| Co-Investigator(Kenkyū-buntansha) |
TANAKA Yukie Fukui Medical School, Research Assistant, 医学部, 教務職員 (10197486)
NAKAMURA Shunーichi Fukui Medical School, Research Associate, 医学部, 助手 (40155833)
TANIGUCHI Takanobu Fukui Medical School, Research Associate, 医学部, 助手 (60217130)
HASHIMOTO Eikichi Fukui Medical School, Associate Professor, 医学部, 助教授 (20116239)
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| Research Abstract |
Recently we are trying to clarify the role of protein phosphorylation in intracellular signal transduction. In numerous protein kinases we focussed on the function of protein-tyrosine kinases and protein kinase C because we believe these kinases are extraordinary important in intracellular signal transduction. A cytosolic protein-tyrosine kinase has been highly purified from porcine spleen and analysis of this kinase by SDS/PAGE revealed a molecular mass of 40 kDa. It had autophosphorylation activity which was carried out by intramolecular catalysis. The activity of phosphorylation of this kinase increased with the amount of phosphate incorporated into the enzyme. A phosphate exchange reaction was observed during the autophosphorylation. Membrane phospholipids differently modulate the activity of this kinase. The effect of phospholipids on this kinase is quite confusing, and it is dependent on the combination of substrates and phospholipids. The effects of poly-basic amino acids, hepar
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in and ionic strength on the activity of this enzyme were also studied. The effect of these factors is mainly on the substrates employed rather than on the enzyme itself.
The ability of thrombin to induce protein-tyrosine phosphorylation in intact human platelets was studied by using antibodies to phosphotyrosine. Upon stimulation by thrombin there was an increase in the amount of protein-tyrosine phosphorylation of three bands with molecular masses of 135, 124, and 76 kDa in a time dependent manner. The time courses for thrombin-induced protein-tyrosine phosphorylation were similar to that for serotonin release. These protein-tyrosine phosphorylations were also seen in human platelets by the addition of vanadate and H_2O_2 which were known as inhibitors of protein-tyrosine phosphatases. Aggregation of platelets was also induced by these agents in a dose dependent manner. The observation that the aggregation was preceded by the increase in protein-tyrosine phosphorylations suggest that tyrosine phosphorylation may be involved in an early stage of platelet aggregation.
In an attempt to understand the molecular mechanism of the down regulation of protein kinase C, we analyzed the proteolytic modification of this enzyme using rat liver plasma membrane. Under higher ionic strength than physiological level (>140mM NaCl) and slightly alkaline pH (7.5-8.0), protease-activated form of protein kinase C with Mr. 50,000 was rapidly released from membrane. The properties of this activated enzyme were essentially same with those of protein kinase M (corresponding to the catalytic fragment of protein kinase C) reported earlier. Based on these results, we supposed that activation of Na^+/H^+ exchanger in plasma membrane may be a trigger for inducing the down regulation of protein kinase C, because Na^+ influx and cytoplasmic alkalinization are usually observed after activation of Na^+/H^+ exchanger by various extracellular stimuli. In contrast, higher molecular weight form of protein kinase C (Mr. 80,000) was generated from membrane under lower ionic strength condition. The activity of this enzyme was stimulated 2-fold by Ca^<2+> and phospholipid. This partially activated kinase was converted to a Ca^<2+>-phospholipid- independent form by further digestion with trypsin without significant change in molecullar mass. However, the physiological significance of these proteolyzed enzyme with Mr. 80,000 is not clear at this time. Further detailed analysis of down regulation of protein kinase C seems to be important for understanding the role of protein kinase C in signal transduction. Less
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