1990 Fiscal Year Final Research Report Summary
Expression of Tyrosine-Phosphorylated Proteins in Cultured Megakaryocytic Cells and its Significance in Signal Transduction.
| Project/Area Number |
63571059
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Kitasato University |
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Principal Investigator |
KOBAYASHI Kitasato Univ. Pharmaceutical Sci. Professor, 薬学部・生理化学, 教授 (90050319)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIHARA N. Kitasato Univ. Pharmaceutical Sci. Instructor, 生理化学, 助手 (10050585)
WATANABE Y. Kitasato Univ. Pharmaceutical Sci. Assist. Prof., 生理化学, 助教授 (80050409)
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| Project Period (FY) |
1988 – 1990
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| Keywords | Bone Marrow Megakaryocytes / Platelets / Tyrosine-phosphorylation / Gp IIIa / Staurosporine / Thrombin / PMA |
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| Research Abstract |
The basal conditions for the short term liquid culture of megakaryocytes from mouse bone marrow were established, and effects of various stimulants on proliferation, maturation and differentiation of the cultured megakaryocytes were tested. Insulin, platelet growth factor and transferrin were shown to stimulate the proliferation phase, while staurosporine, a welknown inhibitor of intracelular protein phosphorylation, prevented selectively the stimulation of megakaryo-cyte proliferation by these agents. Ganglioside GDla stimulated both the proliferation and the maturation phase of megakaryocytes only in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, while the effect on the maturation phase was more evident. On the other hand, GDla stimulated selectively the proliferation phase in the presence of a general megakaryocyte colony stimulating factor, interleukin 3. Furthermore, ganglioside GMI promoted the latest stage of platelet production from mature megakaryo
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cytes. These results showed that gangliosides are involved in platelet production from bone marrow pluripotential stem cell rather selectively at specified phases.
Studies on rabbit platelets have shown that stimulation by phorbol myristate acetate increases phosphorylation of tyrosine residues in some proteins such as the "48K" in addition to the well-documented phosphorylation of serine/threonine residues. Rabbit platelet membrane glycoprotein GPIII a of theGP IIb/IIIa complex which appeared at membrane surface of the stimulated platelets was detected with monoclonal human GP IIIa antibody. Electrophoretic analysis in the presence of sodium dodecyl sulfate revealed that in the phorbol myristate acetate-stimulated platelets GP IIIa appeared as an additional, less dissociable 95K phosphoprotein band in addition to the original 66K band as detected in non-stimulated platelets. Discrimination with a phosphotyrosineseecific monoclonal antibody prepared in our hand revealed that the 95K phosphoprotein band reacted positively. Thus it is highly probable that GP IIIa can be phosphorylated in tyrosine residues and the covalent modification is involved in signal transduction of stimulated platelets. These findings are in accord with the recent report of Golfen (J. Biol. Chem. 111, 3117,1990). Less
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