid:17496916
  36. 36. Patterson KI, Brummer T, O’Brien PM, Daly RJ. Dual-specificity phosphatases: critical regulators with diverse cellular targets. Biochem J. 2009;418:475–489. pmid:19228121
  37. 37. Pawson T, Scott JD. Signaling through scaffold, anchoring, and adaptor proteins. Science. 1997;278:2075–2080. pmid:9405336
  38. 38. Pawson T, Nash P. Assembly of cell regulatory systems through protein interaction domains. Science. 2003;300:445–452. pmid:12702867
  39. 39. Whiteway MS, Wu C, Leeuw T, Clark K, Fourest-Lieuvin A, Thomas DY, et al. Association of the yeast pheromone response G protein beta gamma subunits with the MAP kinase scaffold Ste5p. Science. 1995;269:1572–1575. pmid:7667635
  40. 40. Printen JA, Sprague GF. Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics. 1994;138:609–619. pmid:7851759
  41. 41. Dickens M, Rogers JS, Cavanagh J, Raitano A, Xia Z, Halpern JR, et al. A cytoplasmic inhibitor of the JNK signal transduction pathway. Science. 1997;277:693–696. pmid:9235893
  42. 42. Whitmarsh AJ, Cavanagh J, Tournier C, Yasuda J, Davis RJ. A mammalian scaffold complex that selectively mediates MAP kinase activation. Science. 1998;281:1671–1674. pmid:9733513
  43. 43. Tanoue T, Adachi M, Moriguchi T, Nishida E. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat Cell Biol. 2000;2:110–116. pmid:10655591
  44. 44. Bardwell AJ, Abdollahi M, Bardwell L. Docking sites on mitogen-activated protein kinase (MAPK) kinases, MAPK phosphatases and the Elk-1 transcription factor compete for MAPK binding and are crucial for enzymic activity. Biochem J. 2003;370:1077–1085. pmid:12529172
  45. 45. Kallunki T, Deng T, Hibi M, Karin M. c-Jun can recruit JNK to phosphorylate dimerization partners via specific docking interactions. Cell. 1996;87:929–939. pmid:8945519
  46. 46. Tanoue T, Maeda R, Adachi M, Nishida E. Identification of a docking groove on ERK and p38 MAP kinases that regulates the specificity of docking interactions. EMBO J. 2001;20:466–479. pmid:11157753
  47. 47. Lee T, Hoofnagle AN, Kabuyama Y, Stroud J, Min X, Goldsmith EJ, et al. Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry. Mol Cell. 2004;14:43–55. pmid:15068802
  48. 48. Chang CI, Xu B, Akella R, Cobb MH, Goldsmith EJ. Crystal structures of MAP kinase p38 complexed to the docking sites on its nuclear substrate MEF2A and activator MKK3b. Mol Cell. 2002;9:1241–1249. pmid:12086621
  49. 49. Zhang Y-Y, Wu J-W, Wang Z-X. A distinct interaction mode revealed by the crystal structure of the kinase p38α with the MAPK binding domain of the phosphatase MKP5. Sci Signal. 2011;4:ra88. pmid:22375048
  50. 50. Alexa A, Sok P, Gross F, Albert K, Kobori E, Póti ÁL, et al. A non-catalytic herpesviral protein reconfigures ERK-RSK signaling by targeting kinase docking systems in the host. Nat Commun. 2022;13:472. pmid:35078976
  51. 51. Jacobs D, Glossip D, Xing H, Muslin AJ, Kornfeld K. Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes Dev. 1999;13:163–175. pmid:9925641
  52. 52. Galanis A, Yang SH, Sharrocks AD. Selective targeting of MAPKs to the ETS domain transcription factor SAP-1. J Biol Chem. 2001;276:965–973. pmid:11029469
  53. 53. Mace PD, Wallez Y, Egger MF, Dobaczewska MK, Robinson H, Pasquale EB, et al. Structure of ERK2 bound to PEA-15 reveals a mechanism for rapid release of activated MAPK. Nat Commun. 2013;4:1681. pmid:23575685
  54. 54. Formstecher E, Ramos JW, Fauquet M, Calderwood DA, Hsieh JC, Canton B, et al. PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase. Dev Cell. 2001;1:239–250. pmid:11702783
  55. 55. Sorgeloos F, Peeters M, Hayashi Y, Borghese F, Capelli N, Drappier M, et al. A case of convergent evolution: Several viral and bacterial pathogens hijack RSK kinases through a common linear motif. Proc Natl Acad Sci U S A. 2022;119:e2114647119. pmid:35091472
  56. 56. Back PS, O’Shaughnessy WJ, Moon AS, Dewangan PS, Hu X, Sha J, et al. Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required for Toxoplasma apical complex biogenesis. Proc Natl Acad Sci U S A. 2020;117:12164–12173. pmid:32409604
  57. 57. Back PS, O’Shaughnessy WJ, Moon AS, Dewangan PS, Reese ML, Bradley PJ. Multivalent Interactions Drive the Toxoplasma AC9:AC10:ERK7 Complex To Concentrate ERK7 in the Apical Cap. mBio. 2022:e0286421. pmid:35130732
  58. 58. Aurrecoechea C, Brestelli J, Brunk BP, Fischer S, Gajria B, Gao X, et al. EuPathDB: a portal to eukaryotic pathogen databases. Nucleic Acids Res. 2010;38:D415–D419. pmid:19914931
  59. 59. Behnke MS, Wootton JC, Lehmann MM, Radke JB, Lucas O, Nawas J, et al. Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS ONE. 2010;5:e12354. pmid:20865045
  60. 60. Suvorova ES, Francia M, Striepen B, White MW. A novel bipartite centrosome coordinates the apicomplexan cell cycle. PLoS Biol. 2015;13:e1002093. pmid:25734885
  61. 61. Sugi T, Kawazu S-I, Horimoto T, Kato K. A single mutation in the gatekeeper residue in TgMAPKL-1 restores the inhibitory effect of a bumped kinase inhibitor on the cell cycle. Int J Parasitol Drugs Drug Resist. 2015;5:1–8. pmid:25941623
  62. 62. Lourido S, Shuman J, Zhang C, Shokat KM, Hui R, Sibley LD. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature. 2010;465:359–362. pmid:20485436
  63. 63. Hitz E, Balestra AC, Brochet M, Voss TS. PfMAP-2 is essential for male gametogenesis in the malaria parasite Plasmodium falciparum. Sci Rep. 2020;10:11930. pmid:32681115
  64. 64. Rangarajan R, Bei AK, Jethwaney D, Maldonado P, Dorin D, Sultan AA, et al. A mitogen-activated protein kinase regulates male gametogenesis and transmission of the malaria parasite Plasmodium berghei. EMBO Rep. 2005;6:464–469. pmid:15864297
  65. 65. Dorin-Semblat D, Quashie N, Halbert J, Sicard A, Doerig C, Peat E, et al. Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics. Mol Microbiol. 2007;65:1170–1180. pmid:17651389
  66. 66. Cheong YH, Moon BC, Kim JK, Kim CY, Kim MC, Kim IH, et al. BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor. Plant Physiol. 2003;132:1961–1972. pmid:12913152
  67. 67. Gu YQ, Yang C, Thara VK, Zhou J, Martin GB. Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase. Plant Cell. 2000;12:771–786. pmid:10810149
  68. 68. Modrzynska K, Pfander C, Chappell L, Yu L, Suarez C, Dundas K, et al. A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle. Cell Host Microbe. 2017;21:11–22. pmid:28081440
  69. 69. Radke JB, Lucas O, De Silva EK, Ma Y, Sullivan WJ, Weiss LM, et al. ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst. Proc Natl Acad Sci U S A. 2013;110:6871–6876. pmid:23572590
  70. 70. O’Shaughnessy WJ, Hu X, Henriquez SA, Reese ML. Toxoplasma ERK7 defends the apical complex from premature degradation. bioRxiv. 2021. p. 2021.12.09.471932.
  71. 71. Dos Santos PN, Tosetti N, Krishnan A, Haase R, Maco B, Suarez C, et al. Revisiting the Role of Toxoplasma gondii ERK7 in the Maintenance and Stability of the Apical Complex. mBio. 2021;12:e0205721. pmid:34607461
  72. 72. Kazatskaya A, Kuhns S, Lambacher NJ, Kennedy JE, Brear AG, McManus GJ, et al. Primary Cilium Formation and Ciliary Protein Trafficking Is Regulated by the Atypical MAP Kinase MAPK15 in Caenorhabditis elegans and Human Cells. Genetics. 2017;207:1423–1440. pmid:29021280
  73. 73. Miyatake K, Kusakabe M, Takahashi C, Nishida E. ERK7 regulates ciliogenesis by phosphorylating the actin regulator CapZIP in cooperation with Dishevelled. Nat Commun. 2015;6:6666. pmid:25823377
  74. 74. Henrich LM, Smith JA, Kitt D, Errington TM, Nguyen B, Traish AM, et al. Extracellular signal-regulated kinase 7, a regulator of hormone-dependent estrogen receptor destruction. Mol Cell Biol. 2003;23:5979–5988. pmid:12917323
  75. 75. Dosztányi Z, Csizmók V, Tompa P, Simon I. The pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins. J Mol Biol. 2005;347:827–839. pmid:15769473
  76. 76. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol. 2020;37:1530–1534. pmid:32011700