Mitogen Activated Protein Kinase (MPK) Interacts With Auxin Influx Carrier (OsAux/LAX1) Involved in Auxin Signaling in Plant
© Mohanta et al. 2015
Received: 8 September 2015
Accepted: 21 October 2015
Published: 30 October 2015
Mitogen activated protein kinases (MPKs) are serine/threonine protein kinases that contain characteristic T-x-Y motif in the activation loop region. MPKs are important signaling molecules involved in diverse signaling cascades that regulate plant growth, development and stress responses by conducting phosphorylation events in their target proteins. MPKs phosphorylate their target proteins at either S-P/T-P (Serine/Proline/Threonine) amino acid. To understand, if MPKs are involved in the auxin signaling cascade, we identified probable target proteins of MPKs involved in auxin signaling or transport processes.
A genome-wide search of the rice genome database led us to identification of the OsAux/LAX1 gene as a potential downstream target protein of MPKs. In-silico analysis predicted that MPKs interact with OsAux/LAX1 proteins which were validated by a yeast two-hybrid assay that showed OsMPK3, OsMPK4 and OsMPK6 are physically interact with OsAux/LAX1 protein.
The yeast two-hybrid interaction showed that MPKs are directly involved in auxin signaling events in plants. This is the first study to report direct involvement of MPKs in the auxin signaling pathway.
KeywordsOsAux/LAX Mitogen activated protein kinase (MPK) Phosphorylation Yeast two-hybrid
The plant mitogen activated protein kinases (MPKs) are evolutionarily conserved serine/threonine protein kinases that contain a characteristic T-x-Y motif in the activation loop region and group specific conserved docking domains in the C-terminal region [1, 2]. MPKs are involved in highly conserved signal transduction cascade that consists of at least three kinase modules. The kinase module contains a MP3K (mitogen activated protein kinase kinase kinase), a MP2K (mitogen activated protein kinase kinase) and a MPK . In the event of any environmental or cellular signaling process, plasma membrane activates MAP3Ks, which conserved serine/threonine protein kinases that phosphorylate downstream amino acids at the S/T-X3–5-S/T motif of MP2K in the activation loop domain. The MP2Ks then phosphorylate the downstream MPKs at the threonine and tyrosine residue of T-x-Y motif . Once, MPKs are phosphorylated, they can able to phosphorylate a wide array of downstream substrate proteins including other kinases, proteins and transcription factors to regulate gene expression [1, 3, 4]. The integrity of phosphorylation events of specific MPK with their substrate proteins is mediated by shared docking domains and adaptor proteins [1, 5].
The plant MPKs pathway is a major and well developed pathway involved in growth, development and biotic and abiotic stress responses in plants [2, 6]. This pathway is very complex and involves crosstalk with several other pathways [6–8]; therefore, the present study was conducted to decipher the complex interaction mechanism involved in plant MPKs and their involvement in auxin signaling events. Sorensson et al.,  reported that, MPKs phosphorylates their downstream target proteins either at S-P-R/S-S-P-R/S-P-K/S-S-P-K consensus sequences . Therefore, we investigated whether; OsAux/LAX1 is a suitable interacting partner of MPK as it contains the S-P motif at position 88. Therefore, we planned to conduct interaction analysis of rice OsMPKs and OsAux/LAX1 protein to confirm their physical interaction. An in-silico interaction study (docking interaction) was conducted to determine the details of the interacting amino acids of OsMPK and OsAux/LAX1 proteins (docking and protein-protein interaction). The results obtained by the in-silico interaction study were validated by a yeast two hybrid interaction assay. This is the first study to explain the direct involvement of the MPK pathway in auxin signaling events.
Results and Discussion
Sequence Retrieval, Template Identification, Homology Modeling and Structural Analysis
Protein-protein Docking Studies
Protein-protein Interaction Analysis
After conducting the necessary minimization steps, the protein complexes were submitted to Dimplot to identify the interaction sites (Fig. 3) . Additionally, the hydrogen and hydrophobic bonds formed by the OsMPK-OsAux/LAX1 complexes were analyzed using Pymol (Fig. 3), while Dimplot was used to analyze the dimers and plot the various hydrogen and hydrophobic interactions. Amino acids Lys536 and Cys530 of OsMPK3 form hydrogen bonds with Glu33 and Ser286 of OsAux/LAX respectively at distances of 2.55 and 2.42 Ao (Fig. 3a). Amino acids Cys556, Ala546 and Gly554 of OsMPK4 form hydrogen bonds with Asn164, Thr319, and Thr222 respectively, at distances of OsAux/LAX1 with distance 3.13, 3.20 and 2.25 Ao (Fig. 3b). Amino acids Gly561 and Tyr563 of OsMPK6 form hydrogen bonds with Thr230 and Arg271 of OsAux/LAX1 at distances of 3.31 and 3.16 Ao (Fig. 3c).
Yeast Two-hybrid Interactions of MPKs and LAX1
Auxin is an important hormone that regulates growth, development, tropism, apical dominance and several other processes in plants [21–23] as well as plays a crucial role in root development [6, 24, 25]. Auxin is synthesized in the aerial parts of the plant and transported toward the root tip to facilitate root development . The transport of auxin from the aerial part of the plant to the root tip is conducted by specialized auxin transporter molecules, popularly known as auxin influx and efflux carriers [27–32] in a polarized manner . Auxin influx carrier (Aux/LAX), a transmembrane amino acid transporter infuses the auxin molecule into the cell and the efflux carrier exports the auxin molecule to the adjacent cell in polarized manner [34–36]. Transport of the auxin molecule across the plasma membrane is an active process; therefore, the carrier molecules must be activated for the process to occur [35, 37]. Protein phosphorylation by kinase is one of the most important process that phosphorylates the target protein and leads to activation so it can carry out its active process [1, 38]. Mitogen activated protein kinases are most important family proteins found in plants which enable diverse cellular processes [1, 2]. Mitogen activated protein kinases have been reported to phosphorylate the target protein at the serine/proline (SP) or threonine/proline (TP) amino acid (S/T-P motif) . We found that the auxin signaling protein, OsAux/LAX1 contains an S-P motif at 88th position indicating that MPKs might phosphorylates the OsAux/LAX1 protein. To carry out phosphorylation events in the target protein (OsAux/LAX1), OsMPK first interacts with the protein via a hydrogen bond (Fig. 3) after which it carries out its phosphorylation event. The protein-protein interaction sites are different from the phosphorylation sites.
Auxin signaling event is crucial to growth and development of plants. However, the auxin signaling pathway is complex and involves interactions with several cascades. The result of the present study indicated that the MPK cascade is involved in auxin signaling events. This is the first report regarding involvement of MPK pathway in auxin signaling events.
Sequence Retrieval and Homology Modeling
Prior to homology modeling, the sequences of OsMPK3, and OsMPK4 were retrieved from the NCBI protein sequence database (http://www.ncbi.nlm.nih.gov/protein) in FASTA format. The sequences of OsMPK6 and OsAux/LAX1 were retrieved from the “rice genome annotation database” . The Genebank accession numbers of OsMPK3, and OsMPK4 are DQ826422 and FJ621301 respectively while protein identification numbers of OsMPK6 and OsAux/LAX1 are LOC_Os06g06090 and LOC_Os02g01100 respectively (rice genome annotation project). An excellent relationship to study the protein primary and secondary structure can be achieved by homology based modeling . It is possible to understand the protein function by computational modeling of a target protein using its proper template. This comparative modeling is based on the assumption that two proteins will have tertiary structure that shares a high percentage of similarity .
The models quality was checked by energetic and geometric means. The modeled homology structures were further validated using PROCHECK [11, 46] ERRAT  and VERIFY3D . The PROCHECK software analyzes the stereochemical properties to assess quality of the Ramachandran plot, planarity of the peptide bond, the main chain hydrogen bond energy, Cα chiralities, non bonded interactions, and the overall G factor . The ERRAT algorithm based on the statistical parameters of non-bonded interactions between different types of atoms and subsequently provides the accuracy of the protein model . VERIFY3D checks the compatibility of the atomic models with its own amino acid sequence. A high VERIFY3D profile score indicates a better quality of model . For further evaluation of the models, the ModEval Model Evaluation Server was used to calculate different model scores including z-Dope, GA341, z-pair, z-surf, and z-combi [12, 47, 48].
Protein-protein plays important roles in different biological processes, including signal transduction, gene expression, cellular transport, inhibition of enzyme activities, and the association of multi-domain proteins which leads to creation of stable protein-protein complexes important to meet their biological functions [49–51]. A protein-protein docking study was conducted to analyze the interaction of OsMPK3, OsMPK4 and OsMPK6 with OsAux/LAX1. The modeled structures were submitted to the GRAMM-X docking server  one at a time to achieve solid body docking using the fast Fourier transformation process by employing the smoothed Lennard-Jones potential, refinement stage and knowledge-based scoring, which provides the best surface match. Three dimers were formed after each successful docking, OsMPK3-OsAux/LAX1, OsMPK4-OsAux/LAX1 and OsMPK-OsAux/LAX1. The best dimer orientation found upon protein-protein docking, was again fed to the GRAMM-X server to obtain initial dimer orientations.
Cloning of OsMPK and OsLAX1
The OsMPKs (OsMPK3, OsMPK4 and OsMPK6) sequences were cloned from Oryza sativa using the proper adapter primer sequences for restriction digestion, after which the full length cDNA was amplified (Table 2) . Amplified OsMPKs and OsAux/LAX genes were confirmed by sequencing and all the clones were confirmed to be in the proper reading frame. The OsMPKs (OsMPK1, OsMPK2, OsMPK3) and OsAux/LAX1 genes were then cloned in pGADT7 and pGBKT7 vectors (BD Bioscience, USA) for yeast two-hybrid (Y2H) analysis as previously reported . A match maker yeast two-hybrid assay kit was used to check the protein-protein interactions (BD Bioscience, USA).
Yeast competent cells (AH109) were prepared according to the manufacturer’s instruction for transformation of GADT7 and pGBKT7 vectors (BD Bioscience, USA). The OsMPKs and OsAux/LAX1 constructs were co-transformed for yeast two-hybrid analysis. Transformation was carried out in PEG/LiAc (polyethylene glycol/lithium acetate) solution at 30 °C for one hour in a water bath while shaking at 200 rpm. Transformed cells were then centrifuged at 700 x g for five minutes, after which the pellet was recovered and co-transformed constructs were plated in selected drop out (DO) nutrient medium that lacks -Leu and -Trp (SDO/-Leu/-Trp) amino acid. The colonies obtained from DO media were then streaked on selective double drop out (DDO) media deficient in the amino acids Ade, His, Leu and Trp (SD/-Ade/-His/-Leu/-Trp). Blank pGADT7 and pGBKT7 vectors were used as controls in both selective media.
Mitogen Activated Protein Kinase
Mitogen Activated Protein Kinase Kinase
Mitogen Activated Protein Kinase Kinase Kinase
Oryza sativa Auxin Influx Carrier
Discrete Optimized Protein Energy
Yeast two Hybrid
This work was carried out with the support of the “Cooperative Research Programme for Agriculture Science & Technology Development (PJ01049704)” Rural Development Administration, Republic of Korea. The funder played no role in the study, design, data collection and analysis, decision to publish or preparation of the manuscript.
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