Mitogen-activated protein kinases (MAPKs) have been the focus of many studies over the past several decades, but the understanding of one subgroup of MAPKs, orthologs of MAPK15, known as atypical MAPKs, has lagged behind others. In most organisms, specific activating signals or downstream responses of atypical MAPK signaling pathways have not yet been identified even though these MAPKs are associated with many eukaryotic processes, including cancer and embryonic development. In this Review, we discuss recent studies that are shedding new light on both the regulation and function of atypical MAPKs in different organisms. In particular, the analysis of the atypical MAPK in the amoeba Dictyostelium discoideum has revealed important roles in chemotactic responses and gene regulation. The rapid and transient phosphorylation of the atypical MAPK in these responses suggest a highly regulated activation mechanism in vivo despite the ability of atypical MAPKs to autophosphorylate in vitro. Atypical MAPK function can also impact the activation of other MAPKs in amoeba. These advances are providing new perspectives on possible MAPK roles in animals that have not been previously considered, and this might lead to the identification of potential targets for regulating cell movement in the treatment of diseases.

Mitogen-activated protein kinases (MAPKs) are a large family of protein kinases that serve important roles in eukaryotic signal transduction pathways (Pearson et al., 2001). The first discovered MAPKs were named for roles in mammalian mitogen-stimulated pathways, but the MAPK designation has been retained for related protein kinases that respond to a wide variety of signals and lead to many cellular responses, such as the regulation of cell growth and gene expression (Chen and Thorner, 2007; Hadwiger and Nguyen, 2011; Jonak et al., 2002; Lu and Malemud, 2019; Pearson et al., 2001; Raman et al., 2007). Some MAPKs have been referred to as extracellular signal-regulated kinases (ERKs) and others have been designated as p38 or c-Jun-N kinases, but all of these kinases belong to the same large group of serine/threonine protein kinases based on phylogenetic analysis. Most MAPKs have a common dually phosphorylated activation motif (pTXpY), which is required for full activation of catalytic activity, whereas others have slight modifications to this motif (Cargnello and Roux, 2011). The signals that trigger the activation of these protein kinases are diverse and the receptors are often G-protein-coupled or tyrosine kinase receptors (Raman et al., 2007). These receptors trigger protein kinase cascades upstream of MAPKs that typically include MAPK kinases (also known as MAP2Ks or MEKs) and MAPK kinase kinases (also known as MAP3Ks, RAFs or MEKKs) (Fig. 1) (Raman et al., 2007). A broad range of proteins are downstream substrates of MAPKs, and phosphorylation of these proteins leads to many cellular responses that include mitogenic processes, cellular differentiation, directed cell growth (chemotrophy) and cell movement (e.g. chemotaxis). The mammalian MAPK3 and MAPK1 (also known as Erk1 and Erk2, respectively) and yeast Fus3 and Kss1 MAPKs have been extensively studied and much is known about their regulation, specificity and function. The signaling mechanisms of these and other MAPK-mediated pathways have been used to generate canonical models of how MAPKs operate to control cellular processes, and in most cases these models fit well with experimental data on MAPKs in a wide range of eukaryotes. However, one subgroup of MAPKs, including the mammalian MAPK15 (also known as Erk8 or Erk7) and orthologs in other organisms, has been categorized as atypical MAPKs because their regulation and function appear to be outliers with respect to the canonical signaling model (Bogoyevitch and Court, 2004; Coulombe and Meloche, 2007; Deniz et al., 2022). Other subgroups of MAPKs, such as those represented by mammalian Erk3 and Erk4 (MAPK6 and MAPK4), have also been labeled as atypical due phylogenetic sequence analysis differences; however, in this Review we will only discuss what is known and unknown about the regulation and function of MAPK15-related atypical MAPKs.

Fig. 1.

Typical and atypical MAPK signaling pathways. External signal-stimulated receptors activate signal transduction pathways that can lead to the activation of MAPKs (also known as ERKs). Typical MAPK signaling pathways include upstream protein kinases such as MAPK kinases (MAP2Ks or MEKs), MAPK kinase kinases (MAP3Ks or MEKKs). MAPKs are capable of phosphorylating many substrates, leading to many cellular responses. Thus far, no upstream protein kinases have been identified for atypical MAPKs and the external signals that stimulate these pathways are unknown in most organisms.

Fig. 1.

Typical and atypical MAPK signaling pathways. External signal-stimulated receptors activate signal transduction pathways that can lead to the activation of MAPKs (also known as ERKs). Typical MAPK signaling pathways include upstream protein kinases such as MAPK kinases (MAP2Ks or MEKs), MAPK kinase kinases (MAP3Ks or MEKKs). MAPKs are capable of phosphorylating many substrates, leading to many cellular responses. Thus far, no upstream protein kinases have been identified for atypical MAPKs and the external signals that stimulate these pathways are unknown in most organisms.

The atypical MAPK MAPK15 was the last of the mammalian MAPKs to be identified (Abe et al., 1999). Other members of this MAPK subgroup have been identified in many eukaryotes, but not in nonmotile organisms, such as fungi or plants (Schwebs et al., 2018). The first member of this atypical group of MAPKs was identified and isolated in the amoeba Dictyostelium discoideum but was not recognized as an atypical MAPK until after MAPK phylogeny became more completely defined (Schwebs et al., 2018; Segall et al., 1995). Although it is absent in yeast, atypical MAPKs exist in other eukaryotic model organisms (e.g. mice, frogs, flies, nematodes and amoebae) and recently have also been found in eukaryotic parasitic microbes, such as toxoplasma and trypanosomes (Fig. 2). Based on current knowledge, mammals and most other organisms have only a single atypical MAPK gene, even though other MAPKs can be represented by multiple genes.

Fig. 2.

Phylogenetic tree of atypical and other subgroups of MAPKs. Protein sequence alignment and phylogenetic analysis were conducted using ClustalW and maximum likelihood method based on a JTT matrix-based model (Mega7 software) (Jones et al., 1992; Kumar et al., 2016). Out of 1000 bootstrapped trees the percentage of associated taxa clustered together is shown next to the branches. Branch lengths are drawn to scale and measured in the number of amino acid substitutions per site (scale bar=0.2 substitutions per site). MAPK (atypical in red box) sequences were analyzed from Homo sapiens – human (Hs), Dictyostelium discoideum (Dd), Dictyostelium purpureum (Dp), Cavenderia fasciculata (Cf), Heterostelium album (Ha), Acanthamoeba castellanii (Ac), Toxoplasma gondii (Tg), Plasmodium vivax (Pv), Paramecium tetraurelia (Pt), Tetrahymena thermophila (Tt), Trypanosoma brucei brucei (Tb), Drosophila melanogaster (Dm), Mus musculus – mouse (Mm), Lepisosteus oculatus – gar (Lo), Xenopus tropicalis – frog (Xt), Entamoeba histolytica (Eh), and Saccharomyces cerevisiae – budding yeast (Sc).

Fig. 2.

Phylogenetic tree of atypical and other subgroups of MAPKs. Protein sequence alignment and phylogenetic analysis were conducted using ClustalW and maximum likelihood method based on a JTT matrix-based model (Mega7 software) (Jones et al., 1992; Kumar et al., 2016). Out of 1000 bootstrapped trees the percentage of associated taxa clustered together is shown next to the branches. Branch lengths are drawn to scale and measured in the number of amino acid substitutions per site (scale bar=0.2 substitutions per site). MAPK (atypical in red box) sequences were analyzed from Homo sapiens – human (Hs), Dictyostelium discoideum (Dd), Dictyostelium purpureum (Dp), Cavenderia fasciculata (Cf), Heterostelium album (Ha), Acanthamoeba castellanii (Ac), Toxoplasma gondii (Tg), Plasmodium vivax (Pv), Paramecium tetraurelia (Pt), Tetrahymena thermophila (Tt), Trypanosoma brucei brucei (Tb), Drosophila melanogaster (Dm), Mus musculus – mouse (Mm), Lepisosteus oculatus – gar (Lo), Xenopus tropicalis – frog (Xt), Entamoeba histolytica (Eh), and Saccharomyces cerevisiae – budding yeast (Sc).

The MAPK15-related atypical MAPKs are distinguished from other MAPKs based on sequence differences throughout the kinase domain rather than by sections of sequences that represent unique domains. Some of these atypical MAPKs contain an unusually long C-terminal region, but this feature is not universal among homologs and the importance of this extended region is not well understood. Another distinguishing feature of these atypical MAPKs is that the phosphorylation of the activation motif (TXY) is not mediated by a conventional MAPK kinase (Abe et al., 2001; Schwebs et al., 2018). Thus far, no upstream protein kinase has been identified as the activator of these atypical MAPKs. As discussed later in this Review, some data points to the possibility of an autophosphorylation mechanism for activation. In most organisms, the external signals that stimulate atypical MAPK activation have not been identified and this gap in knowledge has been a challenge to the characterization of atypical MAPK function and regulation.

In this Review, we provide an overview of the associations of atypical MAPK signaling in a wide variety of cellular processes in animals and then discuss the analyses of atypical MAPKs in single-cell organisms including the amoeba Dictyostelium discoideum, where emerging roles for atypical MAPK signaling in chemotactic movement and gene regulation have been found. We will describe the regulation of atypical MAPK signaling and substrate preferences in amoeba and suggest future directions that might uncover the external signaling that regulates atypical MAPK signaling pathways in other organisms.

The mammalian atypical MAPK MAPK15 has been partially characterized in terms of expression, activation and interactions in a variety of mammals and cell lines, but remains poorly characterized in terms of function and regulation. MAPK15 expression during mammalian embryogenesis has not been reported, but it shows wide-spread transcript and protein expression in adult mammalian tissues. While the abundance of endogenously expressed MAPK15 protein is relatively low in most tissues and cell lines, elevated levels have been found in lung, kidney, and reproductive tissues (Abe et al., 1999, 2002). The significance of MAPK15 expression levels is not fully understood in noncancerous tissues. Although phosphorylated MAPK15 can be detected using general phosphoMAPK antibodies or by observing changes in MAPK15 migration in electrophoretic gels (Abe et al., 2001, 1999; Haimovitz and Shinitzky, 2001; Klevernic et al., 2006), specific signals that activate MAPK15 have not been identified. Therefore, many studies of MAPK15 have relied on heterologous expression of wild-type, mutant, or epitope-tagged proteins (Abe et al., 1999). Thus, it has been difficult to distinguish between physiological and non-physiological effects of MAPK15 signaling in mammalian cells.

There is evidence of MAPK15 involvement in several processes such as cell growth, ciliogenesis, nuclear receptor localization, genomic stability and autophagy, and some of these processes have also been associated with typical MAPKs in tumorigenic growth or cancer. In yeast two-hybrid screens, MAPK15 can bind to HIC-5, a co-activator of corticoid receptors, and heterologous expression of MAPK15 suppresses androgen and glucocorticoid receptor regulated gene expression (Saelzler et al., 2006). MAPK15 function is important for the translocation of the nuclear receptor estrogen-related receptor α (ERRα) to the cytoplasm and this process has been proposed to occur through the binding of ERRα to L/IxxxL/I sites in MAPK15 (Rossi et al., 2011). Reduction of MAPK15 expression in murine cell lines can reduce cilia formation and interfere with Hedgehog (Hh) signaling (Kazatskaya et al., 2017; Miyatake et al., 2015; Pietrobono et al., 2021). A few reports have connected MAPK15 function with genomic integrity. Treatment of cells with hydrogen peroxide (H2O2) results in increased levels of MAPK15 phosphorylation, and MAPK15 associates with proliferating cell nuclear antigen (PCNA), an integral component of chromatin, suggesting MAPK15 plays a role in general genome maintenance (Groehler and Lannigan, 2010; Klevernic et al., 2009; Klevernic et al., 2006).

Several studies have linked MAPK15 to the regulation of autophagy, beginning with an initial observation that the MAPK15 C-terminus can bind to ATG8-like proteins, such as LC3B (also known as MAP1LC3B) (Colecchia et al., 2012). The heterologous expression of wild-type MAPK15, but not a kinase-dead variant, has been reported to increase LC3B-positive autophagic structures, and the knockdown of endogenous MAPK15 reduced the number of these structures. Interactions with the oncoprotein BCR-ABL and the phosphorylation of unc-51-like kinase 1 (ULK1) have also been associated with the heterologous expression of MAPK15, and phosphorylation of both MAPK15 and ULK1 show correlations with mitophagy (Colecchia et al., 2018, 2015; Franci et al., 2022; Zhang et al., 2021). The precise role of MAPK15 in autophagy awaits further investigations. Many of the characterizations of mammalian MAPK15 have relied on heterologous expression or in vitro analysis, making it a challenge to understand functions of MAPK15 under normal physiological conditions. As with most MAPKs, MAPK15 might play a role in cell growth and therefore potentially contribute to diseases such as cancer, which we will discuss next in more detail.

Many MAPKs have been investigated for their roles in cancer because MAPKs were originally associated with regulation of cell growth and division (Iavarone et al., 2006; Le Gall et al., 2000; Wagner and Nebreda, 2009). Several studies have reported increased MAPK15 expression in tumors or cancer-derived cell lines, including thyroid cancer cell lines, embryonal carcinomas, colorectal cancer cells and gastric cancer tissues, as well as detectable expression in lung cancer cells (Cai et al., 2017; Iavarone et al., 2006; Jin et al., 2015; Rossi et al., 2016; Xu et al., 2010). Comparisons of gene expression in different tumors have led to the consideration that MAPK15 RNA, and possibly enhancer RNA (eRNA) associated with MAPK15, might serve as signatures that could help with cancer detection or survivability predictions with neuroblastoma, prostate or gallbladder cancers (Fan et al., 2021; Giwa et al., 2020; Roy et al., 2021). Conversely, other studies suggest that MAPK15 expression might be reduced in some cancers, such as estrogen receptor α (ERα)-positive breast cancers (Henrich et al., 2003; Rossi et al., 2011). The expression of a kinase-dead version of MAPK15, assumed to be a dominant-negative allele, decreases ERα degradation and presumably promotes cancer development in human breast cells (Henrich et al., 2003). However, altered gene expression is a common hallmark of most cancers, and changes in expression do not necessarily implicate a specific gene as a driver of cancer progression.

A better assessment of MAPK15 function in cancer can be inferred by observing changes in cancer cell growth when MAPK15 gene expression in cancer cell lines is knocked down using inhibitory RNAs. Lower levels of MAPK15 expression resulted in reduced proliferation of an embryonal carcinoma cell line, a human breast epithelial cell line, colorectal tumor cells, four different gastric cancer cell lines and a germ-cell-tumor-derived cell line (Groehler and Lannigan, 2010; Jin et al., 2015; Rossi et al., 2016; Xu et al., 2010). Furthermore, a reduction of MAPK15 expression can decrease the sensitivity of lung cancer cells to arsenic trioxide (As2O3), but attenuate the resistance of a nasopharyngeal cancer derived cell line to radiotherapy, suggesting that MAPK15 expression might affect the efficacy of some cancer treatments (Li et al., 2018; Wu et al., 2017). Inhibition of MAPK15 kinase activity negatively impacts ciliogenesis and cell proliferation through the Hh signaling pathway in medulloblastoma cells (Pietrobono et al., 2021). Cancer cells typically possess substantial telomeric activity compared to highly differentiated cells, and a decrease in MAPK15 expression can reduce telomerase function (Cerone et al., 2011). Heterologous MAPK15 expression promotes the phosphorylation of MAPK15 and interactions with potential drivers of cell proliferation, including cABL, PTC3, PCNA, ERRα and c-Jun (Groehler and Lannigan, 2010; Iavarone et al., 2006; Rossi et al., 2011; Xu et al., 2010). Besides these interactions, heterologous MAPK15 expression enhances embryonal carcinoma tumorigenicity, limits p53 activation and increases viability in response to irradiation (Li et al., 2018; Rossi et al., 2016). Although a defined role of MAPK15 function in cancer has yet to be determined, the observations of these many studies suggest that MAPK15 contributes to the regulation of cell growth and division in mammals.

Atypical MAPK orthologs have been analyzed in several non-mammalian animal models and associated with functions such as ciliogenesis, hormone release and neuronal signaling. Erk7 knockdown in developing Xenopus laevis embryos resulted in the reduced abundance and size of cilia in multi-ciliated cells (Miyatake et al., 2015). This development resembles the phenotype associated with reduced CapZ-interacting protein (CapZIP; also known as RCSD1). Erk7 was found to be capable of phosphorylating CapZIP in vitro and in cultured cells with heterologous expression of Erk7 and CapZIP. This phosphorylation can be mediated by the scaffold protein Dishevelled (Dvl), and a similar relationship between Erk7 and ciliogenesis was also observed in cultured mouse epithelial cells (Miyatake et al., 2015). In Drosophila melanogaster, the atypical MAPK ortholog Erk7 was identified in a RNAi screen as a protein kinase that regulates secretion of insulin-like peptides (ILPs) from insulin-producing cells (Hasygar and Hietakangas, 2014). Overexpression of the wild-type allele but not a kinase dead (K43R) allele resulted in dispersion of puncta of Sec16, a protein involved in secretory pathways that is normally localized to ER exit sites (Zacharogianni et al., 2011).

MAPK15 has also been found to have roles in neuronal function in the nematode Caenorhabditis elegans. The atypical MAPK15 ortholog in C. elegans (MAPK-15) has been associated with dopamine uptake in ciliated sensory neurons; mutant alleles of MAPK-15 were identified in a screen for swimming-induced paralysis, a phenotype also observed with defects in a dopamine transporter (DAT-1) (Bermingham et al., 2017). A large deletion in the mapk-15 locus alters male mating behavior and the localization of polycistin-2 ortholog (PKD-2), another protein required for proper male mating (Piasecki et al., 2017). More recently, loss of atypical MAPK function in C. elegans has been found to cause overgrowth of the URX sensory neuron, which is important for oxygen sensing, during the adult stage (McLachlan et al., 2018). This neuron has non-ciliated dendrites, suggesting that there are also roles for atypical MAPKs in non-ciliated structures. These genetic studies in animal models indicate that atypical MAPKs can play important roles in development of specialized cells or tissues of multicellular organisms. However, atypical MAPKs also exist in single-cell organisms, including intracellular parasites as discussed below.

Atypical MAPK function has been assessed in several single-cell eukaryotic parasites, such as Trypanosoma, Toxoplasma and Entamoeba. In Trypanosoma brucei, the organism that causes sleeping sickness, the atypical MAPK Erk8 was found to be autophosphorylated and it associated with PCNA in pull-down experiments, through a putative PCNA-interacting protein-box (PIP-box) motif (Valenciano et al., 2016). RNAi-mediated depletion of the atypical MAPK resulted in reduced PCNA phosphorylation; however, a direct phosphorylation of PCNA by the atypical MAPK has not been demonstrated. Interestingly, the trypanosome atypical MAPK was more sensitive than the human homolog to an atypical MAPK inhibitor, opening the possibility that the Trypanosome atypical MAPK might be a target for disease treatment (Nicolae and Moldovan, 2016). In Toxoplasma gondii, the parasite responsible for toxoplasmosis, the atypical MAPK (ERK7) was shown to associate with a scaffold protein AC9 in the apical complex (Back et al., 2020; Lacey et al., 2007). Reduced levels of the Toxoplasma ERK7 prevent maturation of the apical complex and therefore impair motility, invasion and egress of the parasite (O'Shaughnessy et al., 2020).

Furthermore, the only MAPK identified thus far in the parasite Entamoeba histolytica, a known cause of amoebiasis, belongs to the atypical MAPK subgroup (Ray et al., 2005). In Entamoeba invadens, which is associated with a similar pathology to E. histolytica in reptiles, H2O2 exposure results in an increase in atypical MAPK expression, and knockdown of the atypical MAPK expression impairs the efficiency of encystation, a transition to a dormant phase in response to starvation (Singh et al., 2018). Analysis of atypical MAPKs in these parasitic organisms underscores the association of atypical MAPKs with nuclear functions and specialized cell structures, similar to those observed in animals, but the external signals that regulate these atypical MAPK pathways remain to be determined.

Perhaps the greatest advancements in understanding atypical MAPK regulation and function have come from their study in the amoeba Dictyostelium discoideum, an organism often used to study signal transduction mechanisms involved with eukaryotic cell movement and differentiation. In this organism, studies of the atypical MAPK have uncovered significant roles in directed cell movement (chemotaxis) and gene regulation. The atypical MAPK homolog in Dictyostelium was first identified and designated as Erk2 (also known as ErkB) in 1995, but early characterizations of this homolog were based on a hypomorph allele resulting from an insertional mutation near the locus that resulted in a severe reduction of Erk2 expression (Segall et al., 1995). A notable phenotype of this mutant was the inability of cells to aggregate during the starvation-induced developmental life cycle. The underlying defect appeared to be a deficiency in cAMP levels, the intercellular chemoattractant signal that mediates the aggregation process (Fig. 3). This hypomorph mutant was capable of chemotaxis toward cAMP, allowing co-aggregation with wild-type cells, but the mutant cells were defective in cell differentiation during multicellular development (Gaskins et al., 1996; Segall et al., 1995; Wang et al., 1998). The hypomorph allele could also be suppressed by a loss of the cAMP-specific phosphodiesterase RegA, which is consistent with a role of Erk2 negatively regulating RegA to increase cAMP levels (Fig. 4) (Maeda et al., 2004). One study suggested that factors released from wild-type cells can rescue aggregation in clonal populations of the Erk2 mutant, but the identity of such factors has not been determined (Maeda and Kuwayama, 2000). An erk2 null mutant was eventually created from a disruption of the open reading frame, resulting in phenotypes that were much more severe than those of the hypomorph mutant (Nichols et al., 2019; Schwebs et al., 2018). The null mutant was incapable of chemotaxis toward either cAMP (the chemoattractant that mediates aggregation for multicellular development) or folic acid (the chemoattractant that mediates foraging for bacterial food sources) suggesting that the atypical MAPK is a crucial component for directed cell movement (Fig. 3). Complementation of the null mutant with heterologous expression of the wild-type Erk2 allele restores chemotaxis and allows for normal multicellular development (Schwebs et al., 2018). This study also found that loss of Erk2 slows growth on bacterial lawns, but this phenotype can be partly explained by the lack of chemotactic movement toward bacterial cells. Additionally, the growth rate is further reduced in mutants lacking both Erk2 and the only other Dictyostelium MAPK, Erk1 (also known as ErkA), suggesting some overlap in MAPK function for growth on bacteria. The mutant phenotypes of Erk2 indicate critical roles for this atypical MAPK in chemotaxis and multicellular development, and subsequent studies have begun to determine the molecular mechanisms surrounding these roles.

Fig. 3.

Chemotactic movement of Dictyostelium. The atypical MAPK Erk2 is required for chemotaxis toward folate or LPS and cAMP in Dictyostelium. Individual Dictyostelium amoeba can chemotax to folate or LPS as a mechanism to forage for bacterial food sources. The feeding on bacteria allows for growth and proliferation. In response to starvation, Dictyostelium secrete and chemotax to external cAMP as a mechanism to induce neighboring cells (∼105 cells/aggregate) to aggregate. Multicellular aggregates undergo a developmental life cycle to form fruiting bodies (a sphere of spores on top of a stalk). This developmental morphogenesis includes cell movement and differentiation within the aggregate. Bacteria, amoeba and fruiting body are not drawn to scale. Dictyostelium erk2 mutants are defective in chemotaxis, causing reduced growth on bacterial lawns and defective multicellular development.

Fig. 3.

Chemotactic movement of Dictyostelium. The atypical MAPK Erk2 is required for chemotaxis toward folate or LPS and cAMP in Dictyostelium. Individual Dictyostelium amoeba can chemotax to folate or LPS as a mechanism to forage for bacterial food sources. The feeding on bacteria allows for growth and proliferation. In response to starvation, Dictyostelium secrete and chemotax to external cAMP as a mechanism to induce neighboring cells (∼105 cells/aggregate) to aggregate. Multicellular aggregates undergo a developmental life cycle to form fruiting bodies (a sphere of spores on top of a stalk). This developmental morphogenesis includes cell movement and differentiation within the aggregate. Bacteria, amoeba and fruiting body are not drawn to scale. Dictyostelium erk2 mutants are defective in chemotaxis, causing reduced growth on bacterial lawns and defective multicellular development.

Fig. 4.

Atypical MAPK signaling pathways in Dictyostelium. The chemoattractants folate and cAMP stimulate G-protein-coupled receptors (Far1 and cARs, respectively) and associated G-proteins (Gα4, Gα2 and the Gβγ dimer), leading to the activation of the atypical MAPK Erk2 through an unknown mechanism. Erk2 mediates chemotactic movement, inhibits cAMP turnover and enhances activation of the other Dictyostelium MAPK (Erk1) in a secondary response, as well as phosphorylating (marked by ‘P’) and mediating translocation of the transcription factor GtaC. Other signaling components shown are adenylyl cyclase (AC), RegA cAMP-specific phosphodiesterase (PDE), conventional MAPK kinase (MekA). Unknown signaling factors are indicated with a question mark.

Fig. 4.

Atypical MAPK signaling pathways in Dictyostelium. The chemoattractants folate and cAMP stimulate G-protein-coupled receptors (Far1 and cARs, respectively) and associated G-proteins (Gα4, Gα2 and the Gβγ dimer), leading to the activation of the atypical MAPK Erk2 through an unknown mechanism. Erk2 mediates chemotactic movement, inhibits cAMP turnover and enhances activation of the other Dictyostelium MAPK (Erk1) in a secondary response, as well as phosphorylating (marked by ‘P’) and mediating translocation of the transcription factor GtaC. Other signaling components shown are adenylyl cyclase (AC), RegA cAMP-specific phosphodiesterase (PDE), conventional MAPK kinase (MekA). Unknown signaling factors are indicated with a question mark.

Early studies have demonstrated that activated Erk2 from Dictyostelium cells stimulated with cAMP or folate is capable of phosphorylating myelin basic protein (MBP) in in vitro kinase assays (Maeda et al., 1996; Maeda and Firtel, 1997). The stimulation of Erk2 kinase activity required cAMP receptors (Car1 and Car3), but Gα2 and Gβ, the G-protein subunits believed to couple with these receptors, were only partially required, because some kinase activity was observed in strains with disruptions in the genes encoding Gα2 and Gβ (Fig. 4). In contrast, the G-protein subunits that couple to the folate receptor, Gα4 and Gβ, were essential for Erk2 kinase activity in response to folate (Maeda and Firtel, 1997). In later studies, general phospho-MAPK antibodies detected phosphorylated Erk2 after chemoattractant stimulation, supporting the in vitro kinase assay results, and the folate receptor (Far1) was found to be essential for activation of Erk2 in response to folate (Brzostowski and Kimmel, 2006; Pan et al., 2016; Schwebs and Hadwiger, 2015). More recently, lipopolysaccharide (LPS) has also been found to stimulate the folate receptor and activate Erk2 (Pan et al., 2018). Activation of Erk2 in response to lysophosphatidic acid (LPA), a potential chemoattractant, has also been reported (Schenk et al., 2001). LPA activation did not require cAMP receptors (Car1 and Car3) but did require Gβ function. Other external signals that control aggregate size, which might act independently of G-protein coupled pathways (e.g. components of countin), can modulate the levels of Erk2 activation, but the specific mechanisms of this alteration are not understood (Brock et al., 2003). Folate- and cAMP-stimulated cells also activate Ras proteins, and several studies have suggested that Erk2 activation can be altered in a variety Ras mutants (Aubry et al., 1997; Knetsch et al., 1996; Kosaka et al., 1998; Lim et al., 2005). However, the activation of some Ras proteins by chemoattractants occurs in pathways that do not rely on Erk2 function (Pan et al., 2016). Together these studies suggest the Dictyostelium Erk2 is regulated through GPCR-initiated chemotaxis pathways and that alterations in other signaling proteins can impact Erk2 activity.

The Dictyostelium kinome contains only two MAPKs, the atypical MAPK Erk2 and another more typical MAPK, Erk1 (Fig. 2) (Goldberg et al., 2006). The activation of these MAPKs occurs very differently from each other (Goldberg et al., 2006; Schwebs et al., 2018). The chemoattractants cAMP and folate stimulate the rapid activation of Erk2 (within 30 s) that typically persists for 2–3 min (Brzostowski and Kimmel, 2006; Maeda et al., 1996; Maeda and Firtel, 1997; Schwebs and Hadwiger, 2015). Under conditions allowing cell–cell interactions, a burst of Erk1 activation occurs in a secondary response as Erk2 becomes dephosphorylated (Schwebs and Hadwiger, 2015). The sequential activation of Erk2 and then Erk1 in response to chemoattractants roughly correspond to the active chemotactic movement and then the adaptation response (a period of ‘rest’ caused by desensitization to the chemotactic signaling pathway), respectively, consistent with the requirement of Erk2 but not Erk1 in chemotactic movement. The period of Erk2 activation can be extended by increasing the chemoattractant concentration or preventing chemoattractant turnover (Brzostowski and Kimmel, 2006). Dictyostelium possesses only a single conventional MAPK kinase, MekA, and the loss of MekA produces phenotypes similar to the loss of Erk1, namely the formation of small aggregates during multicellular development (Gaskins et al., 1994; Sobko et al., 2002). Activation of Erk1 is dependent on MekA, but the loss of MekA does not impact Erk2 activation, consistent with the inability of conventional MAPK kinases to activate atypical MAPKs in other species (Ma et al., 1997; Schwebs et al., 2018) (Fig. 4). The dephosphorylation of Erk2 is not fully understood, but the loss of MPL1, a phosphatase with leucine-rich repeat regions, extends the duration of the phosphorylation of Erk2 in response to external cAMP (Rodriguez et al., 2008). Loss of MPL1 also reduces cAMP-mediated chemotaxis and impairs aggregation, potentially by not allowing proper desensitization of the chemotactic response, suggesting that this phosphatase plays an important role in early development. The rapid and transient activation of Erk2 after chemoattractant stimulation is consistent with Erk2 mediating chemotaxis and other downstream responses, such as regulation of transcription factors.

Early phosphoprotein labeling studies in Dictyostelium revealed that Erk2 function was required for the phosphorylation of a serine residue at position 250 of the Erk2-dependent phosphoprotein A (EppA) (Chen and Segall, 2006). A proline residue follows this particular serine residue, suggesting that Erk2 recognizes the same substrate motif pS/pTP as that recognized by other groups of MAPKs. Although the primary structure of EppA did not reveal clues about EppA function, loss of this protein or conversion of the S250 residue into alanine delayed development, reduced folate chemotaxis and reduced cAMP accumulation, consistent with the known roles of Erk2 (Chen and Segall, 2006). More recently, an extensive phosphoproteomic analysis conducted on folate- or cAMP-stimulated Dictyostelium revealed many proteins that were phosphorylated on motifs containing serine or threonine followed by proline and then arginine residues (pS/pTPR), and a screen of protein kinase mutants revealed that Erk2 was critical for pTPR phosphorylation in response to chemoattractants. Additional analysis of recombinant Erk2 purified from bacteria showed a preference of Erk2 to phosphorylate pS/pTPR peptides (Nichols et al., 2019). Some putative MAPK15 substrates in animals, such as the Xenopus CapZIP protein, also contain such sites but further studies are needed to determine whether atypical MAPKs in animals and amoeba have similar or different substrate phosphorylation site preferences (our own unpublished observation). A later study of the Dictyostelium Erk2-regulated transcription factor GtaC supports the idea that similar motifs (pS/pTPK/R) are phosphorylated in vivo (Hadwiger et al., 2022). The regulation of transcription factors by Erk2 suggests that it might also have roles in regulating gene expression downstream of chemotactic signals, which we will explore next.

Previous studies have shown that some MAPKs interact with transcription factors to regulate gene expression (Lavaur et al., 2007; Lin et al., 2017; Slone et al., 2016; Yang et al., 1999). For atypical MAPKs, early studies have reported associations between MAPK15 and the ERRα receptor transcription factor, and between MAPK15 and PCNA, a co-factor of DNA polymerase (Groehler and Lannigan, 2010; Rossi et al., 2011; Valenciano et al., 2016). However, these associations were inferred based on protein–protein interactions or in vitro phosphorylation assays. Phosphorylation of these proteins by the atypical MAPK has not been shown in vivo. Our recent study in Dictyostelium revealed that the atypical MAPK Erk2 is required for the translocation of the GATA transcription factor GtaC from the nucleus to the cytoplasm in response to the chemoattractant cAMP (Fig. 4) (Hadwiger et al., 2022). The GtaC transcription factor is a key regulator of Dictyostelium development, and the shuttling of GtaC from the nucleus into the cytoplasm occurs in response to periodic (occurring in ∼7-min cycles) endogenous cAMP signaling during the aggregation of starved cells (Cai et al., 2014; Keller and Thompson, 2008; Santhanam et al., 2015). GtaC contains a nuclear localization sequence and a potential nuclear export sequence, and the repeated nucleocytoplasmic shuttling of GtaC has been proposed to serve as a developmental timer mechanism to coordinate development and gene expression (Cai et al., 2014). Chromatin immunoprecipitation experiments indicate that GtaC can bind to many sites within the genome and that loss of GtaC has profound consequences on developmental gene expression (Keller and Thompson, 2008; Santhanam et al., 2015). The translocation of GtaC was also found to occur in response to the chemoattractant folate (Hadwiger et al., 2022). As mentioned earlier, folate and cAMP regulate Dictyostelium foraging and aggregation, respectively, suggesting that the translocation of GtaC alone is not the only mechanism promoting these two very different cell behaviors. Loss of GtaC promotes folate receptor expression and folate chemotaxis, suggesting that GtaC can function as a repressor of the folate receptor gene and that the translocation from the nucleus releases this repression (Hadwiger et al., 2022; Santhanam et al., 2015). In contrast, GtaC induces the expression of cAMP receptors, and the periodic return of GtaC to the nucleus during aggregation can induce cAMP receptor gene expression (Cai et al., 2014; Santhanam et al., 2015). The regulation of GtaC translocation is complex and requires at least one of four Erk2-preferred phosphorylation sites found within GtaC (Hadwiger et al., 2022). GFP-tagged Erk2 is found both in the nucleus and cytoplasm, and the stimulation of cells with chemoattractants does not result in a noticeable shift in Erk2 location (Adhikari et al., 2021). However, without Erk2 function GFP–GtaC is restricted to the nucleus, suggesting that active Erk2 phosphorylates GtaC in the nucleus (Hadwiger et al., 2022). The studies discussed in this section strongly implicate an important role for an atypical MAPK in the regulation of gene expression in amoeba, and if conserved throughout evolution, this function would be expected to also be present in animal cells.

Despite the above advances in understanding the functions of MAPK15-related atypical MAPKs, the specific mechanisms by which it is activated are still uncertain. Several studies have demonstrated atypical MAPK autophosphorylation in vitro or in heterologous expression systems (Abe et al., 1999; Nichols et al., 2019; Valenciano et al., 2016). Many protein kinases are capable of autophosphorylation under nonphysiological conditions, but most MAPKs are tightly regulated by MAPK kinases in vivo based on genetic analysis (Beenstock et al., 2016). Atypical MAPKs appear to be unique in that their overexpression often results in a phosphorylated state, as opposed to what is seen for other conventional MAPKs, suggesting that elevated expression enhances autophosphorylation or the constitutive phosphorylation by another protein kinase (Abe et al., 2001, 1999, 2002; Kuo et al., 2004). None of the conventional MAPK kinases have been shown to regulate the phosphorylation of atypical MAPKs, leaving many researchers to assume that MAPK kinases do not exist for these atypical MAPKs (Abe et al., 2001, 1999, 2002). There are likely many different mechanisms yet to be verified that could regulate the phosphorylation of atypical MAPKs including those that involve autophosphorylation or phosphorylation by another protein kinase (Fig. 5). Except for chemotactic signaling in Dictyostelium, the external signals that regulate atypical MAPK pathways in other organisms are unknown. Although the Dictyostelium atypical MAPK Erk2 can autophosphorylate in vitro, the chemoattractant stimulation of Erk2 phosphorylation in vivo is highly regulated leading to a rapid and transient phosphorylation of this MAPK (Nichols et al., 2019; Schwebs and Hadwiger, 2015). The ability to regulate atypical MAPK pathways with external signals, such as is the case with Dictyostelium chemotactic responses, will undoubtedly help with understanding the mechanism(s) of atypical MAPK activation.

Fig. 5.

Possible mechanisms for the regulation of atypical MAPK activation through a MAPK kinase or autophosphorylation. (A) An atypical MAPK (marked with a K) could be phosphorylated by an unconventional MAPK kinase (MAP2K*) in a mechanism similar to the activation of other MAPKs. However, a MAP2K* responsible for phosphorylating atypical MAPKs has not yet been identified. (B,C) Autophosphorylation of an atypical MAPK could be mediated by the actions of regulatory subunits (marked with a R) that negatively (B) or positively (C) regulate MAPK–MAPK interactions. Regulatory subunits could also be regulated by yet to be defined intracellular signals (small red circle). (D) An atypical MAPK could show constitutive autophosphorylation and a phosphatase (PPase) could serve as the primary regulator.

Fig. 5.

Possible mechanisms for the regulation of atypical MAPK activation through a MAPK kinase or autophosphorylation. (A) An atypical MAPK (marked with a K) could be phosphorylated by an unconventional MAPK kinase (MAP2K*) in a mechanism similar to the activation of other MAPKs. However, a MAP2K* responsible for phosphorylating atypical MAPKs has not yet been identified. (B,C) Autophosphorylation of an atypical MAPK could be mediated by the actions of regulatory subunits (marked with a R) that negatively (B) or positively (C) regulate MAPK–MAPK interactions. Regulatory subunits could also be regulated by yet to be defined intracellular signals (small red circle). (D) An atypical MAPK could show constitutive autophosphorylation and a phosphatase (PPase) could serve as the primary regulator.

Atypical MAPKs appear to be structurally conserved in a wide range of eukaryotes and do not depend on conventional MAPK kinases for activation, but whether they serve common functions or signal transduction processes in different eukaryotes remains to be determined. Currently, atypical MAPKs have been associated with a variety of cellular processes in animals, but many of these associations are based only on in vitro protein interactions or heterologous expression data (Fig. 6). Genetic analysis (i.e. reduced expression or loss-of-function alleles) suggests that atypical MAPKs play a role in polarized cell development (e.g. in ciliated and neuronal cells) or processes such as chemotactic movement and secretion (Hasygar and Hietakangas, 2014; Miyatake et al., 2015; O'Shaughnessy et al., 2020; Piasecki et al., 2017; Schwebs et al., 2018). If atypical MAPKs are found to also play a role in mammalian cell chemotaxis that is similar to their role in amoeba, then these MAPKs could provide important contributions to cell motility, such as in movement of immune cells during infections or of cancer cells during metastasis. Atypical MAPKs, like other MAPKs, might also have a general role in regulating cell growth as indicated by the growth-impaired phenotypes of cancer cells and Dictyostelium with reduced atypical MAPK expression (Groehler and Lannigan, 2010; Jin et al., 2015; Rossi et al., 2016; Schwebs et al., 2018; Xu et al., 2010). Studies in mammalian cells, trypanosomes and Dictyostelium also suggest that atypical MAPKs can regulate transcription factors and other nuclear proteins (Groehler and Lannigan, 2010; Hadwiger et al., 2022; Valenciano et al., 2016). Although some functional similarities exist for atypical MAPKs in different eukaryotes, determining the extent of common functions in various cell responses will likely require identifying the activating signals in organisms other than amoeba. Likely candidates for such signals might include chemoattractants, morphogens or other signals that can trigger polarized responses. Animal chemoattractants and chemokines should be explored for their ability to activate atypical MAPK pathways in chemotactic cells by monitoring the phosphorylation of the MAPK, but this analysis might need to focus on rapid responses such as those observed in amoeba. Furthermore, animal cells with reduced or inhibited atypical MAPK activity could be assayed for deficiencies in chemotaxis. Given that atypical MAPKs play such an important role in amoeba cell movement and gene expression, the investigation of atypical MAPKs in mammalian development, immune responses and cancer seems warranted. If atypical MAPK signaling pathways are found to be important for immune cell movement, then these pathways might become valuable targets for manipulating immune responses in the treatment of some diseases. If atypical MAPK pathways are determined to be contributors of cell growth or migration in the development of cancer, then understanding the stimuli and cellular responses associated with these pathways could prove useful for generating therapeutic treatments to reduce these cellular activities.

Fig. 6.

Atypical MAPK signaling pathways in animals and amoeba. Chemoattractants stimulate G-protein-coupled receptors to activate atypical MAPK signal transduction pathways in amoeba. Question marks signify components that have not been identified such as external signals, receptors and upstream activating mechanisms. Cellular activities associated with atypical MAPK signaling have not been conclusively identified in most organisms (e.g. animals) because the external signals that activate them are not known.

Fig. 6.

Atypical MAPK signaling pathways in animals and amoeba. Chemoattractants stimulate G-protein-coupled receptors to activate atypical MAPK signal transduction pathways in amoeba. Question marks signify components that have not been identified such as external signals, receptors and upstream activating mechanisms. Cellular activities associated with atypical MAPK signaling have not been conclusively identified in most organisms (e.g. animals) because the external signals that activate them are not known.

Funding

Our work in this area was supported by National Institutes of Health (NIGMS; R15 GM131269-02 to J.A.H.). Deposited in PMC for release after 12 months.

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Competing interests

The authors declare no competing or financial interests.