MAPKKK5 (MEKK1-subfamily MAP kinase kinase kinase, At5g66850; also MAP3Kgamma) is a cytosolic and plasma membrane-associated serine/threonine protein kinase that forms the MAP3K tier of the MAPKKK3/MAPKKK5-MKK4/MKK5-MPK3/MPK6 cascade activated by cell surface pattern recognition receptors. Its central kinase domain phosphorylates the activation-loop Ser/Thr residues of MKK4 and MKK5 (and weakly MKK2). MAPKKK5 is itself activated by phosphorylation by receptor-like cytoplasmic kinases downstream of PRRs: PBL27 phosphorylates its C-terminal region in a CERK1-dependent manner after chitin perception, RLCK VII kinases such as PBL19 phosphorylate Ser-599, and BSK1 phosphorylates Ser-289; MPK6 phosphorylates Ser-682/Ser-692 in a positive feedback loop. MAPKKK5 acts largely redundantly with MAPKKK3 in MPK3/MPK6 activation triggered by chitin, flg22, elf18 and Pep peptides, contributing to defense gene expression, chitin-induced callose deposition and resistance to bacterial and fungal pathogens, but it is not required for the PAMP-induced ROS burst. Together with YODA it also contributes to gamete transmission and embryogenesis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000165 MAPK cascade | IBA GO_REF:0000033 | ACCEPT | Summary: MAPKKK5 is a MEKK-subfamily MAP3K that phosphorylates the activation loops of MKK4 and MKK5, the MAP2Ks for MPK3/MPK6; the IBA places MAP3K-tier participation in MAPK cascades at a deep PANTHER node. Reason: MAPKKK5 performs the MAP3K step of the MKK4/MKK5-MPK3/MPK6 relay, so it executes part of the MAPK cascade. Consistent with direct biochemistry and genetics in Arabidopsis. Supporting Evidence: PMID:27679653 MAPKKK5 interacts with MKK4 and MKK5 in vivo and phosphorylates their activation loops PMID:35652263 In plant immunity, MAPKKK3 and MAPKKK5 function redundantly upstream of the same MKK4/MKK5-MPK3/MPK6 module. |
| GO:0000165 MAPK cascade | IMP PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | ACCEPT | Summary: mapkkk5 single mutants showed reduced chitin-induced MPK3/MPK4/MPK6 activation, rescued by MAPKKK5-FLAG (Yamada 2016); Bi 2018 and Liu 2022 show the requirement is clearest in mapkkk3 mapkkk5 double mutants. Reason: Combined with direct phosphorylation of MKK4/MKK5 activation loops, the mutant phenotype supports participation (not just necessity) in the MAPK cascade. Single-mutant phenotype was not reproduced by Bi et al. 2018, but the double-mutant data confirm the role. Supporting Evidence: PMID:27679653 MAPKKK5 interacts with MKK4 and MKK5 in vivo and phosphorylates their activation loops PMID:27679653 The kinase domain of MAPKKK5 directly phosphorylated MKK4K108R and MKK5K99R (Fig 8B) PMID:29871986 two highly related MAPKKKs, MAPKKK3 and MAPKKK5, mediate MPK3/6 activation by at least four PRRs and confer resistance to bacterial and fungal pathogens file:ARATH/MAPKKK5/MAPKKK5-deep-research-falcon.md The reproducible higher-order-mutant evidence indicates substantial MAPKKK3/MAPKKK5 redundancy; conclusions from single mutants are laboratory- and assay-dependent. |
| GO:0002221 pattern recognition receptor signaling pathway | IGI PMID:29871986 Receptor-Like Cytoplasmic Kinases Directly Link Diverse Patt... | MODIFY | Summary: IGI with MAPKKK3 (AT1G53570): mapkkk3 mapkkk5 double mutants lose MPK3/6 activation by flg22, elf18, chitin and Pep2, i.e. downstream of FLS2, EFR, CERK1 and PEPRs, and RLCK VII kinases phosphorylate MAPKKK5 Ser-599. Reason: MAPKKK5 is an intracellular signal-transducing kinase within PRR signaling, so the process is correct. All PRRs tested (FLS2, EFR, CERK1/LYK5, PEPR1/2) are plasma membrane receptors, so the more specific child term cell surface pattern recognition receptor signaling pathway (GO:0002752), which is also used for the chitin perception module, is more informative. Proposed replacements: cell surface pattern recognition receptor signaling pathway Supporting Evidence: PMID:29871986 two highly related MAPKKKs, MAPKKK3 and MAPKKK5, mediate MPK3/6 activation by at least four PRRs and confer resistance to bacterial and fungal pathogens PMID:29871986 directly phosphorylate MAPKKK5 Ser-599, which is required for pattern-triggered MPK3/6 activation, defense gene expression, and disease resistance |
| GO:0004674 protein serine/threonine kinase activity | HDA PMID:21477822 Autophosphorylation profiling of Arabidopsis protein kinases... | ACCEPT | Summary: High-throughput wheat-germ cell-free autophosphorylation screen of 759 Arabidopsis kinases; MAPKKK5 was among the active kinases. Reason: MAPKKK5 is a catalytically active Ser/Thr kinase (it phosphorylates Thr and Ser residues in MKK4/MKK5 activation loops, and K375M abolishes its activity). The term is correct, though less specific than MAP kinase kinase kinase activity. Supporting Evidence: PMID:21477822 179 of the 759 PKs had autophosphorylation activity PMID:27679653 The kinase domain of MAPKKK5 directly phosphorylated MKK4K108R and MKK5K99R (Fig 8B) |
| GO:0004709 MAP kinase kinase kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for MAP kinase kinase kinase activity from the MEKK-subfamily node; MAPKKK5 itself is among the WITH/FROM sources (AT5G66850 under the MAPK cascade row) and has direct experimental support. Reason: Core activity. The MAPKKK5 kinase domain directly phosphorylates the S/TxxxxxS/T activation-loop motifs of MKK4 and MKK5 in vitro; alanine substitution of these sites abolishes phosphorylation. Supporting Evidence: PMID:27679653 The kinase domain of MAPKKK5 directly phosphorylated MKK4K108R and MKK5K99R (Fig 8B) PMID:27679653 MAPKKK5 interacts with MKK4 and MKK5 in vivo and phosphorylates their activation loops |
| GO:0004709 MAP kinase kinase kinase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 2.7.11.25 (MAP3K) mapping; UniProt assigns EC 2.7.11.25 from Yamada et al. 2016. Reason: Consistent with experimental MAP3K activity toward MKK4/MKK5. Supporting Evidence: PMID:27679653 The kinase domain of MAPKKK5 directly phosphorylated MKK4K108R and MKK5K99R (Fig 8B) |
| GO:0005515 protein binding | IPI PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | REMOVE | Summary: IPI with MKK4 (UniProtKB:O80397). Reason: Bare protein binding is uninformative about molecular function; removal does not dispute the reported interaction. The functionally meaningful content of the MAPKKK5 interactions is captured by MAP kinase kinase kinase activity (MKK4/MKK5 are its substrates) and by its role as a substrate of RLCK VII kinases in the cell surface pattern recognition receptor signaling pathway. Supporting Evidence: PMID:27679653 The BiFC signals indicated that MAPKKK5 interacts with MKK2, MKK4, and MKK5 mainly in the cytosol |
| GO:0005515 protein binding | IPI PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | REMOVE | Summary: IPI with PBL27 (UniProtKB:Q1PDV6). Reason: Bare protein binding is uninformative about molecular function; removal does not dispute the reported interaction. The functionally meaningful content of the MAPKKK5 interactions is captured by MAP kinase kinase kinase activity (MKK4/MKK5 are its substrates) and by its role as a substrate of RLCK VII kinases in the cell surface pattern recognition receptor signaling pathway. Supporting Evidence: PMID:27679653 demonstrating that PBL27 interacts with MAPKKK5 at the PM where CERK1 and PBL27 form the complex |
| GO:0005515 protein binding | IPI PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | REMOVE | Summary: IPI with MKK5 (UniProtKB:Q8RXG3). Reason: Bare protein binding is uninformative about molecular function; removal does not dispute the reported interaction. The functionally meaningful content of the MAPKKK5 interactions is captured by MAP kinase kinase kinase activity (MKK4/MKK5 are its substrates) and by its role as a substrate of RLCK VII kinases in the cell surface pattern recognition receptor signaling pathway. Supporting Evidence: PMID:27679653 The BiFC signals indicated that MAPKKK5 interacts with MKK2, MKK4, and MKK5 mainly in the cytosol |
| GO:0005515 protein binding | IPI PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | REMOVE | Summary: IPI with MKK2 (UniProtKB:Q9S7U9). Reason: Bare protein binding is uninformative about molecular function; removal does not dispute the reported interaction. The functionally meaningful content of the MAPKKK5 interactions is captured by MAP kinase kinase kinase activity (MKK4/MKK5 are its substrates) and by its role as a substrate of RLCK VII kinases in the cell surface pattern recognition receptor signaling pathway. Supporting Evidence: PMID:27679653 The BiFC signals indicated that MAPKKK5 interacts with MKK2, MKK4, and MKK5 mainly in the cytosol |
| GO:0005515 protein binding | IPI PMID:29871986 Receptor-Like Cytoplasmic Kinases Directly Link Diverse Patt... | REMOVE | Summary: IPI with PBL19 (AGI_LocusCode:AT5G47070). Reason: Bare protein binding is uninformative about molecular function; removal does not dispute the reported interaction. The functionally meaningful content of the MAPKKK5 interactions is captured by MAP kinase kinase kinase activity (MKK4/MKK5 are its substrates) and by its role as a substrate of RLCK VII kinases in the cell surface pattern recognition receptor signaling pathway. Supporting Evidence: PMID:29871986 directly phosphorylate MAPKKK5 Ser-599, which is required for pattern-triggered MPK3/6 activation, defense gene expression, and disease resistance |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: ATP binding from InterPro protein kinase domain and ATP-binding-site signatures. Reason: MAPKKK5 has a canonical kinase domain (346-607) with the ATP-binding lysine K375; the K375M mutant is kinase-inactive. ATP binding is a correct, non-core consequence of its kinase activity. Supporting Evidence: PMID:27679653 The kinase domain of MAPKKK5 directly phosphorylated MKK4K108R and MKK5K99R (Fig 8B) |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: IBA for cytoplasm (is_active_in) from the MEKK-subfamily node. Reason: MAPKKK5 interacts with MKK4/MKK5 mainly in the cytosol, consistent with cytoplasmic activity. Supporting Evidence: PMID:27679653 The BiFC signals indicated that MAPKKK5 interacts with MKK2, MKK4, and MKK5 mainly in the cytosol |
| GO:0005829 cytosol | IDA PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | ACCEPT | Summary: MAPKKK5-GFP was found in soluble and microsomal fractions and at the cytosol and plasma membrane after plasmolysis (transient expression in N. benthamiana); BiFC with MKKs mainly cytosolic in Arabidopsis protoplasts. Reason: Direct localization evidence; cytosol is where MAPKKK5 meets its MKK substrates. Supporting Evidence: PMID:27679653 the plasmolysis experiment indicated the GFP fluorescence of MAPKKK5 was detected at cytosol and plasma membrane (PM) in Nb leaves PMID:27679653 The BiFC signals indicated that MAPKKK5 interacts with MKK2, MKK4, and MKK5 mainly in the cytosol |
| GO:0005829 cytosol | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt SubCell mapping (Cytoplasm, cytosol) based on Yamada 2016. Reason: Consistent with IDA. Supporting Evidence: PMID:27679653 the plasmolysis experiment indicated the GFP fluorescence of MAPKKK5 was detected at cytosol and plasma membrane (PM) in Nb leaves |
| GO:0005886 plasma membrane | IDA PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | ACCEPT | Summary: MAPKKK5-GFP detected at the plasma membrane and in microsomal fractions; BiFC places the PBL27-MAPKKK5 complex at the plasma membrane. Reason: MAPKKK5 lacks a transmembrane domain but is peripherally associated with the plasma membrane where it receives input from the CERK1-PBL27 complex. Supporting Evidence: PMID:27679653 the plasmolysis experiment indicated the GFP fluorescence of MAPKKK5 was detected at cytosol and plasma membrane (PM) in Nb leaves PMID:27679653 demonstrating that PBL27 interacts with MAPKKK5 at the PM where CERK1 and PBL27 form the complex |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt SubCell mapping (Cell membrane) based on Yamada 2016. Reason: Consistent with IDA. Supporting Evidence: PMID:27679653 the plasmolysis experiment indicated the GFP fluorescence of MAPKKK5 was detected at cytosol and plasma membrane (PM) in Nb leaves |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | REMOVE | Summary: AtSubP sequence-based prediction of chloroplast localization. Reason: UniProt annotates no transit peptide, and experimental localization places MAPKKK5 in the cytosol and at the plasma membrane, where it interacts with PBL27 and the MKKs. No evidence supports a plastid pool; the prediction is contradicted by direct data. Supporting Evidence: PMID:27679653 the plasmolysis experiment indicated the GFP fluorescence of MAPKKK5 was detected at cytosol and plasma membrane (PM) in Nb leaves PMID:27679653 The BiFC signals indicated that MAPKKK5 interacts with MKK2, MKK4, and MKK5 mainly in the cytosol |
| GO:0045088 regulation of innate immune response | IMP PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | KEEP AS NON CORE | Summary: mapkkk5 mutants have reduced chitin-induced MAPK activation and callose deposition and increased susceptibility to Alternaria brassicicola. Reason: MAPKKK5 contributes positively to PTI through the MAPK cascade; regulation of innate immune response is a correct but broad description of the downstream outcome. The core role is captured by MAP3K activity in the cell surface PRR signaling MAPK cascade. Supporting Evidence: PMID:27679653 callose deposition was significantly reduced in the mapkkk5 mutants PMID:27679653 The mapkkk5 mutants developed larger disease lesions than the wild type |
| GO:0046777 protein autophosphorylation | HDA PMID:21477822 Autophosphorylation profiling of Arabidopsis protein kinases... | KEEP AS NON CORE | Summary: High-throughput cell-free autophosphorylation screen. Reason: MAPKKK5 does autophosphorylate in vitro, but weakly relative to transphosphorylation of MKK4/MKK5; no physiological role for autophosphorylation has been shown. Supporting Evidence: PMID:21477822 179 of the 759 PKs had autophosphorylation activity PMID:27679653 has a low autophosphorylation activity compared with the transphosphorylation of MKK4 and MKK5 |
| GO:0050832 defense response to fungus | IEP PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | KEEP AS NON CORE | Summary: The IEP basis in Yamada 2016 is induction of MAPKKK5 transcripts by chitin oligosaccharide ((GlcNAc)7) treatment, not by fungal infection. Reason: Kept as non-core, consistent with the IMP row for the same term, which independently supports a contribution to fungal resistance. The IEP basis itself (transcript induction by purified chitin elicitor) is better read as response to chitin (GO:0010200) than as participation in a defense response to fungus; expression changes are not evidence of the gene product performing the defense. Supporting Evidence: PMID:27679653 Expression of MAPKKK5 was induced by chitin PMID:27679653 The mapkkk5 mutants developed larger disease lesions than the wild type |
| GO:0050832 defense response to fungus | IMP PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | KEEP AS NON CORE | Summary: Two independent mapkkk5 T-DNA mutants developed larger lesions after Alternaria brassicicola infection; Bi 2018 (double mutant) and Yan 2018 (powdery mildew) also report reduced fungal resistance. Reason: The phenotype shows necessity for full resistance, but MAPKKK5 acts in that defense only by relaying the signal through the MAPK cascade; it does not perform the defensive effector steps. Kept as non-core; the core role is MAP3K activity in PRR-triggered MAPK signaling. Supporting Evidence: PMID:27679653 The mapkkk5 mutants developed larger disease lesions than the wild type PMID:29440595 the mapkkk5 mutant displayed enhanced susceptibility to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000, and to the fungal powdery mildew pathogen Golovinomyces cichoracearum |
| GO:0106310 protein serine kinase activity | EXP PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | ACCEPT | Summary: In vitro kinase assays show MAPKKK5 phosphorylates Ser and Thr residues in the activation loops of MKK4 (T224/S230) and MKK5 (T215/S221). Reason: Directly demonstrated activity; a component of the MAP3K activity, which is the more informative description of the core function. Supporting Evidence: PMID:27679653 The kinase domain of MAPKKK5 directly phosphorylated MKK4K108R and MKK5K99R (Fig 8B) PMID:27679653 MAPKKK5 interacts with MKK4 and MKK5 in vivo and phosphorylates their activation loops |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea-based mapping (RHEA:17989, protein serine phosphorylation). Reason: Consistent with experimental data. Supporting Evidence: PMID:27679653 The kinase domain of MAPKKK5 directly phosphorylated MKK4K108R and MKK5K99R (Fig 8B) |
| GO:1900150 regulation of defense response to fungus | IGI PMID:29871986 Receptor-Like Cytoplasmic Kinases Directly Link Diverse Patt... | KEEP AS NON CORE | Summary: mapkkk3 mapkkk5 double mutants show increased susceptibility to the fungal pathogen Botrytis cinerea (Bi 2018). Reason: Correct downstream consequence of MAPKKK5 action in PRR-triggered MAPK signaling, redundantly with MAPKKK3; not the core activity. Supporting Evidence: PMID:29871986 two highly related MAPKKKs, MAPKKK3 and MAPKKK5, mediate MPK3/6 activation by at least four PRRs and confer resistance to bacterial and fungal pathogens |
| GO:1900424 regulation of defense response to bacterium | IGI PMID:29871986 Receptor-Like Cytoplasmic Kinases Directly Link Diverse Patt... | KEEP AS NON CORE | Summary: mapkkk3 mapkkk5 double mutants show increased susceptibility to Pseudomonas syringae (Bi 2018); Yan 2018 reports the single mapkkk5 mutant is also more susceptible. Reason: Correct downstream consequence of MAPKKK5 relaying PRR signals into the MPK3/MPK6 cascade; not the core activity. Supporting Evidence: PMID:29871986 two highly related MAPKKKs, MAPKKK3 and MAPKKK5, mediate MPK3/6 activation by at least four PRRs and confer resistance to bacterial and fungal pathogens PMID:29440595 the mapkkk5 mutant displayed enhanced susceptibility to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000, and to the fungal powdery mildew pathogen Golovinomyces cichoracearum |
| GO:2000071 regulation of defense response by callose deposition | IMP PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | KEEP AS NON CORE | Summary: Chitin-induced callose deposition was reduced in mapkkk5 mutants and was not fully restored by the PBL27-phosphosite (6xA) variant. Reason: Callose deposition is a downstream output of the chitin-activated MAPK cascade; MAPKKK5 regulates it indirectly via MKK4/MKK5-MPK3/MPK6 and does not participate in callose synthesis. Retained as non-core. Supporting Evidence: PMID:27679653 callose deposition was significantly reduced in the mapkkk5 mutants |
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Download this section (compressed HTML)Q: Yamada et al. (2016) found strong chitin-signaling defects and enhanced flg22-induced MAPK activation in mapkkk5 single mutants, whereas Bi et al. (2018) found near-normal single-mutant responses and a positive requirement for MAPKKK3/MAPKKK5 downstream of FLS2. Is the flg22 effect positive or negative, and does the discrepancy reflect alleles, growth conditions or tissue?
Q: Which RLCK is the physiological activator of MAPKKK5 after chitin perception: PBL27 (C-terminal sites S617-S685) or RLCK VII-4 members such as PBL19 (Ser-599)? Bi et al. reported normal chitin-induced MAPKKK5 phosphorylation in pbl27.
Q: Should the MPK6-mediated feedback phosphorylation of MAPKKK5 (Ser-682/Ser-692) be represented in GO-CAM as a positive feedback edge in the chitin perception and other PRR modules?
Experiment: Side-by-side analysis of mapkkk5 single and mapkkk3 mapkkk5 double mutants (both labs' alleles) for chitin- and flg22-induced MPK3/MPK6 activation under identical conditions to resolve the single-mutant discrepancy.
Experiment: Phosphosite complementation of mapkkk3 mapkkk5 with combined PBL27-site (6xA) and Ser-599 mutations to test whether the two RLCK inputs are additive or alternative routes to MAPKKK5 activation after chitin perception.
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