Abstract
The effect of phosphoinositides on the activity of protein kinase C (PKC) isotypes was investigated. PKC alpha, beta I, beta II, gamma, delta, epsilon, eta, and zeta were expressed in baculovirus-infected insect cells and purified by column chromatography. The calcium-activated PKC isotypes alpha, beta I, beta II, and gamma were not significantly activated by any of the phosphoinositides investigated (phosphatidylinositol-4-phosphate (PtdIns-4-P), PtdIns-3-P, PtdIns-4,5-P2, PtdIns-3,4-P2, and PtdIns-3,4,5-P3) when added in the presence of concentrations of phosphatidylserine that give maximal stimulation. The calcium-insensitive PKC isotypes delta, epsilon, and theta also showed little response to PtdIns-3-P, PtdIns-4-P, or PtdIns-4,5-P2 when these lipids were added in the presence of phosphatidylserine. In contrast, PtdIns-3,4-P2 and PtdIns-3,4,5-P3 caused a 5-15-fold stimulation of these enzymes compared with phosphatidylserine alone. 50% maximal stimulation of PKC epsilon by PtdIns-3,4,5-P3 occurred when this lipid was present at about 1% of the carrier PtdIns-4,5-P2 (about 100 nM). These lipids had little effect on baculovirus-expressed PKC zeta, which was constitutively active. A short chain version of PtdIns-3,4,5-P3, dioctanoyl-PtdIns-3,4,5-P3, activated PKC delta, epsilon, and eta in the absence of other lipids, whereas a short chain version of PtdIns-4,5-P2, dihexanoyl-PtdIns-4,5-P2, did not. Since PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are nominally absent in unstimulated cells and appear within seconds to minutes of stimulation by various cell activators, these lipids could act as second messengers to activate PKC delta, epsilon, or eta in vivo.
Highlights
PI, PII, y, 6, E, 4,and 5 were expressed in baculovirus- lated cells and does not significantly increase in response tocell infected insect cells and purified by column chromatog- activators [2]
Since PtdIns-3,4-Pa2nd PtdIns-3,4,5-P,are nominally absent in unstimulated cells and appear within seconds to minutes of stimulation by various cell activators, these lipids could act as second messengers to activate PKC 6, E, or q in vivo
3,4-P2, and PtdIns-3,4,5-P3 failed to affect the activity of the calcium-dependent PKC isoforms (Fig. lA). These lipids were added in thperesence of a 10-fold excess of their precurphosphoinositide vesicles
Summary
**Financially supported by the MizutaniFoundation for Glyco- PKCs comprising 6, E, ( L ) ,and B (insensitive tocalcium) and science. Horseradish peroxidase anti-mouse conjugate and chemiluforms whose activity is not affected by phorbol ester or the natural activatorDAG [20,21,22] Another subgroupof PKCs may be defined by the recentlydescribed PKC p,with a potential signal peptide and transmembrane domain[23]. Liquiscint was purchasferdom National Diagnostics.Molecular weight standards were purchasferodm Polyphosphoinositides such as PtdIns-4,5-P2 havebeen pre- Bio-Rad. All other chemicals were obtained from Sigma. Viously shown to activate PKC in uitro [24,25,26], though these studies arecompromised by the factthat PtdIns-4,5-P2 can act both asa phospholipidcofactor and as an activator. We report the activationof PKC isoforms 6, E , and q by tification of the lipids was determinedon the basisof the specific activity of the [y-32P]ATP.The radioactivity in thephospholipids was negligible relative to that used inPKC assays. 1,2-dihexanoyl-sn-glycero-3-phosphoriaccid (DiC,PA) [1]and 3,6-diobenzyl-D-myo-inositol 4,5-bis(dibenzylphosphate) [2] to yield 1-(1',2'-
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