Atypical MAP kinases - new insights and directions from amoeba.

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.

An atypical MAPK regulates translocation of a GATA transcription factor in response to chemoattractant stimulation.

The Dictyostelium atypical mitogen-activated protein kinase (MAPK) Erk2 is required for chemotactic responses to cAMP as amoeba undergo multicellular development. In this study, Erk2 was found to be essential for the cAMP-stimulated translocation of the GATA transcription factor GtaC as indicated by the distribution of a GFP-GtaC reporter. Erk2 was also found to be essential for the translocation of GtaC in response to external folate, a foraging signal that directs the chemotaxis of amoeba to bacteria. Erk1, the only other Dictyostelium MAPK, was not required for the GtaC translocation to either chemoattractant, indicating that GFP-GtaC is a kinase translocation reporter specific for atypical MAPKs. The translocation of GFP-GtaC in response to folate was absent in mutants lacking the folate receptor Far1 or the coupled G-protein subunit Gα4. Loss of GtaC function resulted in enhanced chemotactic movement to folate, suggesting that GtaC suppresses responses to folate. The alteration of four Erk2-preferred phosphorylation sites in GtaC impacted the translocation of GFP-GtaC in response to folate and the GFP-GtaC-mediated rescue of aggregation and development of gtaC- cells. The ability of different chemoattractants to stimulate Erk2-regulated GtaC translocation suggests that atypical MAPK-mediated regulation of transcription factors can contribute to different cell fates.

Mitogen-activated protein kinase regulation of the phosphodiesterase RegA in early Dictyostelium development.

Mitogen-activated protein kinase (MAPK) regulation of cAMP-specific phosphodiesterase function has been demonstrated in mammalian cells and suspected to occur in other eukaryotes. Epistasis analysis in the soil amoeba Dictyostelium discoideum suggests the atypical MAPK Erk2 downregulates the function of the cAMP-specific phosphodiesterase RegA to regulate progression of the developmental life cycle. A putative MAPK docking motif located near a predicted MAPK phosphorylation site was characterized for contributions to RegA function and binding to Erk2 because a similar docking motif has been previously characterized in the mammalian PDE4D phosphodiesterase. The overexpression of RegA with alterations to this docking motif (RegAD-) restored RegA function to regA- cells based on developmental phenotypes, but low-level expression of RegAD- from the endogenous regA promoter failed to rescue wild-type morphogenesis. Co-immunoprecipitation analysis indicated that Erk2 associates with both RegA and RegAD-, suggesting the docking motif is not required for this association. Epistasis analysis between regA and the only other Dictyostelium MAPK, erk1, suggests Erk1 and RegA can function in different pathways but that some erk1- phenotypes may require cAMP signalling. These results imply that MAPK downregulation of RegA in Dictyostelium is accomplished through a different mechanism than MAPK regulation of cAMP-specific phosphodiesterases in mammalian cells and that the regulation in Dictyostelium does not require a proximal MAPK docking motif.

Role of the Vps9-domain protein RgfA in Dictyostelium chemotaxis and development.

Proteins with a Vps9 domain function as guanine nucleotide exchange factors for Rab proteins and can mediate the uptake of cell surface receptors or other molecules through endocytosis. However, genes encoding these proteins have not been previously studied in cells with robust chemotactic capabilities. Several genes encoding Vps9 domains were identified in the genome of Dictyostelium discoideum, and one of the genes, designated as rgfA (DDB_G0272038), was examined for functions in cell growth, development, and chemotaxis. The rgfA gene was expressed during vegetative growth and throughout development, but disruption of this gene resulted in no major alterations in cell growth, macropinocytosis, developmental morphology, or chemotactic movement. However, heterologous expression of RgfA resulted in a delay of developmental morphogenesis and impaired chemotaxis of cells to folate but did not affect macropinocytosis. These results suggest that RgfA might share redundant functions with other Dictyostelium Vps9-domain proteins and that heterologous expression of this protein can alter processes that depend on the reception of external signals.

MAPK docking motif in the Dictyostelium Gα2 subunit is required for aggregation and transcription factor translocation.

Some G protein alpha subunits contain a mitogen-activated protein kinase (MAPK) docking motif (D-motif) near the amino terminus that can impact cellular responses to external signals. The Dictyostelium Gα2 G protein subunit is required for chemotaxis to cAMP during the onset of multicellular development and this subunit contains a putative D-motif near the amino terminus. The Gα2 subunit D-motif was altered to examine its potential role in chemotaxis and multicellular development. In gα2- cells the expression of the D-motif mutant (Gα2D-) or wild-type subunit from high copy number vectors rescued cell aggregation but blocked the transition of mounds into slugs. This phenotype was also observed in parental strains with a wild-type gα2 locus indicating that the heterologous Gα2 subunit expression interferes with multicellular morphogenesis. Expression of the Gα2D- subunit from a low copy number vectors in gα2- cells did not rescue aggregation whereas the wild-type Gα2 subunit rescued aggregation efficiently and allowed wild-type morphological development. The Gα2D- and Gα2 subunit were both capable of restoring comparable levels of cAMP stimulated motility and the ability to co-aggregate with wild-type cells implying that the aggregation defect of Gα2D- expressing cells is due to insufficient intercellular signaling. Expression of the Gα2 subunit but not the Gα2D- subunit fully restored the ability of cAMP to stimulate the translocation of the GtaC transcription factor suggesting the D-motif is important for transcription factor regulation. These results suggest that the D-motif of Gα2 plays a role in aggregation and other developmental responses involved with cAMP signaling.

The Galpha4 G protein subunit interacts with the MAP kinase ERK2 using a D-motif that regulates developmental morphogenesis in Dictyostelium.

G protein Galpha subunits contribute to the specificity of different signal transduction pathways in Dictyostelium discoideum but Galpha subunit-effector interactions have not been previously identified. The requirement of the Dictyostelium Galpha4 subunit for MAP kinase (MAPK) activation and the identification of a putative MAPK docking site (D-motif) in this subunit suggested a possible interaction between the Galpha4 subunit and MAPKs. In vivo association of the Galpha4 subunit and ERK2 was demonstrated by pull-down and co-immunoprecipitation assays. Alteration of the D-motif reduced Galpha4 subunit-ERK2 interactions but only slightly altered MAPK activation in response to folate. Expression of the Galpha4 subunit with the altered D-motif in galpha4(-)cells allowed for slug formation but not the morphogenesis associated with culmination. Expression of this mutant Galpha4 subunit was sufficient to rescue chemotactic movement to folate. Alteration of the D-motif also reduced the aggregation defect associated with constitutively active Galpha4 subunits. These results suggest Galpha4 subunit-MAPK interactions are necessary for developmental morphogenesis but not for chemotaxis to folate.

G{alpha}5 subunit-mediated signalling requires a D-motif and the MAPK ERK1 in Dictyostelium.

The Dictyostelium Galpha5 subunit has been shown to reduce cell viability, inhibit folate chemotaxis and accelerate tip morphogenesis and gene expression during multicellular development. Alteration of the D-motif (mitogen-activated protein kinase docking site) at the amino terminus of the Galpha 5 subunit or the loss of extracellular signal-regulated kinase (ERK)1 diminished the lethality associated with the overexpression or constitutive activation of the Galpha5 subunit. The amino-terminal D-motif of the Galpha5 subunit was also found to be necessary for the reduced cell size, small aggregate formation and precocious developmental gene expression associated with Galpha5 subunit overexpression. This D-motif also contributed to the aggregation delay in cells expressing a constitutively active Galpha5 subunit, but the D-motif was not necessary for the inhibition of folate chemotaxis. These results suggest that the amino-terminal D-motif is required for some but not all phenotypes associated with elevated Galpha5 subunit functions during growth and development and that ERK1 can function in Galpha5 subunit-mediated signal transduction.

Multiple phosphorylation sites on the RegA phosphodiesterase regulate Dictyostelium development.

In Dictyostelium, the intracellular cAMP-specific phosphodiesterase RegA is a negative regulator of cAMP-dependent protein kinase (PKA), a key determinant in the timing of developmental morphogenesis and spore formation. To assess the role of protein kinases in the regulation of RegA function, this study identified phosphorylation sites on RegA and characterized the role of these modifications through the analysis of phospho-mimetic and phospho-ablative mutations. Mutations affecting residue T676 of RegA, a presumed target of the atypical MAP kinase Erk2, altered the rate of development and impacted cell distribution in chimeric organisms suggesting that phosphorylation of this residue reduces RegA function and regulates cell localization during multicellular development. Mutations affecting the residue S142 of RegA also impacted the rate developmental morphogenesis but in a manner opposite of changes at T676 suggesting the phosphorylation of the S142 residue increases RegA function. Mutations affecting residue S413 residue altered aggregate sizes and delayed developmental progression suggesting that PKA operates in a negative feedback mechanism to increase RegA function. These results suggest that the phosphorylation of different residues on RegA can lead to increased or decreased RegA function and therefore in turn regulate developmental processes such as aggregate formation, cell distribution, and the kinetics of developmental morphogenesis.

Developmental morphology and chemotactic responses are dependent on G alpha subunit specificity in Dictyostelium.

Dictyostelium discoideum expresses multiple G alpha subunits but only a single G beta and G gamma subunit suggesting that the specific response to an external signal depends largely on G alpha subunit function or G protein-independent signaling from the receptor. To test the contribution of G alpha subunit functional specificity, the chimeric G alpha subunits, G alpha2/4 and G alpha5/4, were created and analyzed along with wild-type subunits for the ability to substitute for the G alpha4 subunit in mediating responses from folate receptors. The G alpha2/4 subunit, but not the G alpha2 or G alpha5/4 subunits, partly rescued chemotaxis and cGMP accumulation in folate-stimulated g alpha4(-) cells. Expression of the G alpha5/4 or G alpha5 subunits resulted in an inhibition of g alpha4- and wild-type cell movement and a reduced aggregate size in developing wild-type and g alpha5- cells suggesting these subunits mediate similar responses. Only the G alpha4 subunit was capable of correcting developmental morphology in g alpha4- multicellular aggregates suggesting that the chimeric G alpha2/4 or G alpha5/4 subunits were insufficient to provide the G alpha4 function necessary for proper development. These results indicate that Dictyostelium G alpha subunit specificity is not limited to receptor coupling and that G alpha subunit sequences outside of the carboxyl terminus are important for cell movement and developmental processes.

Dictyostelium Erk2 is an atypical MAPK required for chemotaxis.

The Dictyostelium genome encodes only two MAPKs, Erk1 and Erk2, and both are expressed during growth and development. Reduced levels of Erk2 expression have been shown previously to restrict cAMP production during development but still allow for chemotactic movement. In this study the erk2 gene was disrupted to eliminate Erk2 function. The absence of Erk2 resulted in a complete loss of folate and cAMP chemotaxis suggesting that this MAPK plays an integral role in the signaling mechanisms involved with this cellular response. However, folate stimulation of early chemotactic responses, such as Ras and PI3K activation and rapid actin filament formation, were not affected by the loss of Erk2 function. The erk2- cells had a severe defect in growth on bacterial lawns but assays of bacterial cell engulfment displayed only subtle changes in the rate of bacterial engulfment. Only cells with no MAPK function, erk1-erk2- double mutants, displayed a severe proliferation defect in axenic medium. Loss of Erk2 impaired the phosphorylation of Erk1 in secondary responses to folate stimulation indicating that Erk2 has a role in the regulation of Erk1 activation during chemotaxis. Loss of the only known Dictyostelium MAPK kinase, MekA, prevented the phosphorylation of Erk1 but not Erk2 in response to folate and cAMP confirming that Erk2 is not regulated by a conventional MAP2K. This lack of MAP2K phosphorylation of Erk2 and the sequence similarity of Erk2 to mammalian MAPK15 (Erk8) suggest that the Dictyostelium Erk2 belongs to a group of atypical MAPKs. MAPK activation has been observed in chemotactic responses in a wide range of organisms but this study demonstrates an essential role for MAPK function in chemotactic movement. This study also confirms that MAPKs provide critical contributions to cell proliferation.

Acanthamoeba and Dictyostelium Use Different Foraging Strategies.

Amoeba often use cell movement as a mechanism to find food, such as bacteria, in their environment. The chemotactic movement of the soil amoeba Dictyostelium to folate or other pterin compounds released by bacteria is a well-documented foraging mechanism. Acanthamoeba can also feed on bacteria but relatively little is known about the mechanism(s) by which this amoeba locates bacteria. Acanthamoeba movement in the presence of folate or bacteria was analyzed in above agar assays and compared to that observed for Dictyostelium. The overall mobility of Acanthamoeba was robust like that of Dictyostelium but Acanthamoeba did not display a chemotactic response to folate. In the presence of bacteria, Acanthamoeba only showed a marginal bias in directed movement whereas Dictyostelium displayed a strong chemotactic response. A comparison of genomes revealed that Acanthamoeba and Dictyostelium share some similarities in G protein signaling components but that specific G proteins used in Dictyostelium chemotactic responses were not present in current Acanthamoeba genome sequence data. The results of this study suggest that Acanthamoeba does not use chemotaxis as the primary mechanism to find bacterial food sources and that the chemotactic responses of Dictyostelium to bacteria may have co-evolved with chemotactic responses that facilitate multicellular development.

The Dictyostelium MAPK ERK1 is phosphorylated in a secondary response to early developmental signaling.

Previous reports have suggested that the two mitogen-activated protein kinases (MAPKs) in Dictyostelium discoideum, ERK1 and ERK2, can be directly activated in response to external cAMP even though these MAPKs play different roles in the developmental life cycle. To better characterize MAPK regulation, the levels of phosphorylated MAPKs were analyzed in response to external signals. Only ERK2 was rapidly phosphorylated in response to the chemoattractants, cAMP and folate. In contrast, the phosphorylation of ERK1 occurred as a secondary or indirect response to these stimuli and this phosphorylation was enhanced by cell-cell interactions, suggesting that other external signals can activate ERK1. The phosphorylation of ERK1 or ERK2 did not require the function of the other MAPK in these responses. Folate stimulation of a chimeric population of erk1- and gα4- cells revealed that the phosphorylation of ERK1 could be mediated through an intercellular signal other than folate. Loss of ERK1 function suppressed the developmental delay and the deficiency in anterior cell localization associated with gα5- mutants suggesting that ERK1 function can be down regulated through Gα5 subunit-mediated signaling. However, no major changes in the phosphorylation of ERK1 were observed in gα5- cells suggesting that the Gα5 subunit signaling pathway does not regulate the phosphorylation of ERK1. These findings suggest that the activation of ERK1 occurs as a secondary response to chemoattractants and that other cell-cell signaling mechanisms contribute to this activation. Gα5 subunit signaling can down regulate ERK1 function to promote prestalk cell development but not through major changes to the level of phosphorylated ERK1.

Loss of cAMP-specific phosphodiesterase rescues spore development in G protein mutant in dictyostelium.

Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well-understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Gα2 and Gα4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP-specific phosphodiesterase RegA. The regA gene was disrupted in gα2(−) and gα4(−) cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2(−) mutants. There gA(−) mutation had no major effects on developmental morphology but enriched the distribution of the Gα mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Gα4- mediated folate chemotaxis. However, the regA gene disruption in gα4(−) cells, but not in gα2(−) cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA(−) strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from the gα4(−)regA(−) strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Gα4(HC) (Gα4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Gα4 subunit expression can mask regA(−) associated phenotypes even when provided with wild-type intercellular signaling. These findings indicate that the Gα2 and Gα4-mediated pathways provide different contributions to the development of spores and stalk cells and that the absence of RegA function can bypass some but not all defects in G protein regulated spore development.

MAPKs in development: insights from Dictyostelium signaling pathways.

Mitogen activated protein kinases (MAPKs) play important roles in the development of eukaryotic organisms through the regulation of signal transduction pathways stimulated by external signals. MAPK signaling pathways have been associated with the regulation of cell growth, differentiation, and chemotaxis, indicating MAPKs contribute to a diverse set of developmental processes. In most eukaryotes, the diversity of external signals is likely to far exceed the diversity of MAPKs, suggesting that multiple signaling pathways might share MAPKs. Do different signaling pathways converge before MAPK function or can MAPKs maintain signaling specificity through interactions with specific proteins? The genetic and biochemical analysis of MAPK pathways in simple eukaryotes such as Dictyostelium offers opportunities to investigate functional specificity of MAPKs in G protein-mediated signal transduction pathways. This review considers the regulation and specificity of MAPK function in pathways that control Dictyostelium growth and development.

MAP kinases have different functions in Dictyostelium G protein-mediated signaling.

Extracellular signal regulated kinases (ERKs) are a class of MAP kinases that function in many signaling pathways in eukaryotic cells and in some cases, a single stimulus can activate more than one ERK suggesting functional redundancy or divergence from a common pathway. Dictyostelium discoideum encodes only two MAP kinases, ERK1 and ERK2, that both function during the developmental life cycle. To determine if ERK1 and ERK2 have overlapping functions, chemotactic and developmental phenotypes of erk1(-) and erk2(-) mutants were assessed with respect to G protein-mediated signal transduction pathways. ERK1 was specifically required for Galpha5-mediated tip morphogenesis and inhibition of folate chemotaxis but not for cAMP-stimulated chemotaxis or cGMP accumulation. ERK2 was the primary MAPK phosphorylated in response to folate or cAMP stimulation. Cell growth was not altered in erk1(-), erk2(-) or erk1(-)erk2(-) mutants but each mutant displayed a different pattern of cell sorting in chimeric aggregates. The distribution of GFP-ERK1 or GFP-ERK2 fusion proteins in the cytoplasm and nucleus was not grossly altered in cells stimulated with cAMP or folate. These results suggest ERK1 and ERK2 have different roles in G protein-mediated signaling during growth and development.

A cAMP receptor-like G protein-coupled receptor with roles in growth regulation and development.

Dictyostelium discoideum uses G protein-mediated signal transduction for many vegetative and developmental functions, suggesting the existence of G protein-coupled receptors (GPCRs) other than the four known cyclic adenosine monophosphate (cAMP) receptors (cAR1-4). Sequences of the cAMP receptors were used to identify Dictyostelium genes encoding cAMP receptor-like proteins, CrlA-C. Limited sequence identity between these putative GPCRs and the cAMP receptors suggests the Crl receptors are unlikely to be receptors for cAMP. The crl genes are expressed at various times during growth and the developmental life cycle. Disruption of individual crl genes did not impair chemotactic responses to folic acid or cAMP or alter cAMP-dependent aggregation. However, crlA(-) mutants grew to a higher cell density than did wild-type cells and high-copy-number crlA expression vectors were detrimental to cell viability, suggesting that CrlA is a negative regulator of cell growth. In addition, crlA(-) mutants produce large aggregates with delayed anterior tip formation indicating a role for the CrlA receptor in the development of the anterior prestalk cell region. The scarcity of GFP-expressing crlA(-) mutants in the anterior prestalk cell region of chimeric organisms supports a cell-autonomous role for the CrlA receptor in prestalk cell differentiation.