MAPK-associated proteins in Arabidopsis thaliana

Abstract Mitogen-Activated Protein Kinases (MAPKs) are signalling proteins that are highly conserved among eukaryotes. They are involved in many biological pathways including cell differentiation and response to different stress situations. The MAPK cascade is composed of three signalling kinases, MAP3K, MAP2K and MAPK, which phosphorylate each other in a sequential manner. The different MAPK substrates are regulated by phosphorylation on serine or threonine. This thesis presents a new method for purification of phosphoproteins from a complex protein extract. Subsequent studies using this method identified AtPHOS34 to be phosphorylated upon biotic stress. The phosphorylation site of AtPHOS34 was also determined. Several Arabidopsis thaliana MPK3/6 in vitro substrates were identified by a peptide and bioinformatics screen and the predicted phosphorylation sites were confirmed with kinase assays using mutated and wild-type proteins. MAPK Substrate 80 (here denoted MKS80) was characterised as an in planta MAPK substrate and in detail analyses of MKS80 linked its function to stomatal patterning and regulation of carbohydrate balance. The MKS80 interaction partner α-glucan phosphorylase (PHS2) was established by a yeast two-hybrid screen, in vitro pull-down and in planta co-immunoprecipitation assays. PHS2 catalyses the reversible reaction between glucose-1-phosphate and highly branched soluble heteroglycans and was shown to be important in senescence. Biochemical analyses revealed a stable level of PHS2 and light-dependent accumulation of MKS80. Arabidopsis over-expressing wild-type MKS80 had a lower starch content, whereas plants over-expressing the phosphomimetic form of MKS80 died at the seedling stage, clearly demonstrating the importance of this phosphorylation. In plants over-expressing MKS80, the anthocyanin level was lowered in different stress situations. Slower senescence was detected in detached leaves over-expressing MKS80, indicating a change in metabolism. A system for delayed senescence can be of interest for the agricultural industry

Enrichment of phosphoproteins and phosphopeptide derivatization identify universal stress proteins in elicitor-treated Arabidopsis

Protein phosphorylation is a key biological process that regulates reactions involved in plant-microbe interactions. The phosphorylated form of a protein often represents only a small fraction of the total population and can be problematic to analyze in a mass spectrometer. We demonstrate how a titanium dioxide (TiO2) resin can be employed for the enrichment of phosphoproteins, as well as a method to derivatize TiO2-purified phosphopeptides to facilitate determination of the exact site of phosphorylation. The use of these methods was exemplified by the identification of two plant proteins that were shown to be phosphorylated after the elicitation of Arabidopsis cells with Phytophthora infestans zoospores and xylanase. Both of the proteins that were identified, At5g54430.1 and At4g27320.1, were found to contain a universal stress protein domain with conserved residues for ATP binding

Arabidopsis cytosolic alpha-glycan phosphorylase, PHS2, is important during carbohydrate imbalanced conditions.

Arabidopsis thaliana has two isoforms of alpha-glycan phosphorylase (EC 2.4.1.1), one residing in the plastid and the other in the cytosol. The cytosolic phosphorylase, PHS2, acts on soluble heteroglycans that constitute a part of the carbohydrate pool in a plant. This study aimed to define a physiological role for PHS2. Under standard growth conditions phs2 knock-out mutants do not show any clear growth phenotype, and we hypothesised that during low-light conditions where carbohydrate imbalance is perturbed, this enzyme is important. Soil-grown phs2 mutant plants developed leaf lesions when placed in very low light. Analysis of soluble heteroglycan (SHG) levels showed that the amount of glucose residues in SHG was higher in the phs2 mutant compared to wild-type plants. Furthermore, a standard senescence assay from soil-grown phs2 mutant plants showed that leaves senesced significantly faster in darkness than the wild-type leaves. We also found decreased hypocotyl extension in in vitro-grown phs2 mutant seedlings when grown for long time in darkness at 6 °C. We conclude that PHS2 activity is important in the adult stage during low-light conditions and senescence, as well as during prolonged seedling development when carbohydrate levels are unbalanced

Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates

MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. in the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all proteins tested more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that AtIg80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of AtIg80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of AtIg80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning

Mutations In Emp2 Cause Childhood-Onset Nephrotic Syndrome

Nephrotic syndrome (NS) is a genetically heterogeneous group of diseases that are divided into steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS). SRNS inevitably leads to end-stage kidney disease, and no curative treatment is available. To date, mutations in more than 24 genes have been described in Mendelian forms of SRNS; however, no Mendelian form of SSNS has been described. To identify a genetic form of SSNS, we performed homozygosity mapping, whole-exome sequencing, and multiplex PCR followed by next-generation sequencing. We thereby detected biallelic mutations in EMP2 (epithelial membrane protein 2) in four individuals from three unrelated families affected by SRNS or SSNS. We showed that EMP2 exclusively localized to glomeruli in the kidney. Knockdown of emp2 in zebrafish resulted in pericardial effusion, supporting the pathogenic role of mutated EMP2 in human NS. At the cellular level, we showed that knockdown of EMP2 in podocytes and endothelial cells resulted in an increased amount of CAVEOLIN-1 and decreased cell proliferation. Our data therefore identify EMP2 mutations as causing a recessive Mendelian form of SSNS.WoSScopu