| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
We have studies the function of the cytoarchitecture of the human keratinocytes in the aspect of the binding proteins to the keratin intermediate filament sand desmosomal apparatus. To identify the novel function of these well characterized structural proteins, we used the yeast two hybrid system to clone the keratin and desmosome binding proteins.
Desmocollins constitute the extracellular part of desmosomal complexes with another desmosomal cadherin, desmogleins. These two desmosomal cadherins consist of three distinct isoforms, Dsc 1-3 and Dsg 1-3, and each desmocollin isoform has two splicing forms, the longer Dsc1a-3a and the shorter Dsc1b-3b. However, the function of the intracellular domain of shorter Dsc is yet unknown. Again, we used the yeast two-hybrid system to find out proteins binding to the carboxyl end of Dsc 2b from human skin cDNA library. Rack1, receptor for activated C kinase, was isolated as an interacting protein with the intracellular region of all Dsc b forms. Thu
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s the cytoplasmic tail of desmocollins was suggested to act as an anchoring site of PKC and to have a function modifying the cell adhesion through the phosphorylation of desmosomal proteins.
We next attempted to clone and characterize a novel protein, which interact with KIF at the cytoplasmic region of the human skin by yeast two-hybrid system. Employing the head domain of type I keratin K14, K16 as a bait, we screened the human skin cDNA and could obtain a candidate clone. The cloned gene consists of approx. 4.0kbp and has relatively large 1.0kbp 5' untranslating region. The predicted gene product is 105 kD protein encoded by 970 amino acids with no significant homology with known proteins. This protein contains several nuclear localization signals as well as one putative transmembrane segment in its carboxyl half. The isolated clone showed the obvious binding with type I keratin, K14 and K18, but no interaction with type II keratin, K6 and K8 at least in the yeast system. When the cloned gene was fused with green fluorescence protein (GFP) and used for the forced expression into cultured keratinocytes, the GFP signals are observed predominantly along with the nuclear membrane. These nuclear signals were overlapped with keratin localization around the nuclei in the confocal-microscopic analysis. These data suggest that this newly identified clone encodes a cross linking protein, which binds the KIF to the nuclear membrane. Less
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