Calcium-dependent protein kinase 16 phosphorylates and activates the aquaporin PIP2;2 to regulate reversible flower opening in Gentiana scabra.

Flower opening is important for successful pollination in many plant species, and some species repeatedly open and close their flowers. This is thought to be due to turgor pressure changes caused by water influx/efflux, which depends on osmotic oscillations in the cells. In some ornamental plants, water-transporting aquaporins, also known as plasma membrane intrinsic proteins (PIPs), may play an important role in flower opening. However, the molecular mechanism(s) involved in corolla movement are largely unknown. Gentian (Gentiana spp.) flowers undergo reversible movement in response to temperature and light stimuli; using gentian as a model, we showed that the Gentiana scabra aquaporins GsPIP2;2 and GsPIP2;7 regulate repeated flower opening. In particular, phosphorylation of a C-terminal serine residue of GsPIP2;2 is important for its transport activity and relates closely to the flower re-opening rate. Furthermore, GsPIP2;2 is phosphorylated and activated by the calcium (Ca2+)-dependent protein kinase GsCPK16, which is activated by elevated cytosolic Ca2+ levels in response to temperature and light stimuli. We propose that GsCPK16-dependent phosphorylation and activation of GsPIP2;2 regulate gentian flower re-opening, with stimulus-induced Ca2+ signals acting as triggers.

Validity of the cell-extracted proteome as a substrate pool for exploring phosphorylation motifs of kinases.

Three representative protein kinases with different substrate preferences, ERK1 (Pro-directed), CK2 (acidophilic), and PKA (basophilic), were used to investigate phosphorylation sequence motifs in substrate pools consisting of the proteomes from three different cell lines, MCF7 (human mammary carcinoma), HeLa (human cervical carcinoma), and Jurkat (human acute T-cell leukemia). Specifically, recombinant kinases were added to the cell-extracted proteomes to phosphorylate the substrates in vitro. After trypsin digestion, the phosphopeptides were enriched and subjected to nanoLC/MS/MS analysis to identify their phosphorylation sites on a large scale. By analyzing the obtained phosphorylation sites and their surrounding sequences, phosphorylation motifs were extracted for each kinase-substrate proteome pair. We found that each kinase exhibited the same set of phosphorylation motifs, independently of the substrate pool proteome. Furthermore, the identified motifs were also consistent with those found using a completely randomized peptide library. These results indicate that cell-extracted proteomes can provide kinase phosphorylation motifs with sufficient accuracy, even though their sequences are not completely random, supporting the robustness of phosphorylation motif identification based on phosphoproteome analysis of cell extracts as a substrate pool for a kinase of interest.

Chronic hyperglycemia reduces the expression of intercellular adhesion molecules and increases intercellular hyperpermeability in the periodontal epithelium.

BACKGROUND/AIMS: Hyperglycemia in diabetes is closely associated with periodontal disease progression. This study aimed to investigate the effect of hyperglycemia on the barrier function of gingival epithelial cells as a cause of hyperglycemia-exacerbated periodontitis in diabetes mellitus. METHODS: The abnormal expression of adhesion molecules in gingival epithelium in diabetes was compared between db/db and control mice. To study the effects of hyperglycemia on interepithelial cell permeability, the mRNA and protein expressions of adhesion molecules were investigated using a human gingival epithelial cell line (epi 4 cells) in the presence of either 5.5 mM glucose (NG) or 30 mM glucose (HG). Immunocytochemical and histological analyses were performed. We also studied HG-related intracellular signaling to assess abnormal adhesion molecule expression in the cultured epi 4 cells. RESULTS: The results of the proteomic analysis implied the abnormal regulation of cell-cell adhesion, and mRNA and protein expression assessments revealed the significant downregulation of Claudin1 expression in the gingival tissues of db/db mice (p < .05 vs control). Similarly, the mRNA and protein expressions of adhesion molecules were lower in epi 4 cells cultured under HG conditions than in those cultured under NG conditions (p < .05). Three-dimensional culture and transmission electron microscopy revealed reduced thickness of the epithelial cell layers with no flattened apical cells and heterogeneously arranged intercellular spaces among adjacent epi 4 cells under the HG. These results were consistent with the increased permeability of epi 4 cells under the HG relative to that of cells under the NG. This abnormal expression of intercellular adhesion molecules under the HG was related to the increased expression of receptors for advanced glycation end products (AGEs) and oxidative stress relative to that seen under the NG, along with stimulation of ERK1/2 phosphorylation in epi 4 cells. CONCLUSIONS: High glucose-induced impairment of intercellular adhesion molecule expression in gingival epithelial cells was related to the intercellular permeability of gingival cells, representing a possible link to hyperglycemia-related AGE signaling, oxidative stress, and ERK1/2 activation.

Identification of Endogenous Kinase Substrates by Proximity Labeling Combined with Kinase Perturbation and Phosphorylation Motifs.

Mass-spectrometry-based phosphoproteomics can identify more than 10,000 phosphorylated sites in a single experiment. But, despite the fact that enormous phosphosite information has been accumulated in public repositories, protein kinase-substrate relationships remain largely unknown. Here, we describe a method to identify endogenous substrates of kinases by using a combination of a proximity-dependent biotin identification method, called BioID, with two other independent methods, kinase-perturbed phosphoproteomics and phosphorylation motif matching. For proof of concept, this approach was applied to casein kinase 2 (CK2) and protein kinase A (PKA), and we identified 24 and 35 putative substrates, respectively. We also show that known cancer-associated missense mutations near phosphosites of substrates affect phosphorylation by CK2 or PKA and thus might alter downstream signaling in cancer cells bearing these mutations. This approach extends our ability to probe physiological kinase-substrate networks by providing new methodology for large-scale identification of endogenous substrates of kinases.

The jPOST environment: an integrated proteomics data repository and database.

Rapid progress is being made in mass spectrometry (MS)-based proteomics, yielding an increasing number of larger datasets with higher quality and higher throughput. To integrate proteomics datasets generated from various projects and institutions, we launched a project named jPOST (Japan ProteOme STandard Repository/Database, https://jpostdb.org/) in 2015. Its proteomics data repository, jPOSTrepo, began operations in 2016 and has accepted more than 10 TB of MS-based proteomics datasets in the past two years. In addition, we have developed a new proteomics database named jPOSTdb in which the published raw datasets in jPOSTrepo are reanalyzed using standardized protocol. jPOSTdb provides viewers showing the frequency of detected post-translational modifications, the co-occurrence of phosphorylation sites on a peptide and peptide sharing among proteoforms. jPOSTdb also provides basic statistical analysis tools to compare proteomics datasets.

Mass Spectrometry-Compatible Subcellular Fractionation for Proteomics.

We found that nuclear envelopes stabilize against surfactants in the presence of ethylene glycol (EG). We, therefore, developed a novel subcellular fractionation approach for proteomics using RIPA buffer containing EG and phase transfer surfactants. This method involves separating the cells into the cytoplasm, organelles, and nucleus, including intermediate filaments without ultracentrifugation. These fractions are directly applicable to sample preparation for shotgun proteomics as they have no mass spectrometry (MS)-incompatible chemicals, whereas those separated by traditional fractionation protocols require desalting. This protocol is successfully applied to subcellular fractionation with only 3.5 × 105 cells. Here, it was combined with phosphoproteomics and proteomics to identify phosphorylation sites regulating protein subcellular localization. In total, 59 phosphorylation sites on 42 phosphopeptides and 32 proteins showing different enrichment patterns between phosphoproteomics and the corresponding proteomics were identified, which are potential candidate sites to regulate the protein subcellular localization, including serine 706 on CD44 and serine 22 on lamin A/C.

Comparative proteomics of Helicobacter pylori strains reveals geographical features rather than genomic variations.

Helicobacter pylori, a pathogen of various gastric diseases, has many genome sequence variants. Thus, the pathogenesis and infection mechanisms of the H. pylori-driven gastric diseases have not been elucidated. Here, we carried out a large-scale proteome analysis to profile the heterogeneity of the proteome expression of 7 H. pylori strains by using an LC/MS/MS-based proteomics approach combined with a customized database consisting of nonredundant tryptic peptide sequences derived from full genome sequences of 52 H. pylori strains. The nonredundant peptide database enabled us to identify more peptides in the database search of MS/MS data compared with a simply merged protein database. Using this approach, we carried out proteome analysis of genome-unknown strains of H. pylori at as large a scale as genome-known ones. Clustering of the H. pylori strains using proteome profiling slightly differed from the genome profiling and more clearly divided the strains into two groups based on the isolated area. Furthermore, we identified phosphorylated proteins and sites of the H. pylori strains and obtained the phosphorylation motifs located in the N-terminus that are commonly observed in bacteria.

Nucleation and growth of water ice on oxide surfaces: the influence of a precursor to water dissociation.

We have investigated how nucleation and growth processes of ice are influenced by interfacial molecular interactions on some oxide surfaces, such as rutile TiO2(110), TiO2(100), MgO(100), and Al2O3(0001), based on the diffraction patterns of electrons transmitted through ice crystallites under the experimental configuration of reflection high energy electron diffraction (RHEED). The cubic ice Ic grows on the TiO2(110) surface with the epitaxial relationship of (110)Ic//(110)TiO2 and [001]Ic//[11[combining macron]0]TiO2. The epitaxial ice growth tends to be disturbed on the TiO2(110) surface under the presence of oxygen vacancies and adatoms. The result is not simply ascribable to small misfit values between TiO2 and ice Ic lattices (∼2%) because ice grains are formed randomly on TiO2(100). No template effects are identified during ice nucleation on the pristine MgO(100) and Al2O3(0001) surfaces either. The water molecules are chemisorbed weakly on these surfaces as a precursor to dissociation via the acid-base interaction. Such anchored water species act as an inhibitor of epitaxial ice growth because the orientation flexibility of physisorbed water during nucleation is hampered at the interface by the preferential formation of hydrogen bonds.

Phosphoproteomic profiling reveals ABA-responsive phosphosignaling pathways in Physcomitrella patens.

Abscisic acid (ABA) and its signaling system are important for land plants to survive in terrestrial conditions. Here, we took a phosphoproteomic approach to elucidate the ABA signaling network in Physcomitrella patens, a model species of basal land plants. Our phosphoproteomic analysis detected 4630 phosphopeptides from wild-type P. patens and two ABA-responsive mutants, a disruptant of group-A type-2C protein phosphatase (PP2C; ppabi1a/b) and AR7, a defective mutant in ARK, identified as an upstream regulator of SnRK2. Quantitative analysis detected 143 ABA-responsive phosphopeptides in P. patens. The analysis indicated that SnRK2-mediated phosphorylation and target motifs were partially conserved in bryophytes. Our data demonstrate that the PpSnRK2B and AREB/ABF-type transcription factors are phosphorylated in vivo in response to ABA under the control of ARK. On the other hand, our data also revealed the following: (i) the entire ABA-responsive phosphoproteome in P. patens is quite diverse; (ii) P. patens PP2C affects additional pathways other than the known ABA signaling pathway; and (iii) ARK is mainly involved in ABA signaling. Taken together, we propose that the core ABA signaling pathway is essential in all land plants; however, some ABA-responsive phosphosignaling uniquely developed in bryophytes during the evolutionary process.

Algal Protein Kinase, Triacylglycerol Accumulation Regulator 1, Modulates Cell Viability and Gametogenesis in Carbon/Nitrogen-Imbalanced Conditions.

Nutrient-deprived microalgae accumulate triacylglycerol (TAG) in lipid droplets. A dual-specificity tyrosine phosphorylation-regulated kinase, TAG accumulation regulator 1 (TAR1) has been shown to be required for acetate-dependent TAG accumulation and the degradation of chlorophyll and photosynthesis-related proteins in photomixotrophic nitrogen (N)-deficient conditions (Kajikawa et�al. 2015). However, this previous report only examined particular condition. Here, we report that in photoautotrophic N-deficient conditions, tar1-1 cells, with a mutation in the TAR1 gene, maintained higher levels of cell viability and lower levels of hydrogen peroxide generation and accumulated higher levels of TAG and starch compared with those of wild type (WT) cells with bubbling of air containing 5% carbon dioxide. Transcriptomic analyses suggested that genes involved in the scavenging of reactive oxygen species are not repressed in tar1-1 cells. In contrast, the mating efficiency and mRNA levels of key regulatory genes for gametogenesis, MID, MTD and FUS, were suppressed in tar1-1 cells. Among the TAR1-dependent phosphopeptides deduced by phosphoproteomic analysis, protein kinases and enzymes related to N assimilation and carbon (C) metabolism are of particular interest. Characterization of these putative downstream factors may elucidate the molecular pathway whereby TAR1 mediates cellular propagation and C and N metabolism in C/N-imbalanced stress conditions.

jPOSTrepo: an international standard data repository for proteomes.

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.

Uncovering Phosphorylation-Based Specificities through Functional Interaction Networks.

Protein kinases are an important class of enzymes involved in the phosphorylation of their targets, which regulate key cellular processes and are typically mediated by a specificity for certain residues around the target phospho-acceptor residue. While efforts have been made to identify such specificities, only ∼30% of human kinases have a significant number of known binding sites. We describe a computational method that utilizes functional interaction data and phosphorylation data to predict specificities of kinases. We applied this method to human kinases to predict substrate preferences for 57% of all known kinases and show that we are able to reconstruct well-known specificities. We used an in vitro mass spectrometry approach to validate four understudied kinases and show that predicted models closely resemble true specificities. We show that this method can be applied to different organisms and can be extended to other phospho-recognition domains. Applying this approach to different types of posttranslational modifications (PTMs) and binding domains could uncover specificities of understudied PTM recognition domains and provide significant insight into the mechanisms of signaling networks.

Identifications of Putative PKA Substrates with Quantitative Phosphoproteomics and Primary-Sequence-Based Scoring.

Protein kinase A (PKA or cAMP-dependent protein kinase) is a serine/threonine kinase that plays essential roles in the regulation of proliferation, differentiation, and apoptosis. To better understand the functions of PKA, it is necessary to elucidate the direct interplay between PKA and their substrates in living human cells. To identify kinase target substrates in a high-throughput manner, we first quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drug stimulations. LC-MS/MS analyses identified 2755 and 3191 phosphopeptides from experiments with activator or inhibitor of PKA. To exclude potential indirect targets of PKA, we built a computational model to characterize the kinase sequence specificity toward the substrate target site based on known kinase-substrate relationships. Finally, by combining the sequence recognition model with the quantitative changes in phosphorylation measured in the two drug perturbation experiments, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 previously known substrates. Moreover, 18 of these sites were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed a confident list of PKA substrate candidates, expanding our knowledge of PKA signaling network.

Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn.

We developed a novel approach to enlarge phosphoproteome coverage by selective elution depending on the number of phosphoryl group of peptides from a single titanium dioxide (TiO2) microcolumn using hydrophilic interaction chromatography (HILIC). In this approach, acidic methylphosphonate buffer including organic solvent is used for selective elution of singly phosphorylated peptides from an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) microcolumn and basic elution conditions with phosphate, ammonium hydroxide, and pyrrolidine are then employed for eluting multiply phosphorylated peptides retained by the HAMMOC microcolumn. Finally, we successfully identified 11 300 nonredundant phosphopeptides from triplicate analyses of 100 µg of HeLa cell lysates using this approach. This simple strategy made it possible to accomplish comprehensive and efficient phosphoproteome analysis from limited sample amounts loaded onto a single HAMMOC microcolumn without additional fractionation or enrichment approaches.

Large-scale identification of phosphorylation sites for profiling protein kinase selectivity.

Protein kinase selectivity is largely governed by direct binding to the target site(s) on the substrate. Thus, substrate determinants identified from sequences around phosphorylation sites are desirable resources for matching kinases to their substrates. In this study, we tried to identify kinase-selective substrate determinants, including motif sequences, based on large-scale discovery of kinase/substrate pairs. For this purpose, we employed a combination strategy of in vitro kinase reaction followed by LC-MS/MS analysis and applied it to three well-studied kinases: c-AMP regulated protein kinase A (PKA), extracellular signal-regulated kinase 1 (ERK1), and RAC-alpha serine/threonine-protein kinase (AKT1). Cellular proteins were fractionated, dephosphorylated with thermosensitive alkaline phosphatase, phosphorylated with the target kinase, and digested with Lys-C/trypsin, and then phosphopeptides were enriched using TiO2-based hydroxy acid-modified metal oxide chromatography (HAMMOC) and subjected to LC-MS/MS. As a result, 3585, 4347, and 1778 in vitro phosphorylation sites were identified for PKA, ERK1, and AKT1, respectively. As expected, these extensive identifications of phosphorylation sites enabled extraction of both known and novel motif sequences, and this in turn permitted fine discrimination of the specificities of PKA and AKT1, which both belong to the AGC kinase family. Other unique features of the kinases were also characterized, including phospho-acceptor preference (Ser or Thr) and bias ratio of singly/multiply phosphorylated peptides. More motifs were found with this methodology as compared with target kinase phosphorylation of peptides obtained by predigestion of proteins with Lys-C/trypsin. Thus, this approach to characterization of kinase substrate determinants is effective for identification of kinases associated with particular phosphorylation sites.

Phosphoproteome analysis of formalin-fixed and paraffin-embedded tissue sections mounted on microscope slides.

Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC-MS sensitivity by miniaturizing nanoLC columns to 25 µm inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 ± 5.4 µg of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases.

Temporal profiling of lapatinib-suppressed phosphorylation signals in EGFR/HER2 pathways.

Lapatinib is a clinically potent kinase inhibitor for breast cancer patients because of its outstanding selectivity for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). However, there is only limited information about the in vivo effects of lapatinib on EGFR/HER2 and downstream signaling targets. Here, we profiled the lapatinib-induced time- and dose-dependent phosphorylation dynamics in SKBR3 breast cancer cells by means of quantitative phosphoproteomics. Among 4953 identified phosphopeptides from 1548 proteins, a small proportion (5-7%) was regulated at least twofold by 1-10 µm lapatinib. We obtained a comprehensive phosphorylation map of 21 sites on EGFR/HER2, including nine novel sites on HER2. Among them, serine/threonine phosphosites located in a small region of HER2 (amino acid residues 1049-1083) were up-regulated by the drug, whereas all other sites were down-regulated. We show that cAMP-dependent protein kinase is involved in phosphorylation of this particular region of HER2 and regulates HER2 tyrosine kinase activity. Computational analyses of quantitative phosphoproteome data indicated for the first time that protein-protein networks related to cytoskeletal organization and transcriptional/translational regulation, such as RNP complexes (i.e. hnRNP, snRNP, telomerase, ribosome), are linked to EGFR/HER2 signaling networks. To our knowledge, this is the first report to profile the temporal response of phosphorylation dynamics to a kinase inhibitor. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib.

Strain visualization using large-angle convergent-beam electron diffraction.

In this study, we report a strain visualization method using large-angle convergent-beam electron diffraction (LACBED).1 We compare the proposed method with the strain maps acquired via STEM-NBD, a combination of scanning transmission electron microscopy (STEM) and nanobeam electron diffraction (NBD). Although STEM-NBD can precisely measure the lattice parameters, it requires a large amount of data and personal computer (PC) resources to obtain a two-dimensional strain map. Deficiency lines in the transmitted disk of LACBED reflect the crystalline structure information and move, curve, or disappear in the deformed area. Properly setting the optical conditions makes it possible to acquire real-space images over a broad area in conjunction with deficiency lines on the transmitted disk. The proposed method acquires images by changing the relative position between the specimen and the deficiency line and can grasp the strain information with a small number of images. In addition, the proposed method does not require high-resolution images. It can reduce the required PC memory or storage consumption in comparison with that of STEM-NBD, which requires a high-resolution diffraction pattern (DP) from each point of the region of interest. Compared with the two-dimensional maps of LACBED and NBD, NBD could detect large distortions in the area where the deficiency line curved, moved, or disappeared. The curving or moving direction of the deficiency line is qualitatively consistent with the NBD results. If quantitative strain values are not essential, strain visualization using LACBED can be considered an effective technique. We believe that the strain information of a sample can be obtained effectively using both methods.

Light-induced stomatal opening requires phosphorylation of the C-terminal autoinhibitory domain of plasma membrane H+-ATPase.

Plasma membrane H+-ATPase provides the driving force for light-induced stomatal opening. However, the mechanisms underlying the regulation of its activity remain unclear. Here, we show that the phosphorylation of two Thr residues in the C-terminal autoinhibitory domain is crucial for H+-ATPase activation and stomatal opening in Arabidopsis thaliana. Using phosphoproteome analysis, we show that blue light induces the phosphorylation of Thr-881 within the C-terminal region I, in addition to penultimate Thr-948 in AUTOINHIBITED H+-ATPASE 1 (AHA1). Based on site-directed mutagenesis experiments, phosphorylation of both Thr residues is essential for H+ pumping and stomatal opening in response to blue light. Thr-948 phosphorylation is a prerequisite for Thr-881 phosphorylation by blue light. Additionally, red light-driven guard cell photosynthesis induces Thr-881 phosphorylation, possibly contributing to red light-dependent stomatal opening. Our findings provide mechanistic insights into H+-ATPase activation that exploits the ion transport across the plasma membrane and light signalling network in guard cells.

Muscle-specific knock-out of NUAK family SNF1-like kinase 1 (NUAK1) prevents high fat diet-induced glucose intolerance.

NUAK1 is a member of the AMP-activated protein kinase-related kinase family. Recent studies have shown that NUAK1 is involved in cellular senescence and motility in epithelial cells and fibroblasts. However, the physiological roles of NUAK1 are poorly understood because of embryonic lethality in NUAK1 null mice. The purpose of this study was to elucidate the roles of NUAK1 in adult tissues. We determined the tissue distribution of NUAK1 and generated muscle-specific NUAK1 knock-out (MNUAK1KO) mice. For phenotypic analysis, whole body glucose homeostasis and muscle glucose metabolism were examined. Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype. Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle. On a high fat diet, MNUAK1KO mice had a lower fasting blood glucose level, greater glucose tolerance, higher insulin sensitivity, and higher concentration of muscle glycogen than control mice. Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle. Consistent with this, insulin signaling was enhanced in the soleus muscle of MNUAK1KO mice, as evidenced by increased phosphorylation of IRS1 Tyr-608, AKT Thr-308, and TBC1D4 Thr-649. These observations suggest that a physiological role of NUAK1 is to suppress glucose uptake through negative regulation of insulin signaling in oxidative muscle.