MAP KINASE KINASE 4 (MKK4; At1g51660) is a group C plant MAPK kinase (MAP2K) of 366 aa that, redundantly with its close paralog MKK5, phosphorylates the activation-loop Thr and Tyr of MPK3 and MPK6. MKK4/MKK5 are the shared middle tier of several MAPK modules: downstream of the MAPKKK YODA they repress stomatal lineage entry and spacing (and, downstream of ERECTA, promote localized cell proliferation in inflorescences); downstream of IDA-HAE/HSL2 they control floral organ abscission; and downstream of cell surface pattern-recognition receptors (via MAPKKK3/MAPKKK5, including the chitin CERK1-PBL27-MAPKKK5 branch and the flagellin receptor FLS2) they activate MPK3/MPK6 for immune gene expression, resistance to bacterial and fungal pathogens, camalexin and ethylene production. Upstream kinases such as YODA and MAPKKK5 phosphorylate its activation loop, and its own activity is inhibited indirectly by BIN2 acting on YODA. MKK4 acts mainly in the cytoplasm, with nuclear and minor chloroplast-stromal pools.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000165 MAPK cascade | IC PMID:9878570 Isolation of ATMEKK1 (a MAP kinase kinase kinase)-interactin... | ACCEPT | Summary: Curator inference of MAPK cascade participation from MAPKK activity. Reason: MKK4 is the MAPKK of YDA/MAPKKK5/MEKK-MKK4/5-MPK3/6 cascades. Supporting Evidence: PMID:17259259 We further establish that the MKK4/MKK5-MPK3/MPK6 module is downstream of YODA, a MAPKKK. |
| GO:0004672 protein kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automated protein kinase activity from the kinase domain. Reason: Correct but general; the specific activity is MAP kinase kinase activity. |
| GO:0004674 protein serine/threonine kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of protein serine/threonine kinase activity for the MAP2K family. Reason: MKK4 is a dual-specificity MAPKK that phosphorylates the Thr (and Tyr) of the MPK3/MPK6 activation loop; correct parent of MAP kinase kinase activity. Supporting Evidence: PMID:19516975 one well-characterized activation module involves dual phosphorylation of AtMPK3 and AtMPK6 by AtMKK4/5 |
| GO:0004708 MAP kinase kinase activity | IDA PMID:9878570 Isolation of ATMEKK1 (a MAP kinase kinase kinase)-interactin... | ACCEPT | Summary: MAP kinase kinase activity from an early study of Arabidopsis MAPK cascade components (abstract-only; abstract centres on MKK2/MEK1). Reason: The cached abstract does not mention MKK4, but MKK4 MAP kinase kinase activity is independently well established (YDA phosphorylates MKK4, MKK4 phosphorylates MPK3/MPK6); accepted, deferring to the curator who read the full text. Supporting Evidence: PMID:19516975 one well-characterized activation module involves dual phosphorylation of AtMPK3 and AtMPK6 by AtMKK4/5 PMID:22307275 BIN2 phosphorylates YDA to inhibit YDA phosphorylation of its substrate MKK4 |
| GO:0004708 MAP kinase kinase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 2.7.12.2 mapping to MAP kinase kinase activity. Reason: Core molecular function. Supporting Evidence: PMID:19516975 one well-characterized activation module involves dual phosphorylation of AtMPK3 and AtMPK6 by AtMKK4/5 |
| GO:0004713 protein tyrosine kinase activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea-based protein tyrosine kinase activity. Reason: MAPKKs are dual-specificity kinases that phosphorylate the activation-loop tyrosine of their MAPK targets; consistent, though MAP kinase kinase activity is the informative term. Supporting Evidence: PMID:19516975 one well-characterized activation module involves dual phosphorylation of AtMPK3 and AtMPK6 by AtMKK4/5 |
| GO:0005515 protein binding | IPI PMID:19513235 Comprehensive analysis of protein-protein interactions betwe... | MODIFY | Summary: Interaction with MPK3/MPK6, the downstream MAP kinases. Reason: MKK4/MKK5 bind and phosphorylate MPK3/MPK6; MAP kinase binding is the informative replacement for generic protein binding. Proposed replacements: mitogen-activated protein kinase binding Supporting Evidence: PMID:19513235 we used the yeast two-hybrid system to conduct a directed protein-protein interaction screen between all the Arabidopsis MPKs and their upstream activators (MAPK kinases; MKK) |
| GO:0005515 protein binding | IPI PMID:19513235 Comprehensive analysis of protein-protein interactions betwe... | MODIFY | Summary: Interaction with MPK3/MPK6, the downstream MAP kinases. Reason: MKK4/MKK5 bind and phosphorylate MPK3/MPK6; MAP kinase binding is the informative replacement for generic protein binding. Proposed replacements: mitogen-activated protein kinase binding Supporting Evidence: PMID:19513235 we used the yeast two-hybrid system to conduct a directed protein-protein interaction screen between all the Arabidopsis MPKs and their upstream activators (MAPK kinases; MKK) |
| GO:0005515 protein binding | IPI PMID:21798944 Evidence for network evolution in an Arabidopsis interactome... | REMOVE | Summary: Interactome-map interaction with BREVIS RADIX (Q17TI5). Reason: Generic protein binding is uninformative; the partner is not part of a characterized function of this MAPKK that a specific molecular-function term would capture. Removal does not imply the interaction is false. |
| GO:0005515 protein binding | IPI PMID:21798944 Evidence for network evolution in an Arabidopsis interactome... | MODIFY | Summary: Interaction with MPK3/MPK6, the downstream MAP kinases. Reason: MKK4/MKK5 bind and phosphorylate MPK3/MPK6; MAP kinase binding is the informative replacement for generic protein binding. Proposed replacements: mitogen-activated protein kinase binding Supporting Evidence: PMID:19516975 one well-characterized activation module involves dual phosphorylation of AtMPK3 and AtMPK6 by AtMKK4/5 |
| GO:0005515 protein binding | IPI PMID:21798944 Evidence for network evolution in an Arabidopsis interactome... | MODIFY | Summary: Interaction with MPK3/MPK6, the downstream MAP kinases. Reason: MKK4/MKK5 bind and phosphorylate MPK3/MPK6; MAP kinase binding is the informative replacement for generic protein binding. Proposed replacements: mitogen-activated protein kinase binding Supporting Evidence: PMID:19516975 one well-characterized activation module involves dual phosphorylation of AtMPK3 and AtMPK6 by AtMKK4/5 |
| GO:0005515 protein binding | IPI PMID:27679653 The Arabidopsis CERK1-associated kinase PBL27 connects chiti... | MODIFY | Summary: MKK4 interacts with MAPKKK5 (Q9C5H5), which phosphorylates its activation loop. Reason: Upstream MAPKKK-MAPKK interaction; MAPKKK binding is informative. Proposed replacements: mitogen-activated protein kinase kinase kinase binding Supporting Evidence: PMID:27679653 In addition, MAPKKK5 interacts with MKK4 and MKK5 in vivo and phosphorylates their activation loops. |
| GO:0005515 protein binding | IPI PMID:32612234 Extensive signal integration by the phytohormone protein net... | REMOVE | Summary: Hormone-network interaction with MYB73. Reason: Generic protein binding is uninformative; the partner is not part of a characterized function of this MAPKK that a specific molecular-function term would capture. Removal does not imply the interaction is false. |
| GO:0005515 protein binding | IPI PMID:32612234 Extensive signal integration by the phytohormone protein net... | REMOVE | Summary: Hormone-network interaction with KNAT1. Reason: Generic protein binding is uninformative; the partner is not part of a characterized function of this MAPKK that a specific molecular-function term would capture. Removal does not imply the interaction is false. |
| GO:0005515 protein binding | IPI PMID:32612234 Extensive signal integration by the phytohormone protein net... | REMOVE | Summary: Hormone-network interaction with BREVIS RADIX. Reason: Generic protein binding is uninformative; the partner is not part of a characterized function of this MAPKK that a specific molecular-function term would capture. Removal does not imply the interaction is false. |
| GO:0005515 protein binding | IPI PMID:32612234 Extensive signal integration by the phytohormone protein net... | MODIFY | Summary: Interaction with MPK3/MPK6, the downstream MAP kinases. Reason: MKK4/MKK5 bind and phosphorylate MPK3/MPK6; MAP kinase binding is the informative replacement for generic protein binding. Proposed replacements: mitogen-activated protein kinase binding Supporting Evidence: PMID:19516975 one well-characterized activation module involves dual phosphorylation of AtMPK3 and AtMPK6 by AtMKK4/5 |
| GO:0005515 protein binding | IPI PMID:32612234 Extensive signal integration by the phytohormone protein net... | MODIFY | Summary: Interaction with MPK3/MPK6, the downstream MAP kinases. Reason: MKK4/MKK5 bind and phosphorylate MPK3/MPK6; MAP kinase binding is the informative replacement for generic protein binding. Proposed replacements: mitogen-activated protein kinase binding Supporting Evidence: PMID:19516975 one well-characterized activation module involves dual phosphorylation of AtMPK3 and AtMPK6 by AtMKK4/5 |
| GO:0005515 protein binding | IPI PMID:32612234 Extensive signal integration by the phytohormone protein net... | REMOVE | Summary: Hormone-network interaction with TIFY8. Reason: Generic protein binding is uninformative; the partner is not part of a characterized function of this MAPKK that a specific molecular-function term would capture. Removal does not imply the interaction is false. |
| GO:0005515 protein binding | IPI PMID:39923935 Identifying Receptor Kinase Substrates Using an 8000 Peptide... | REMOVE | Summary: Interaction with the receptor-like kinase ILK4 (AT3G58760) in a split-luciferase/BiFC assay. Reason: Generic protein binding is uninformative; the partner is not part of a characterized function of this MAPKK that a specific molecular-function term would capture. Removal does not imply the interaction is false. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: ATP binding inferred from the kinase domain. Reason: Required for catalysis. |
| GO:0005634 nucleus | HDA PMID:15610358 High-throughput protein localization in Arabidopsis using Ag... | KEEP AS NON CORE | Summary: Nuclear (and cytoplasmic) GFP signal in a high-throughput GFP-ORF localization screen. Reason: Nuclear and cytoplasmic distribution is typical of MAPKKs acting on MPK3/MPK6, and the observation is credible, but the activating MAPKKK-MKK4 step is mainly cytosolic; the nucleus is retained as a secondary pool rather than the principal site of the core activity. Supporting Evidence: PMID:27679653 MAPKKK5 interacts with MKK2, MKK4, and MKK5 mainly in the cytosol |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt subcellular location mapping (based on the HDA data). Reason: Consistent with HDA data; secondary pool, see HDA row. Supporting Evidence: PMID:27679653 MAPKKK5 interacts with MKK2, MKK4, and MKK5 mainly in the cytosol |
| GO:0005634 nucleus | ISM GO_REF:0000122 | KEEP AS NON CORE | Summary: Nucleus predicted by AtSubP. Reason: Prediction alone would be weak, but it is consistent with the HDA observation; secondary pool. Supporting Evidence: PMID:27679653 MAPKKK5 interacts with MKK2, MKK4, and MKK5 mainly in the cytosol |
| GO:0005737 cytoplasm | HDA PMID:15610358 High-throughput protein localization in Arabidopsis using Ag... | ACCEPT | Summary: GFP-fusion localization screen. Reason: Cytoplasmic localization fits a soluble cascade kinase. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Automated cytoplasm assignment. Reason: Consistent with HDA data. |
| GO:0007165 signal transduction | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of signal transduction. Reason: Correct; the specific process is the MAPK cascade. Supporting Evidence: PMID:17259259 We further establish that the MKK4/MKK5-MPK3/MPK6 module is downstream of YODA, a MAPKKK. |
| GO:0007165 signal transduction | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA prediction of signal transduction. Reason: Correct, general. |
| GO:0009570 chloroplast stroma | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT inference of chloroplast stroma seeded by MKK4 itself. Reason: Based on MKK4 stromal import data; a non-canonical, minor pool whose functional significance is unclear. Supporting Evidence: PMID:19516975 Unexpectedly, however, the MKK4 polypeptide recovered from the chloroplasts retained the same electrophoretic mobility as the full-length in vitro -translated product |
| GO:0009570 chloroplast stroma | IDA PMID:19516975 Surviving the passage: Non-canonical stromal targeting of an... | KEEP AS NON CORE | Summary: In vitro chloroplast import shows MKK4 reaching the stroma. Reason: Direct evidence of non-canonical stromal targeting, but only from in vitro import into isolated pea chloroplasts; no stromal substrate or in vivo function is known. Secondary to its cytosolic signalling role. Supporting Evidence: PMID:19516975 Unexpectedly, however, the MKK4 polypeptide recovered from the chloroplasts retained the same electrophoretic mobility as the full-length in vitro -translated product file:ARATH/MKK4/MKK4-deep-research-falcon.md Chloroplast residence should therefore be recorded as a hypothesis, not its established principal site of action. |
| GO:0009570 chloroplast stroma | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt subcellular location mapping to chloroplast stroma. Reason: As for the IDA; minor pool. |
| GO:0009875 pollen-pistil interaction | IGI PMID:32890733 Stigma Receptivity Is Controlled by Functionally Redundant M... | UNDECIDED | Summary: Stigma receptivity study placing a highly redundant MKK-MPK3/MPK4 cascade in compatible pollination. The abstract names MKK1/2/3/7/9 (quintuple mutant) and MPK3/MPK4, not MKK4; the WITH/FROM list contains MKK1, MKK2, MKK7, MKK9, MPK3 and AT1G51660 (MKK4 itself) but not MKK3 (AT5G40440). Reason: The full text could not be obtained (not in PMC; Europe PMC lists it as subscription-only; the Cell Press page returned 403; the bioRxiv preprint 10.1101/2020.03.09.983767 was rate-limited (HTTP 429) on every attempt), so the genetic evidence for MKK4 cannot be checked. A QuickGO query for this PMID (recorded in MKK4-notes.md) shows TAIR made the same IGI pollen-pistil interaction annotation to exactly the six loci in this WITH/FROM list (MKK1, MKK2, MKK4, MKK7, MKK9, MPK3), so MKK4 appearing in its own WITH/FROM reflects one shared genotype list rather than a self-reference. The RNAi-targeted genes named in the abstract (MKK3, MPK4) were not annotated, which weakens but does not exclude the earlier concern of an MKK3/MKK4 identifier swap; MKK4 may instead have been part of a genotype or RNAi construct described only in the full text. Because the abstract names MKK1/2/3/7/9 and not MKK4, and the full text is inaccessible, the row is left UNDECIDED (not removed) per the rule for unverifiable experimental annotations. Supporting Evidence: PMID:32890733 an extremely functionally redundant mitogen-activated protein kinase (MAPK) cascade is required for maintaining stigma receptivity to accept compatible pollen |
| GO:0010227 floral organ abscission | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA prediction of floral organ abscission. Reason: Supported experimentally (IDA-HAE/HSL2-MKK4/5-MPK3/6); a developmental output of the cascade. Supporting Evidence: PMID:18809915 the Mitogen-Activated Protein Kinase Kinase 4 (MKK4) and MKK5, and a dominant-negative form of Mitogen-Activated Protein Kinase 6 (MPK6) in a mpk3 mutant background all have abscission-defective phenotypes |
| GO:0010227 floral organ abscission | IMP PMID:18809915 Regulation of floral organ abscission in Arabidopsis thalian... | KEEP AS NON CORE | Summary: MKK4/MKK5 RNAi lines are abscission defective; constitutively active MKKs rescue hae hsl2. Reason: Genuine developmental role downstream of HAE/HSL2; non-core relative to the kinase activity. Supporting Evidence: PMID:18809915 the Mitogen-Activated Protein Kinase Kinase 4 (MKK4) and MKK5, and a dominant-negative form of Mitogen-Activated Protein Kinase 6 (MPK6) in a mpk3 mutant background all have abscission-defective phenotypes |
| GO:0010227 floral organ abscission | IMP PMID:25730871 Floral organ abscission is regulated by a positive feedback ... | KEEP AS NON CORE | Summary: MKK4/5-dependent phosphorylation of AGL15 in the HAE positive feedback loop. Reason: Abstract-level evidence consistent with the established abscission role. Supporting Evidence: PMID:25730871 AGL15 is then differentially phosphorylated through development in floral receptacles in a MITOGEN-ACTIVATED PROTEIN KINASE KINASE 4/5-dependent manner. |
| GO:0010229 inflorescence development | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA prediction of inflorescence development. Reason: Supported by ERECTA-YDA-MKK4/5 genetics; developmental output. Supporting Evidence: PMID:23263767 Loss-of-function mutants of MPK3 / MPK6 and MKK4 / MKK5 phenocopy the er mutant, whereas the gain-of-function MKK4 and MKK5 transgenes can rescue the er mutant at both morphological and cellular levels |
| GO:0010229 inflorescence development | IGI PMID:23263767 A MAPK cascade downstream of ERECTA receptor-like protein ki... | KEEP AS NON CORE | Summary: mkk4 mkk5 loss phenocopies erecta; active MKK4/5 rescue er. Reason: Valid developmental output of the ERECTA-YDA-MKK4/5-MPK3/6 module. Supporting Evidence: PMID:23263767 Loss-of-function mutants of MPK3 / MPK6 and MKK4 / MKK5 phenocopy the er mutant, whereas the gain-of-function MKK4 and MKK5 transgenes can rescue the er mutant at both morphological and cellular levels |
| GO:0098542 defense response to other organism | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of defense response for the MKK4/MKK5/MKK9-type node. Reason: Immune signalling downstream of pattern-recognition receptors is a principal role of the MKK4/MKK5 pair; MKK4 is itself one of the experimental donors for this PAINT node. Supporting Evidence: PMID:35652263 MAPKKK3/MAPKKK5 function upstream of MKK4/MKK5‐MPK3/MPK6 in plant immunity PMID:11875555 Here we identify a complete plant MAP kinase cascade (MEKK1, MKK4/MKK5 and MPK3/MPK6) and WRKY22/WRKY29 transcription factors that function downstream of the flagellin receptor FLS2 |
| GO:0098542 defense response to other organism | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA prediction of defense response to other organism. Reason: Consistent with experimental evidence. Supporting Evidence: PMID:11875555 Here we identify a complete plant MAP kinase cascade (MEKK1, MKK4/MKK5 and MPK3/MPK6) and WRKY22/WRKY29 transcription factors that function downstream of the flagellin receptor FLS2 |
| GO:0098542 defense response to other organism | IMP PMID:11875555 MAP kinase signalling cascade in Arabidopsis innate immunity... | ACCEPT | Summary: MKK4/MKK5 act in the flagellin-triggered MAPK cascade conferring resistance. Reason: Principal immune role of the cascade. Supporting Evidence: PMID:11875555 Here we identify a complete plant MAP kinase cascade (MEKK1, MKK4/MKK5 and MPK3/MPK6) and WRKY22/WRKY29 transcription factors that function downstream of the flagellin receptor FLS2 PMID:11875555 Activation of this MAPK cascade confers resistance to both bacterial and fungal pathogens |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | MODIFY | Summary: Protein serine kinase activity from the Rhea:17989 mapping of EC 2.7.12.2. Reason: EC 2.7.12.2 expansion artefact. The characterized activity of MKK4 is dual-specificity phosphorylation of the Thr and Tyr residues of the MPK3/MPK6 activation loop, which is captured precisely by MAP kinase kinase activity; no serine-directed substrate activity is documented for MKK4. Proposed replacements: MAP kinase kinase activity Supporting Evidence: PMID:27679653 MKK4 and MKK5 redundantly function as MAPKKs for MPK3 and MPK6 |
| GO:2000122 negative regulation of stomatal complex development | IGI PMID:17259259 Stomatal development and patterning are regulated by environ... | NEW | Summary: Loss of MKK4/MKK5 causes stomatal clustering; activation suppresses stomatal fate. Reason: MKK4 (with MKK5) performs the phosphorylation step that relays YODA signalling to MPK3/MPK6, which inhibit SPEECHLESS; it does part of the work of this regulatory process. Supporting Evidence: PMID:17259259 Loss of function of MKK4/MKK5 or MPK3/MPK6 disrupts the coordinated cell fate specification of stomata versus pavement cells, resulting in the formation of clustered stomata. PMID:17259259 Conversely, activation of MKK4/MKK5-MPK3/MPK6 causes the suppression of asymmetric cell divisions and stomatal cell fate specification, resulting in a lack of stomatal differentiation. |
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Download this section (compressed HTML)Q: How is output specificity achieved when the same MKK4/MKK5 pair relays YODA (stomata, embryo, inflorescence), HAE/HSL2 (abscission) and immune-receptor signals to MPK3/MPK6?
Q: Are MKK4 and MKK5 fully redundant in the stomatal lineage, or do expression-level or docking differences give one paralog a predominant role?
Q: Should the MAPK-cascade members downstream of pattern recognition receptors (MKK4, MKK5, MPK3, MPK6) carry cell surface pattern recognition receptor signaling pathway (GO:0002752)? MAPKKK5 carries it via a curator IGI row, but no same-role MAP2K or MAPK currently does, so it is not proposed here.
Q: Camalexin induction by MKK4 is supported mainly by gain-of-function (GVG-MKK4DD) data; does mkk4 mkk5 loss of function compromise pathogen-induced camalexin, which would justify positive regulation of camalexin biosynthetic process (GO:1901183) as on MPK3?
Q: Is the pollen-pistil interaction IGI (PMID:32890733) correctly attributed to MKK4? The abstract names MKK1/2/3/7/9, and the WITH/FROM list contains the MKK4 locus (AT1G51660) but not MKK3 (AT5G40440), suggesting a possible MKK3/MKK4 identifier swap.
Q: Does chloroplast-imported MKK4 have a physiological stromal substrate or function, or is the in vitro import a minor pool without signaling consequence?
Experiment: Generate clean mkk4 mkk5 CRISPR double mutants with cell-type-specific complementation (SPCH or MUTE promoters) to test where in the stomatal lineage MKK4/5 activity is required, avoiding RNAi silencing variability.
Type: genetics / cell-type-specific complementation
Experiment: Conditional (estradiol-inducible amiRNA) depletion of MKK4 in an mkk5 null background to separate immune (chitin, flg22, Botrytis) from developmental requirements, assaying MPK3/MPK6 activation, camalexin accumulation and resistance.
Experiment: Live-cell imaging of functional MKK4-fluorescent protein fusions in stable Arabidopsis lines under oxidative stress to test whether a chloroplast stromal pool exists in vivo.
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