Biology subjects

Niinae, T.

Publications and source records attributed to Niinae, T..

4 recordsLinked to original sources

bioRxiv2023

Activity-based tyrosine phosphatomics using F2Pmp probes

We showed that the F2Pmp probe binds to PTP in a sequence-dependent manner. In addition, this study is the first successful example of comprehensive enrichment of classical PTP at the protein level. Furthermore, we found that F2Pmp probe can enrich PTPs in a PTP activity dependent manner. Using the F2Pmp probe, the fluctuation of PTPN1 and PTPN2 activities were revealed. This enrichment approach using the F2Pmp probe could be a generic tool for activity-based tyrosine phosphatomics.

biochemistry↗

bioRxiv2023

Evaluation of the use of different cell types as a substrate pool for in vitro kinase reaction

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.

biochemistry↗

bioRxiv2021

Escherichia coli S2P family intramembrane protease RseP is engaged in the regulated sequential cleavages of FecR in the ferric citrate signaling

Escherichia coli RseP, a member of the S2P family of intramembrane proteases, is involved in the activation of the {sigma}E extracytoplasmic stress response and elimination of remnant signal peptides. However, whether RseP has additional cellular functions is unclear. In this study, we attempted to identify new RseP substrates to explore still unknown physiological roles of this protease. Our mass spectrometry-based quantitative proteomic analysis revealed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with the fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate-dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. Ferric citrate signal-dependent generation of this cleavage product is the essential and sufficient role of RseP in the transcriptional activation of the fec operon. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.

molecular biology↗

bioRxiv2020

Proximity Labeling-assisted Identification of Endogenous Kinase Substrates

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 means of proximity labeling. We used a proximity-dependent biotin identification approach, called BioID, in combination with kinase-perturbed phosphoproteomics profiling and phosphorylation sequence motifs derived from in vitro kinase assay to find molecules that interact with a target kinase, that show altered phosphorylation in response to kinase perturbation, and that are directly phosphorylated by the kinase in vitro; i.e., endogenous kinase substrates. Application of this methodology to casein kinase 2 (CK2) and protein kinase A (PKA) identified 33 and 52 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 study extends our knowledge of kinase-substrate networks by proposing a new large-scale approach to identify endogenous substrates of kinases.

molecular biology↗