ABSTRACT
Mitogen-activated protein kinase (MAPKs) constitute a major component in plant cellular signaling considering the sheer number of MAPKKKKs, MAPKKKs, MAPKKs and MAPKs when compared to yeast and animal systems. Nevertheless, only few complete MAPK cascades have been deciphered and the same hold true for their substrates. Furthermore, cascades often share kinase components, but little is known about their specific interactions and domains. The Arabidopsis thaliana MAP kinase kinase kinase 20 (MKKK20) was recently showed to interact with MKK3 and MPK18 in two non-complementary signaling cascades involved in root cortical microtubule functions. Here, MKKK20 and MKK3 proteins where dissected and tested in yeast two-hybrid assays followed by an in planta validation through bimolecular fluorescence complementation (BiFC) assays and showed that the MKKK20 C-terminal region interacted with MKK3 that comprised a typical DEF domain akin to MAPKs docking domains.
KEYWORDS: MKKK20, MKK3, docking domains, yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC)
Introduction
Signal transduction cascades use protein kinases in a sequential series of phosphorylation steps leading to functional modification of their targeted protein. Signaling specificity within these cascades relies on numerous docking motifs to ensure proper kinase to kinase interactions (mainly MKK to MPK), as well as downstream substrates (MPK to substrates), leading to specific cellular responses. As for yeast1 and mammalian2 MKKs, plant MKKs harbor at their N-terminal a motif known as the D-domain3. The MKK D-domain is a MPK docking site characterized by a cluster of basic residues [R/K] followed by hydrophobic residues [L/I] with a general consensus sequence of [K/R][K/R][K/R]X1-5 [L/I]X[L/I]3. D-domains (or D-sites) bind to a C-terminal complementary region on MPKs, the CD domain (common docking domain), located outside the MPK catalytic domain. The CD domain contains acidic and hydrophobic residues to establish proper electrostatic and hydrophobic interactions with the MKK D-domain4. In plant MPKs, group A and B harbor such a typical CD domain in their C-terminal extension [LH][LHY]DX2[DE]X2[DE]EPXC (X represents any amino acid)3. Interestingly, D-sites are not only found on MKKs but also in MPK substrates. Thus, MPKs CD domain can interact with upstream MKKs and downstream substrates since both harbor a D-domain, suggesting that MKKs and MPK substrates may compete for MPKs CD domain5, while sequential establishment of protein interactions in the MAPK cascades might rely on MPK phosphorylation status.
Contrary to the MKKs and MPKs that have well conserved docking domains, MAPKKKs (MKKKs or MEKKs) docking interaction sites are scarce in animals literature6, and have yet to be uncovered in plants, a surprising situation considering that numerous MKKKs interact directly with MKKs in heterologous systems, like in Y2H screens7,8, and phosphorylate MKKs in kinase assays8, suggesting that scaffolds might not be required for their specific interactions. With 21 MEKKs in A. thaliana and much more in other species9, a major question is how these MEKKs achieve the specificity required to interact with their ten downstream MEKs. Here, the A. thaliana MKKK20 and MKK3 were finely dissected and used in Y2H assays to uncover potential docking domains interacting between these two kinases. Surprisingly, a short MKKK20 C-terminal sequence not only interacted with MKK3 but also harbored docking domain sites similar to MAPK substrates.
Results and discussion
MKKK20 – MKK3 interaction domain
We recently showed that mkkk20, mkk3 and mpk18 mutant plants showed microtubule defect phenotypes and were involved in two separate signaling pathways, a canonical one with MKKK20-MKK3 and an unknown downstream MPK(s), and a non-canonical one with direct MKKK20-MPK18 interaction and [T-D-Y] phosphorylation of the catalytic domain activation loop of MPK188. Here, MKKK20 and MKK3 were dissected into nine and five fragments, respectively, and used for pairwise interactions in a yeast two hybrid system, including a complete bait and prey plasmid swap for a total of 104 interactions tested at high stringency with a 100 mM concentration of 3-AT (3-amino-1,2,4-triazole), retaining only high-affinity binding interactions. Has shown previously8, full length MKKK20 and MKK3 interacted strongly (Figure 1A, yeast colony 6; Figure 1B, yeast colony 2). MKKK20 was then dissected in three sections: N-terminal, Mid and C-terminal. While the initial dissection of MKKK20 quickly pointed to the C-terminal half (amino acids 172-342) as the fragment encompassing the MKKK20 interaction domain (Figure 1A, yeast colony 21; Figure 1B, yeast colony 20), only the full length MKK3 construct interacted with MKKK20. Nonetheless, all segments from MKK3 were also tested against all MKKK20 constructs. Further deletions from the MKKK20 C-terminal part (CS1, 172-267; CS2, 172-284; CS3, 172-318) showed no interaction with the full length MKK3 nor with its major segments encompassing the N-terminal and MKK3 kinase domain (1-344), the NTF2 (Nuclear Transport Factor 2) domain, that facilitates transport of protein into the nucleus, and the C-terminal segment. Since the MKKK20 C-terminal half was sufficient to interact with MKK3 while the CS3 construct was not, this suggested that Box II was important for the interaction, although Box II itself was insufficient (Figure 1A, yeast colony 46; Figure 1B, yeast colony 50). Since the CS3 construct included Box I (285-318), and that Box II alone (amino acids 319-342) did not interact with MKK3, this suggested that Box I and Box II (amino acids 285-342) together might be sufficient to interact with MKK3. Indeed, this was fully validated with the Box I & II construct that still interacted strongly with MKK3 (Figure 1A, yeast colony 41; Figure 1B, yeast colony 44). Although sufficient, interaction of Box I & II with MKK3 was weaker than the MKKK20 C-terminal half, which was also weaker than the MKKK20 full length construct, most probably due to overall protein conformation, stability or variable expression of these smaller constructs in yeast. Nonetheless, the three positive interactions were corroborated in both prey and bait plasmid swaps.
Table 1.
Pairwise interactions between MKK3 and selected MKK3 subdomains as bait (in pDEST32 as the GAL4 DNA-Binding-Domain), and MKKK20 and selected MKKK20 subdomains as prey (in pDEST22 as the GAL4-Activating-Domain).
| Bait MKK3/Prey MKKK20 | Bait MKK3/Prey MKKK20 | ||
|---|---|---|---|
| 1 | MKK3/Empty vector | 26 | MKK3/MKKK20-CS1 |
| 2 | MKK3-N/Empty vector | 27 | MKK3-N/MKKK20-CS1 |
| 3 | MKK3-HNT/Empty vector | 28 | MKK3-HNT/MKKK20-CS1 |
| 4 | MKK3-NTF2/Empty vector | 29 | MKK3-NTF2/MKKK20-CS1 |
| 5 | MKK3-C/Empty vector | 30 | MKK3-C/MKKK20-CS1 |
| 6 | MKK3/MKKK20 | 31 | MKK3/MKKK20-CS2 |
| 7 | MKK3-N/MKKK20 | 32 | MKK3-N/MKKK20-CS2 |
| 8 | MKK3-HNT/MKKK20 | 33 | MKK3-HNT/MKKK20-CS2 |
| 9 | MKK3-NTF2/MKKK20 | 34 | MKK3-NTF2/MKKK20-CS2 |
| 10 | MKK3-C/MKKK20 | 35 | MKK3-C/MKKK20-CS2 |
| 11 | MKK3/MKKK20-N | 36 | MKK3/MKKK20-CS3 |
| 12 | MKK3-N/MKKK20-N | 37 | MKK3-N/MKKK20-CS3 |
| 13 | MKK3-HNT/MKKK20-N | 38 | MKK3-HNT/MKKK20-CS3 |
| 14 | MKK3-NTF2/MKKK20-N | 39 | MKK3-NTF2/MKKK20-CS3 |
| 15 | MKK3-C/MKKK20-N | 40 | MKK3-C/MKKK20-CS3 |
| 16 | MKK3/MKKK20-M | 41 | MKK3/MKKK20-Box I & II |
| 17 | MKK3-N/MKKK20-M | 42 | MKK3-N/MKKK20-Box I & II |
| 18 | MKK3-HNT/MKKK20-M | 43 | MKK3-HNT/MKKK20-Box I & II |
| 19 | MKK3-NTF2/MKKK20-M | 44 | MKK3-NTF2/MKKK20-Box I & II |
| 20 | MKK3-C/MKKK20-M | 45 | MKK3-C/MKKK20-Box I & II |
| 21 | MKK3/MKKK20-C | 46 | MKK3/MKKK20-Box II |
| 22 | MKK3-N/MKKK20-C | 47 | MKK3-N/MKKK20-Box II |
| 23 | MKK3-HNT/MKKK20-C | 48 | MKK3-HNT/MKKK20-Box II |
| 24 | MKK3-NTF2/MKKK20-C | 49 | MKK3-NTF2/MKKK20-Box II |
| 25 | MKK3-C/MKKK20-C | 50 | MKK3-C/MKKK20-Box II |
| A | pEXP32:Krev1/pEXP22:RalGDS-wt (strong positive interaction control) |
| B | pEXP32:Krev1/pEXP22:RalGDSm1 (weak to moderate positive interaction control) |
| C | pEXP32:Krev1/pEXP22:RalGDSm2 (negative interaction control) |
| D | pDEST32/pDEST22 (negative activation control) |
| Abbreviations | |
| MKKK20 | MKKK20 Full length |
| MKKK20-N | MKKK20 N-terminal half |
| MKKK20-M | MKKK20 Middle segment |
| MKKK20-C | MKKK20 C-terminal half |
| MKKK20-CS1 | MKKK20 C-terminal Segment 1 |
| MKKK20-CS2 | MKKK20 C-terminal Segment 2 |
| MKKK20-CS3 | MKKK20 C-terminal Segment 2 |
| MKKK20-Box I & II | MKKK20 C-terminal Box I & II |
| MKKK20-Box II | MKKK20 C-terminal Box II |
| MKK3 | MKK3 Full length |
| MKK3-HNT | MKK3 HNT half kinase domain and half NTF2 |
| MKK3-NTF2 | MKK3 Nuclear transport factor 2 domain |
| MKK3-C | MKK3 C-terminal segment |
Table 2.
Pairwise interactions between MKKK20 and selected MKKK20 subdomains as bait (in pDEST32 as the GAL4 DNA-Binding-Domain), and MKK3 and selected MKK3 subdomains as prey (in pDEST22 as the GAL4-Activating-Domain).
| Bait MKKK20/Prey MKK3 | Bait MKKK20/Prey MKK3 | ||
|---|---|---|---|
| 1 | MKKK20/Empty vector | 28 | MKKK20-CS1/MKK3-HTN |
| 2 | MKKK20/MKK3 | 29 | MKKK20-CS1/MKK3-NTF2 |
| 3 | MKKK20/MKK3-N | 30 | MKKK20-CS1/MKK3-C |
| 4 | MKKK20/MKK3-HNT | 31 | MKKK20-CS2/Empty vector |
| 5 | MKKK20/MKK3-NTF2 | 32 | MKKK20-CS2/MKK3 |
| 6 | MKKK20/MKK3-C | 33 | MKKK20-CS2/MKK3-N |
| 7 | MKKK20-N/Empty vector | 34 | MKKK20-CS2/MKK3-HNT |
| 8 | MKKK20-N/MKK3 | 35 | MKKK20-CS2/MKK3-NTF2 |
| 9 | MKKK20-N/MKK3-N | 36 | MKKK20-CS2/MKK3-C |
| 10 | MKKK20-N/MKK3-HNT | 37 | MKKK20-CS3/Empty vector |
| 11 | MKKK20-N/MKK3-NTF2 | 38 | MKKK20-CS3/MKK3 |
| 12 | MKKK20-N/MKK3-C | 39 | MKKK20-CS3/MKK3-N |
| 13 | MKKK20-M/Empty vector | 40 | MKKK20-CS3/MKK3-HNT |
| 14 | MKKK20-M/MKK3 | 41 | MKKK20-CS3/MKK3-NTF2 |
| 15 | MKKK20-M/MKK3-N | 42 | MKKK20-CS3/MKK3-C |
| 16 | MKKK20-M/MKK3-HNT | 43 | MKKK20-Box I & II/Empty vector |
| 17 | MKKK20-M/MKK3-NTF2 | 44 | MKKK20-Box I & II/MKK3 |
| 18 | MKKK20-M/MKK3-C | 45 | MKKK20-Box I & II/MKK3-N |
| 19 | MKKK20-C/Empty vector | 46 | MKKK20-Box I & II/MKK3-HNT |
| 20 | MKKK20-C/MKK3 | 47 | MKKK20-Box I & II/MKK3-NTF2 |
| 21 | MKKK20-C/MKK3-N | 48 | MKKK20-Box I & II/MKK3-C |
| 22 | MKKK20-C/MKK3-HNT | 49 | MKKK20-Box II/Empty vector |
| 23 | MKKK20-C/MKK3-NTF2 | 50 | MKKK20-Box II/MKK3 |
| 24 | MKKK20-C/MKK3-C | 51 | MKKK20-Box II/MKK3-N |
| 25 | MKKK20-CS1/Empty vector | 52 | MKKK20-Box II/MKK3-HNT |
| 26 | MKKK20-CS1/MKK3 | 53 | MKKK20-Box II/MKK3-NTF2 |
| 27 | MKKK20-CS1/MKK3-N | 54 | MKKK20-Box II/MKK3-C |
| A | pEXP32:Krev1/pEXP22:RalGDS-wt (strong positive interaction control) |
| B | pEXP32:Krev1/pEXP22:RalGDSm1 (weak positive interaction control) |
| C | pEXP32:Krev1/pEXP22:RalGDSm2 (negative interaction control) |
| D | pDEST32/pDEST22 (negative activation control) |
| Abbreviations | |
| MKKK20 | MKKK20 Full length |
| MKKK20-N | MKKK20 N-terminal half |
| MKKK20-M | MKKK20 Middle segment |
| MKKK20-C | MKKK20 C-terminal half |
| MKKK20-CS1 | MKKK20 C-terminal Segment 1 |
| MKKK20-CS2 | MKKK20 C-terminal Segment 2 |
| MKKK20-CS3 | MKKK20 C-terminal Segment 2 |
| MKKK20-Box I & II | MKKK20 C-terminal Box I & II |
| MKKK20-Box II | MKKK20 C-terminal Box II |
| MKK3 | MKK3 Full length |
| MKK3-HNT | MKK3 HNT half kinase domain and half NTF2 |
| MKK3-NTF2 | MKK3 Nuclear transport factor 2 domain |
| MKK3-C | MKK3 C-terminal segment |
Figure 1.
Dissection of MKKK20 and MKK3 interacting domains. All kinase constructs were cloned and sequence-verified prior to their transfer into Gateway™ yeast two-hybrid bait and prey vectors (pDEST32 and pDEST22; Invitrogen™). Two pairwise assays were conducted with bait and prey proteins permutation in the yeast strain MaV203. For yeast two hybrid bait and prey combinations, refer to Tables 1 and 2. A. Bait vectors: MKK3 in pDEST32 as the GAL4 DNA-Binding-Domain (DBD). Prey vectors: MKKK20 in pDEST22 as the GAL4-Activating-Domain (AD). B. Bait vectors: MKKK20 in pDEST32 as the GAL4 DNA-Binding-Domain (DBD). Prey vectors: MKK3 in pDEST22 as the GAL4-Activating-Domain (AD). Interaction strength for HIS3 and URA3 activation assays was scored visually by comparison to the controls. Control A (Krev1/RalGDS-wt): strong positive interaction control. Control B (Krev1/RalGDSm1): weak to moderate interaction control. Control C (Krev1/RalGDSm2): negative interaction control. Control D (pDEST32/pDEST22 empty vectors): negative activation control. C. Schematic illustration of domain dissection from MKKK20. MKKK20 kinase catalytic domain spans from amino acid position 3 to 268. D. Schematic illustration of domain dissection from MKK3. MKK3 kinase catalytic domain spans from amino acid position 80 to 344. MKK3 N-terminal D-domain (docking site for MAPKK; [K/R] [K/R] [K/R] x (1–5) [L/I] x [L/I]) spans amino acid 8 to14. E. Conservation of the MKKK20 C-terminal Box I & II amino acid motifs interacting with the MKK3 and its closest protein kinases, MKKK19 and MKKK21. Black arrow separate Box I from Box II segments. F. Bimolecular fluorescence complementation (BiFC). The corresponding segments of MKKK20 and the full-length coding sequence of MKK3 were cloned into pSPYNE-35SGW (MKKK20-nYFP, MKKK20-C-nYFP, MKKK20-Box I & II-nYFP) and pSPYCE-35SGW (MKK3-cYFP) respectively through gateway cloning (Invitrogen™). AtbZIP63 and AtCNX6 were used as positive controls, while pSPYNE-35SGW (nYFP) and pSPYCE-35SGW (cYFP), and pSPYNE-35SGW (nYFP) and MKK3-cYFP were used as negative controls. Agrobacterium-mediated transient expression was performed as described by Shah et al.10 with slight modifications.
To further validate the positive interactions from the Y2H experiment, the bimolecular fluorescence complementation system (BiFC) was chosen. Unlike the classical in vitro pull-down technique, Y2H and BiFC are in vivo systems. While Y2H has higher throughput practicability, in the current context BiFC has the advantage of being an in planta homologous system. Thus, the three positive MKKK20 segments (MKKK20 full length; MKKK20 C-ter half; and C-ter Box I & II), were cloned into pSPYNE-35SGW (MKKK20-nYFP, MKKK20 C-ter nYFP, MKKK20-Box I & II-nYFP), while the full length MKK3 was cloned in pSPYCE-35SGW (MKK3-cYFP), respectively, through gateway cloning (Invitrogen™). As shown in Figure 1F, the BiFC assay fully validated the previous three MKKK20 interacting segments with MKK3 (MKKK20/MKK3; MKKK20 C-ter half/MKK3; MKKK20-Box I & II/MKK3), showing clear fluorescence in the cytoplasm. Interestingly, the BiFC interaction strength was similar to the one observed in the Y2H screen with stronger interaction from the full length MKKK20 and weaker with the MKKK20 C-ter half and MKKK20-Box I & II. Known positive controls also validated the BiFC assay, with the homodimerizing basic leucine zipper 63 (AtbZIP63)11, with a nuclear localization and the homodimerizing AtCNX6 (cofactor of nitrate reductase and xanthine dehydrogenase 6) with a cytoplasmic localization12. As expected, the two negative controls, empty vectors (pSPYNE-35SGW-nYFP and pSPYCE-35SGW-cYFP) and empty vector/MKK3 (pSPYNE-35SGW-nYFP/pSPYCE-35SGW-MKK3-cYFP) did not show any fluorescence.
MKKK20 Box I & II contains docking motifs typical of MAPKs
The first docking motif involved in MAPK interaction was the D domain (also known as D site or DEJL domain)2,4 found at the N-terminal of numerous MKKs. D domains can also be found in MAPK regulatory proteins and substrates4. On the MAPKs side, the CD domain functions as a MKK docking site. In mammalian systems, the CD domain was also shown as a docking site for MKPs (MAPK-specific phosphatases) and MAPKAPKs (MAPK-activated protein kinase)13. Another major MAPK docking site is the DEF domain (Docking site for ERK, FXFP). DEF domains are generally characterized by a FXFP sequence located between 6 and 20 amino acids C-terminal to the phosphoacceptor site [S/T]-P4. Although most DEF domain harbor a proline following the FXF motif, others have negatively charged amino acids like glutamatic14 and aspartic15 acids instead of proline. Interestingly, the MKKK20 Box I harbor two SP phosphoacceptor sites, the first being followed by a FXFD motif (Figure 1E). Furthermore, MKKK20 and its closest MKKKs in A. thaliana, e. g. MKKK19 and MKKK21, also harbor at the same position the two SP phosphoacceptor sites and a FXFP motif. The presence of the DEF domain is most probably not fortuitous since none of the other 18 Arabidopsis MEKKs have an FXF [P/D/E)] motif, like MKKK5 (FRFN motif), or have a [S/T]-P phosphoacceptor site too far from the FXF motif (MKKK14 FLFG and MKKK15 FFFS, from 100 and 42, amino acids respectively), or being imbedded in the kinase domain, like MKKK11 (FAFK motif). Interestingly, although MKKK20 was found to strongly autophosphorylate while expressed in bacteria, phosphorylation was only observed in the protein kinase domain (amino acids 3-268) in around 25 Ser (S), Thr (T) and Tyr (Y) sites8. The only two SP phosphoacceptor sites were found in Box I and were not phosphorylated, suggesting that the MKKK20 DEF domain might be a bona fide docking site for an upstream protein kinase. As expected, the downstream MKK3 itself could not phosphorylate a kinase null MKKK208. Since the N-terminal region is too small to harbor a docking site and that docking sites are not found in the catalytic protein kinase domain, interaction with MKK3 and with upstream kinases, like MKKKKs and receptor kinases or with other proteins like phosphatases, the MKKK20 C-terminal region most probably harbor at least two docking sites. Since MKKK20 was also shown recently to interact with MKK516, further experiments combining precise dissections and site-directed mutagenesis of the MKKK20 C-terminal domain should revealed minimalistic docking sites and new interactions with upstream and downstream substrates.
Funding Statement
This work was supported by the Natural Sciences and Engineering Research Council of Canada [RGPIN-2014-03883];
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