|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000)
The aim of the present project was to elucidate the role of cytoskeletal proteins in the secretory process using the parietal cell as a model. The actual plans were to determine the functional phosphorylation site of ezrin and to develop a model system which allows an introduction of peptides into the cell in order to make a direct analysis of the function of proteins. In the first year, human ezrin was cloned and the partial sequences were treated with PKA, and no phosphorylation was observed. However, purified rabbit ezrin was phosphorylated by PKA suggesting species difference in the sequence of ezrin. In the separate experiments, we showed that many of pharmacological probes commonly used to analyze signal transduction were useless, and suggested that the establishment of permeabilized cell system was indispensable.
During the next year's research, Professor Goldenring in the University of Georgia informed us that he sequenced rabbit ezrin and found no PKA sites, namely, no species
difference in the PKA site of ezrin. In the meantime, we obtained some data suggesting that ezrin itself is not phosphorylated by PKA, whereas gastric mucosa contains an unidentified kinase which is activated by PKA and phosphorylates C-terminal site of ezrin. This direction is different from the plan at the beginning, but it has now become much more interesting project. At present, experiments are in progress to identify this kinase. For the other project, we successfully developed a new model, the beta-escin permeabilized rabbit gastric gland, which allows the introduction of peptides into the cell. Using this system, it was shown that not only the PKA-inhibitory peptide but also the inhibitory peptide for myosin light chain kinase inhibited cAMP-stimulated acid secretory response. We could also show the possible involvement of small GTP-binding proteins in the secretory process applying functional peptides to the system. This model was thus demonstrated to be a powerful tool to analyze the interaction between cytoskeletal proteins in the parietal cell vesicular transport. Less