Regulation of Phosphatidylinositol 4-phosphate 5-kinase by Protein Kinase C in Human Platelet Membranes.
Phorbol myristate acetate (PM A) increased the formation of |(32)P | PI 4,5-P, in (32)P-prelabeled human platelet. In saponin-permeabilized platelets, in which (32)P from exogenous |γ-(32)P| ATP was incorporated into PI 4-P and PI 4,5-P(2), addition of 10 nM PMA resulted in increased formation of |(32)P|PI 4,5-P(2) and |(32)P|PI 4-P. In order to distinguish whether increased [(32)P]PI 4,5-P(2) formation by PMA reflected merely an increase of [(32)P]PI 4-P, the substrate for PI 4-P 5-kinase, or activation of PI 4-P Skinase, we examined the membrane fraction in which most of the kinase activity was located. Although PMA itself did not affect the PI 4-P 5-kinase activity in the control membranes, the kinase activity was increased nearly 2-fold in membranes pretreated with 10 nM PMA but not 4α-phorbol didecanoate which does not activate protein kinase C (PKC). These results suggested that membrane PI 4-P 5-kinase activity was stimulated by the activation of PKC. However, 100 nM PMA did not stimulate [(32)P]PI 4,s-P(2) formation in saponin-permeabilized platelets, and the PI 4-P 5-kinase activity in membranes from platelets pretreated with 100 nM PMA was almost the same as that in control membranes. This can be explained by product inhibition, since PI 4,5-P(2) inhibited concentration-dependently the membrane PI 4-P 5-kinase activity. The Ca(2+) -dependent PKC fraction partially purified from the platelet cytosol stimulated the membrane PI 4-P 5-kinase activity, whereas the Ca(2)'-independent PKC fraction inhibited the kinase activity. Taken together, the present results suggest that the platelet membrane PI 4-P 5-kinase activity is stimulated by Ca(2+) -dependent PKC (cPKC) and is negatively regulated by PI 4,s-p(2) and Ca(2+) -independent PKC(nPKC).
Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu, 500, Japan, (81)-582-65-1241 (Ext. 2228), (81)-582-65-9002.
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Suzuki
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Nakashima
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Nozawa