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Atsushi Hijikata

Publications and source records attributed to Atsushi Hijikata.

6 recordsLinked to original sources

Alternative splicing in human transcriptome: functional and structural influence on proteins.

Alternative splicing is a molecular mechanism that produces multiple proteins from a single gene, and is thought to produce variety in proteins translated from a limited number of genes. Here we analyzed how alternative splicing produced variety in protein structure and function, by using human full-length cDNAs on the assumption that all of the alternatively spliced mRNAs were translated to proteins. We found that the length of alternatively spliced amino acid sequences, in most cases, fell into a size shorter than that of average protein domain. We evaluated comprehensively the presumptive three-dimensional structures of the alternatively spliced products to assess the impact of alternative splicing on gene function. We found that more than half of the products encoded proteins which were involved in signal transduction, transcription and translation, and more than half of alternatively spliced regions comprised interaction sites between proteins and their binding partners, including substrates, DNA/RNA, and other proteins. Intriguingly, 67% of the alternatively spliced isoforms showed significant alterations to regions of the protein structural core, which likely resulted in large conformational change. Based on those findings, we speculate that there are a large number of cases that alternative splicing modulates protein networks through significant alteration in protein conformation.

Alternative Splicing↗

Construction of quantitative proteome reference maps of mouse spleen and lymph node based on two-dimensional gel electrophoresis.

Quantitative features of the proteome are extremely useful for studying cellular processes at a molecular level. In this study, we attempted to construct quantitative reference proteome maps of the mouse spleen and lymph node based on 2-DE followed by protein identification using MS. We analyzed more than 1000 spots on the 2-DE images and consequently were able to determine that 919 spots were derived from 328 different genes. To obtain statistically reliable information of the protein levels from these 2-DE images, we measured the volumes of the respective spots on 2-DE images obtained by four to six independent experimental runs. These measurements were used to calculate the variability of the volumes of the respective spots on 2-DE following subcellular fractionation, which enabled us to discriminate differentially produced proteins from those within the range of intrinsic variability. More importantly, while the 2-DE data have been traditionally collected in a gel image-based manner, the resultant quantitative 2-DE data could be analyzed using the same procedure as that for mRNA expression profiles. This greatly assists in bridging the gap between the analyses of transcriptomes and proteomes and enables the integration of this data on the same informational platform.

Amino Acid Sequence↗

Disulfide linkages and a three-dimensional structure model of the extracellular ligand-binding domain of guanylyl cyclase C.

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys7-Cys94, Cys72-Cys77, Cys101-Cys128 and Cys179-Cys226. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101-104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.

Amino Acid Sequence↗

Post-transcriptional effects of phorbol 12-myristate 13-acetate on transcriptome of U937 cells.

To identify post-transcriptionally modulated genes at the translational level by phorbol 12-myristate 13-acetate (PMA), we investigated mRNA profiles in the polysomal and the cytoplasmic fractions of U937 cells before and after PMA stimulation using microarrays with 15017 oligonucleotide probes. Global comparison of the profiles showed that the cytoplasmic distribution of mRNAs was considerably modulated upon PMA stimulation. The results also indicate that PMA post-transcriptionally regulated at least 0.7% of detectable genes in U937 cells. Thus, besides transcriptional modulation by PMA, changes in the translational state of transcripts seem to play a critical role in PMA-induced differentiation of U937 cells.

Gene Expression Profiling↗

Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation.

The Ras signaling pathway plays a critical role in B lymphocyte development and activation, but its activation mechanism has not been well understood. At least one mode of Ras regulation in B cells involves a Ras-guanyl nucleotide exchange factor, RasGRP3. We demonstrate here that RasGRP3 undergoes phosphorylation at Thr-133 upon B cell receptor cross-linking, thereby resulting in its activation. Deletion of phospholipase C-gamma2 or pharmacological interference with conventional PKCs resulted in marked reduction in both Thr-133 phosphorylation and Ras activation. Moreover, mutation of Thr-133 in RasGRP3 alone severely impaired its ability to activate Ras in B cell receptor signaling. Hence, our data suggest that PKC, after being activated by diacylglycerol, phosphorylates RasGRP3, thereby contributing to its full activation.

Amino Acid Sequence↗

Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942.

In the cyanobacterium Synechococcus elongatus PCC 7942, KaiA, KaiB, and KaiC are essential proteins for the generation of a circadian rhythm. KaiC is proposed as a negative regulator of the circadian expression of all genes in the genome, and its phosphorylation is regulated positively by KaiA and negatively by KaiB and shows a circadian rhythm in vivo. To study the functions of KaiC phosphorylation in the circadian clock system, we identified two autophosphorylation sites, Ser-431 and Thr-432, by using mass spectrometry (MS). We generated Synechococcus mutants in which these residues were substituted for alanine by using site-directed mutagenesis. Phosphorylation of KaiC was reduced in the single mutants and was completely abolished in the double mutant, indicating that KaiC is also phosphorylated at these sites in vivo. These mutants lost circadian rhythm, indicating that phosphorylation at each of the two sites is essential for the control of the circadian oscillation. Although the nonphosphorylatable mutant KaiC was able to form a hexamer in vitro, it failed to form a clock protein complex with KaiA, KaiB, and SasA in the Synechococcus cells. When nonphosphorylatable KaiC was overexpressed, the kaiBC promoter activity was only transiently repressed. These results suggest that KaiC phosphorylation regulates its transcriptional repression activity by controlling its binding affinity for other clock proteins.

Bacterial Proteins↗