Toshiyuki Fujiwara

Fukuoka University, Hukuoka, Fukuoka, Japan

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Publications (7)18.68 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We have identified an alternative splicing variant in the Cdc42-interacting protein 4 (CIP4) gene in patients with renal cell carcinoma (RCC); almost 50% of the RCCs examined showed an aberrant splicing event in reverse transcription-PCR and the insertion of 19 nucleotides derived from intron9 based on a sequence analysis. This variant (CIP4-V) encodes a premature stop codon, resulting in the loss of a tyrosine phosphorylation site, the Cdc42 binding domain, and the SH3 domain. In this report, we show that overexpression of CIP4-V causes the formation of ubiquitinated aggresomes and a loss of cell-cell adhesion. We determined that CIP4-V increased the beta-catenin tyrosine phosphorylation levels that mediate Fer/Fyn tyrosine kinases and induced beta-catenin mistrafficking from cell membrane to cytoplasmic aggresome. These results indicate that CIP4 is critical for beta-catenin-mediated cell-cell adhesion and may be an important aspect of its functional contribution to RCC, especially with regard to metastasis and invasiveness.
    Biochemical and Biophysical Research Communications 02/2006; 339(4):1083-8. · 2.28 Impact Factor
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    ABSTRACT: GCP170, a member of the golgin family associated with the cytoplasmic face of the Golgi membrane, was found to have a Golgi localization signal at the NH2-terminal region (positions 137-237). Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein that interacted with GCP170. The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed. Immunofluorescence microscopy showed that GCP16 was co-localized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein. Labeling experiments with [3H]palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys69 and Cys72, accounting for its tight association with the membrane. A mutant without potential acylation sites (C69A/C72A) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16. Although the mutant GCP16, even when overexpressed, had no effect on protein transport, overexpression of the wild type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface. Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport from the Golgi to the cell surface.
    Journal of Biological Chemistry 01/2004; 278(51):51957-67. · 4.60 Impact Factor
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    ABSTRACT: To elucidate its effect on proinsulin processing, we introduced the expression of a Pittsburgh type-mutant, alpha1-protease inhibitor M/R (alpha1-PIM/R) and its chimera protein with growth hormone (GH) (GHalpha1-PIM/R) into MIN6 cells. In metabolic labeling and chasing experiments with [3H]-Leu and [35S]-Met, proinsulin appeared in the medium during stimulatory secretion only from MIN6 clones expressing GHalpha1-PIM/R and, surprisingly, alpha1-PIM/R, but not from the clones of either the control or alpha1-PI. The major part of alpha1-PIM/R was secreted through the constitutive pathway and about 10% of total secreted alpha1-PIM/R in the chase periods entered the regulated pathway. On the other hand, GHalpha1-PIM/R was mainly transported to the secretory granules and about 80% of the total secreted GHalpha1-PIM/R in the chase periods was secreted during stimulatory secretion. In the first 3 h chase periods without stimulation, only alpha1-PIM/R and no GHalpha1-PIM/R appeared in the medium, thus suggesting that alpha1-PIM/R might be transported through a constitutive-like pathway for those periods. The alpha1-PI, which had no inhibitory effect on proinsulin processing, showed similar secretion pathways to those of alpha1-PIM/R. This implies that some part of alpha1-PIM/R and alpha1-PI entered the regulated pathway, not due to any specific interaction between the processing endoproteases and serine protease inhibitors, but due to some type of passive transport in a nonselective manner. The inhibitory effect of alpha1-PIM/R in the regulated secretory pathway was slightly but clearly evident when it was expressed in MIN6 beta-cells.
    Endocrine Journal 03/2003; 50(1):9-20. · 2.02 Impact Factor
  • Toshiyuki Fujiwara, Yoshio Misumi, Yukio Ikehara
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    ABSTRACT: Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, blocks protein transport from the endoplasmic reticulum (ER) to the Golgi complex and induces the redistribution of Golgi proteins into the ER. We investigated characteristics of NDGA-induced retrograde movement of the Golgi proteins to the ER. At an early stage of incubation of cells with NDGA, the Golgi complex formed convoluted membrane aggregates. Electron microscopy revealed that these aggregates directly interact en bloc with the ER membrane. The direct interaction and subsequent incorporation of the Golgi proteins into the ER were found to be temperature-dependent. The protein of ER-Golgi intermediate compartment (ERGIC), ERGIC53, was rapidly accumulated in the Golgi upon treatment with NDGA. This accumulation was significantly inhibited by low temperature at 15 degrees C. Under the condition, the redistribution of the Golgi proteins into the ER as well as the direct interaction between the ER and the Golgi by NDGA were also inhibited, suggesting an important role of the ERGIC in the retrograde movement. In contrast, the low temperature did not inhibit formation of the Golgi aggregates by NDGA. Taken together, these results suggest that NDGA causes the redistribution of the Golgi proteins into the ER through the direct connections between the Golgi, the ERGIC, and the ER.
    Biochemical and Biophysical Research Communications 03/2003; 301(4):927-33. · 2.28 Impact Factor
  • Endocrine Journal 01/2003; 50(1):9-20. · 2.02 Impact Factor
  • Biochemistry 12/1992; 31(47):11921-11927. · 3.19 Impact Factor
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    ABSTRACT: Synthesis and processing of rat albumin were investigated in COS-1 cells transiently expressing rat albumin. Analysis using isoelectric focusing revealed that serum-type albumin, which is indistinguishable from the counterpart isolated from rat hepatocyte cuture medium, was secreted from the transfected COS-1 cells, indicating that proalbumin is effectively converted into serum albumin in the COS-1 cells, if not completely. Furthermore methylamine was found to cause the diminution of serum albumin released from the cells, substantiating that the proteolytical conversion of proalbumin occurs in the Golgi complex before discharge from the COS-1 cells.
    Biochemical and Biophysical Research Communications 09/1989; 163(1):194-200. · 2.28 Impact Factor