MAP Kinases
From Purdue Genomics Database Facility
Maria Cristina Suarez mcs@bio.ku.dk
Simon Bressendorff simonb@bio.ku.dk
Prof. John Mundy, Copenhagen Biocenter, Department of Molecular Biology, University of Copenhagen, Denmark
Contents |
MAP kinase cascade signaling components in Selaginella moellendorfii
MAP kinase cascades integrate extra-cellular signals and responses to environmental and developmental cues. Via sequential, reversible phosphorylations, MEKKs activate downstream MEKs (MKK), leading to activation of MPKs that target cytoplasmic and nuclear effectors including transcription factors. MEKKs phosphorylate MKKs on conserved S/T-X3-5-S/T motifs, while MKKs phosphorylate MPKs at a conserved T-X-Y motif (Chang and Karin, 2001). In Selaginella, we found 9 putative MPKs, 4 MKKs, and 23 MEKKs with conserved consensus motifs (MAPK Group, 2002; Fig. 1). In comparison, Arabidopsis encodes some 20 MPKs, 10 MKKs and ~60 MEKKs, with some orthologs identified in other species (Hardin and Wolniak, 2001; del Pozo et al., 2004; Pedley and Martin, 2004). The relatedness of Selaginella MAP kinases was established in phylogenetic trees including homologs from Physcomitrella patens, Oyza sativa and Chlamydomonas reinhardtii.
Fig. 1. WebLogo (Crooks et al., 2004) of consensus motifs of families of MAP kinase components from ClustalW alignments with all MPK, MKKs and MAP3K components used in the alignments. Stack heights represent conservation at a position, and symbol heights within a stack the relative frequency of each residue.
MPKs
A few plant MPKs have a TDY phosphorylation motif, while most have a TEY motif as in animal ERK kinases. Thus far, no annotated plant MPK has a TGY as in animal or yeast p38-like kinases (MAPK Group, 2002). Figure 2 shows that MPKs of ancient plants (Chlamydomonas, Physcomitrella and Selaginella) contain TDY and TEY phosphorylation motifs, as well as one from Selaginella with a TNY motif related to Arabidopsis MAK (male germ cell-associated kinase). Arabidopsis MPKs have been divided into four groups (MAPK group, 2002; Fig.3). The TEY MPKs constitute groups A, B and C, whereas group D contains the TDY MPKs. The putative Selaginella MPKs distribute among groups B, C, and D, but there are no apparent Selaginella or Physcomitrella homologs in group A. Group A includes Arabidopsis AtMPK3 and AtMPK6 and their tobacco and alfalfa orthologs which are involved in developmental processes and are activated in response to biotic and abiotic stresses (Wang et al., 2007; Zhang and Klessig, 2001; Munnik et al., 1999). The closest Selaginella MPK to AtMPK3 & 6 is in group B and may be orthologous to Arabidopsis AtMPK4 which is implicated in pathogen defense and abiotic stress responses (Petersen et al., 2000; Droillard et al., 2004; Andreasson et al., 2005; Brodersen et al., 2006). It would therefore be interesting to establish which of the Selaginella MPKs are activated in response to stresses that challenge early land plant lineages.
Fig. 2 ClustalW alignment of MPKs of Selaginella, Chlamydomonas, and Physcomitrella. Only TEY and TDY MPK phosphorylation motifs are apparently found in these species. The Selaginella MPK model names are highlighted in red.
A characteristic distinguishing groups A, B and C from D is the presence of a C-terminal CD domain that may act as a docking site for MKKs (Tanoue, et al. 2000). This feature is also present in group A, B and C MPKs from Chlamydomonas, Physcomitrella and Selaginella. Homologs of Arabidopsis MAK kinase (MHK) are included in the tree. The Chlamydomonas, homolog CrLF4 regulates flagellar length (Berman et al., 2003) and homologs from Caenorhabditis elegans and Leishmania mexicana may have similar functions in cilia length and morphology (Burghoorn et al., 2007). Thus, it has been proposed that plant MAKs function in meiotic cell division and maturation of male germ cells (Shinkai et al., 2002). In this subfamily, MAK has a TY motif characteristic of Cdc2/Cdk2 family cell cycle regulators, but MAK proteins also have a TxY as do MAP kinases (Miyata and Nishida, 1999). Selaginella MAK homologs were placed in groups I-III in our analysis (Fig. 3).
Fig 3. MPK tree. Sequences were aligned with ClustalW (pairwise alignment–gap opening 35.0, gap extension 0.75, multiple alignment-gap opening 15.0, gap extension 0.30; Hamel et al., 2006), and a tree calculated by bootstrapping 1000 replicates. Phylip output was visualized with TreeView (Page, 1996). Selaginella MPKs are in red. Pp: Physcomitrella, Os: Oryza, Cr: Chlamydomonas. Human ERK and Sc: Saccharomyces cerevisiae, HOG1 were used as outliers.
Plant responses to biotic and abiotic stresses involve changes in MPK genes expression (Seo et al, 1995; Jonak et al 1996; Mizoguchi et al, 1996; Navarro et al., 2004). We measured by quantitative RT-PCR levels of mRNAs of the annotated MPK genes in Selaginella organs and plants exposed to biotic and abiotic stress. Figure 4 summarizes expression levels of the nine putative Selaginella MPKs identified by their phylogenetic group (Fig.3). Overall higher levels of MPKs D1 and D2 mRNAs were detected, and were notably increased in response to bacterial elicitor (flg22) and downregulated in response to heat. In general, significant changes were detected in the levels of mRNAs of MPKs B, C2, D1 and D2 in response to stress. These results suggest regulatory signaling functions for the Selaginella members of groups C and D, in contrast to the predominant role of the Arabidopsis groups A and B MPKs.
Fig 4. Relative expression of MPK genes in Selaginella. Plants were treated with flg22 (bacterial elicitor), mechanical wounding, cold (4ºC), heat stress (37ºC), and 300mM NaCl. MPK gene names correspond to their position in the tree (Fig.3). Selaginella Actin 2 homolog was used as control.
MKKs
MKKs or MEKs fall in four groups as described for Arabidopsis and Oryza (Hammel et al., 2006). We found Selaginella MKKSs represented in groups A and B (Fig. 5) which include Arabidopsis AtMKK1 and AtMKK2 that act upstream of AtMPK4 (Mizoguchi et al., 1998; Ichimura et al., 1998). AtMKK2 is also involved in responses to cold and salinity, and both AtMKK1 & 2 may invoke innate immunity responses (Meszaros et al., 2006). It should therefore be tested whether the Selaginella orthologs interact and form cascades responding to abiotic and biotic threats. Interestingly, Selaginella apparently lacks close homologs of AtMKK4 & 5. This is consistent with the apparent lack in Selaginella of close homologs of AtMPK3 & 6 that participate in one or more cascades with AtMKK4 &5. However, two Selaginella MKKs cluster with two Physcomitrella homologs in group I that are more related to groups C and D from Arabidopsis and rice. The Selaginella MKK in cluster B is related to Arabidopsis MKK3. These cluster B MKKs have an unusual nuclear transfer factor (NTF) domain (Hamel et al., 2006), a feature we also found in the Chlamydomonas and Phsycomitrella homologs.
Fig 5. MKK tree. See Fig. 3 for methods and designations. Human MEK1 was used as an outlier.
MAP3Ks
This diverse family is divided in two large sub-families: The MEKK-like sub-family formed by MEKKs similar to the mammalian MEKK1 and yeast STE11 and BCK1 (Fig. 6; MAPK Group, 2002), and the RAF-like kinases that share more characteristics with mammalian RAF (Fig.7).
MEKK-like kinases - The Selaginella sub-family has 6 members. Interestingly, only two cluster in the plant MEKK-like groups A1-A4, and an independent branch is formed by Chlamydomonas, Physcomitrella and rice. This may indicate an early lineage conserved in monocots and shown as group I (Fig. 6). Most of the Arabidopsis, Brassica and tobacco MEKK-like proteins seem to participate in canonical MAP kinase cascades activating downstream MKKs. In contrast, group A4 MEKK-like members have not been proven to act in MAP kinase cascades and are very similar to the Cdc proteins of fission yeast (Champion et al., 2004).
Fig 6. MEKK-like tree. See Fig. 3 for methods and designations. Human MEKK1 was used as an outlier.
RAF-like MAP3K- There is at least one Selaginella representative for all groups, with the exception of Group C4 which includes the ATN1-like RAF-like MAP3K (Fig. 7. Some of the Group C3 and C4 proteins contain Ankyrin repeats (MAPK Group, 2002). Two of the best studied Arabidopsis MAP3Ks, CTR1 and EDR1, have easily distinguishable homologs in Selaginella. However, neither of these putative MAP3Ks has been confirmed to participate in a canonical MAP kinase cascade in Arabidopsis, and thus the functional characterization of the group B and C MAP3Ks in plants is still largely unexplored.
Fig 7. MA3PK RAF-like tree. See Fig. 3 for methods and designations. Human RAF was used as an outlier.
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List of excluded genes
We have excluded in our trees sequences from protein/gene models in Physcomitrella and Chlamydomonas, which presented dubious annotation, missing signature motifs. Only unique rice paralogs were included in the phylogenetic analysis to avoid over-representation of these sequences in the trees.
