Phosphatidylinositol 3-Kinase-dependent Activation of Protein Kinase C-ζ in Bacterial Lipopolysaccharide-treated Human Monocytes

Abstract

The isoform identity of activated protein kinase C (PKC) and its regulation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with isoform-specific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-. In addition to primary monocytes, activation of PKCin response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-, the kinase did not depend upon the presence of lipids, Ca 2؉ , or diacylglycerol for activity. In addition, the kinase phosphorylates peptide ⑀ and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-from the cytosolic to the particulate membrane fraction upon exposure of monocytes to LPS provided further evidence for activation of the kinase.Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-. Furthermore, activation of PKC-failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P 3 . These findings are consistent with a model in which PKC-is activated downstream of PI 3-kinase in monocytes in response to LPS.

Protein Kinase C ζ Plays a Central Role in Activation of the p42/44 Mitogen-Activated Protein Kinase by Endotoxin in Alveolar Macrophages

“…Sajan et al (40) have linked PDK-1 and PKC to ERK activation by LPS in rat adipocytes. In a model closer to ours, Herrara-Velit et al (41) have shown that in LPS-treated monocytes, PKC activation is PDK-1 dependent. PKC activation requires autophosphorylation, phosphorylation of Thr 402 in the activation loop (probably by PDK-1), removal of the pseudosubstrate from the catalytic domain, and subcellular localization near a substrate (42).…”

Surfactant Protein A Activation of Atypical Protein Kinase C ζ in IκB-α-Dependent Anti-Inflammatory Immune Regulation

“…PKC can be activated in vitro by phosphatidylinositol-3,4,5-triphosphat (42,56), phosphatidic acid (57), ceramide, and arachidonic acid (58,59), and/or by direct interaction with binding proteins (60). To activate PKC , phosphorylation of the activation loop consensus threonine residue Thr 410 by PI3K-induced phosphoinositide-dependent kinase 1 is substantial (41).…”

Ca2+-Evoked Serotonin Secretion by Parafollicular Cells: Roles in Signal Transduction of Phosphatidylinositol 3′-kinase, and the γ and ζ Isoforms of Protein Kinase C

“…When the effects of PI3Ј-kinase inhibitors were studied, cells were incubated with 100 M LY294002, 100 nM wortmannin, or 40 M pseudoZ for 30 min before the addition of C a 2ϩ . PKC and / activities were measured by using minor modifications of previously published methods (Gomez et al, 1997;Herrera-Velit et al, 1997). After treatment, cells were washed in PBS and lysed for 1 hr at 4°C in 150 l of lysis buffer (1% Triton X-100, 20 mM Tris, pH 7.4, 150 mM NaC l, 1 mM EDTA, 2 mM EGTA, 1 mM Na-vanadate, 100 nM okadaic acid, 100 g /ml aprotinin, 100 g /ml leupeptin, 0.5 mM phenylmethanesulfonyl fluoride, 12.5 g /ml pepstatin, and 1 mM benzamide).…”

“…Recent studies have demonstrated that both nPKCs and aPKCs are activated by products of PI3Ј-kinase Toker et al, 1994;Derman et al, 1997;Herrera-Velit et al, 1997;Standaert et al, 1997;Chou et al, 1998). It is thus possible that PI3Ј-kinase is responsible for activating the pseudoZ-sensitive isoform of PKC (such as PKC ) after stimulation of the CaR.…”