Naoyuki Sugiyama

Abscisic acid (ABA) signaling is important for stress responses and developmental processes in plants. A subgroup of protein phosphatase 2C (group A PP2C) or SNF1-related protein kinase 2 (subclass III SnRK2) have been known as major negative or positive regulators of ABA signaling, respectively. Here, we demonstrate the physical and functional linkage between these two major signaling factors. Group A PP2Cs interacted physically with SnRK2s in various combinations, and efficiently inactivated ABA-activated SnRK2s via dephosphorylation of multiple Ser/Thr residues in the activation loop. This step was suppressed by the RCAR/PYR ABA receptors in response to ABA. However the abi1-1 mutated PP2C did not respond to the receptors and constitutively inactivated SnRK2. Our results demonstrate that group A PP2Cs act as 'gatekeepers' of subclass III SnRK2s, unraveling an important regulatory mechanism of ABA signaling.

Major advancements have recently been made in mass spectrometry-based proteomics, yielding an increasing number of datasets from various proteomics projects worldwide. In order to facilitate the sharing and reuse of promising datasets, it is important to construct appropriate, high-quality public data repositories. jPOSTrepo (https://repository.jpostdb.org/) has successfully implemented several unique features, including high-speed file uploading, flexible file management and easy-to-use interfaces. This repository has been launched as a public repository containing various proteomic datasets and is available for researchers worldwide. In addition, our repository has joined the ProteomeXchange consortium, which includes the most popular public repositories such as PRIDE in Europe for MS/MS datasets and PASSEL for SRM datasets in the USA. Later MassIVE was introduced in the USA and accepted into the ProteomeXchange, as was our repository in July 2016, providing important datasets from Asia/Oceania. Accordingly, this repository thus contributes to a global alliance to share and store all datasets from a wide variety of proteomics experiments. Thus, the repository is expected to become a major repository, particularly for data collected in the Asia/Oceania region.

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and beta-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and beta-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.

Abscisic acid (ABA) is a phytohormone that regulates diverse plant processes, including seed germination and the response to dehydration. In Arabidopsis thaliana, protein kinases of the SNF1-related protein kinase 2 (SnRK2) family are believed to transmit ABA- or dehydration-induced signals through phosphorylation of downstream substrates. By mass spectrometry, we identified proteins that were phosphorylated in Arabidopsis wild-type plants, but not in mutants lacking all three members of the SnRK2 family (srk2dei), treated with ABA or subjected to dehydration stress. The number of differentially phosphorylated peptides was greater in srk2dei plants treated with ABA than in the ones subjected to dehydration, suggesting that SnRK2 was mainly involved in ABA signaling rather than dehydration. We identified 35 peptides that were differentially phosphorylated in wild-type but not in srk2dei plants treated with ABA. Biochemical and genetic studies of candidate SnRK2-regulated phosphoproteins showed that SnRK2 promoted the ABA-induced activation of the mitogen-activated protein kinases AtMPK1 and AtMPK2; that SnRK2 mediated phosphorylation of Ser(45) in a bZIP transcription factor, AREB1 (ABA-responsive element binding protein 1), and stimulated ABA-responsive gene expression; and that a previously unknown protein, SnRK2-substrate 1 (SNS1), was phosphorylated in vivo by ABA-activated SnRK2s. Reverse genetic analysis revealed that SNS1 inhibited ABA responses in Arabidopsis. Thus, by integrating genetics with phosphoproteomics, we identified multiple components of the ABA-responsive protein phosphorylation network.