ACTIVATION OF PROTEIN-KINASE-C FAMILY MEMBERS BY THE NOVEL POLYPHOSPHOINOSITIDES PTDINS-3,4-P-2 AND PTDINS-3,4,5-P-3

The effect of phosphoinositides on the activity of protein kinase C (P KC) isotypes was investigated. PKC alpha, beta I, beta II, gamma, delt a, epsilon, eta, and zeta were expressed in baculovirus-infected insec t cells and purified by column chromatography. The calcium-activated P KC isotypes alpha, beta I, beta II, and gamma were not significantly a ctivated by any of the phosphoinositides investigated (phosphatidylino sitol-4-phosphate (PtdIns-4-P), PtdIns-3-P, PtdIns-4,5-P-2, PtdIns-3,4 -P-2, and PtdIns-3,4,5-P-3) when added in the presence of concentratio ns of phosphatidylserine that give maximal stimulation. The calcium-in sensitive PKC isotypes delta, epsilon, and eta also showed little resp onse to PtdIns3-P, PtdIns-4-P, or PtdIns-4,5-P-2 when these lipids wer e added in the presence of phosphatidylserine. In contrast, PtdIns-3,4 -P-2 and PtdIns-3,4,5-P-3 caused a 5-15-fold stimulation of these enzy mes compared with phosphatidylserine alone. 50 % maximal stimulation o f PKC epsilon by PtdIns-3,4,5-P-3 occurred when this lipid was present at about 1% of the carrier PtdIns-4,5-P-2 (about 100 nM). These lipid s had little effect on baculovirus-expressed PKC zeta, which was const itutively active. A short chain version of PtdIns-3,4,5-P-3, dioctanoy l-PtdIns-3,4,5-P-3, activated PKC delta, epsilon, and eta in the absen ce of other lipids, whereas a short chain version of PtdIns-4,5-P-2, d ihexanoyl-PtdIns-4,5-P-2, did not. Since PtdIns-3,4-P-2 and PtdIns-3,4 ,5-P-3 are nominally absent in unstimulated cells and appear within se conds to minutes of stimulation by various cell activators, these Lipi ds could act as second messengers to activate PHC delta, epsilon, or e ta in vivo.