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

Patricia Herrera-Velit,Keith L Knutson,Neil E Reiner

doi:10.1074/jbc.272.26.16445

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-zeta. In addition to primary monocytes, activation of PKC-zeta in response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-zeta, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide epsilon and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-zeta 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-zeta. Furthermore, activation of PKC-zeta failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-zeta activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P3. These findings are consistent with a model in which PKC-zeta is activated downstream of PI 3-kinase in monocytes in response to LPS.

Highlights

Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of protein kinase C (PKC)-z
This subfamily of PKC isoforms differs from cPKC (a, bI, bII, g) and nPKC (d, e, h, u) subfamily members in that aPKC isoforms are neither receptors for phorbol esters nor are regulated by Ca21 or diacylglycerols [11,12,13,14]
The results presented show that PKC-z is rapidly activated in LPS-treated human monocytes, and this occurs downstream of activated PI 3-kinase

Summary

Introduction

Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-z. Activation of PI 3-kinase results in the production of PIP3, which is known to be an activator of the PKC isoforms z, e, and d [9, 10] This is of interest in the context of LPS signaling since evidence has recently been provided to show that a PKC activity is increased in LPS-treated monocytes [3, 4]. This activity appears to be related to one or more PKC isoforms, the activation of which is sustained in the absence of phosphatidylserine, Ca21, and diacylglycerol [3]. APKCs exhibit activator-independent activity which is increased upon exposure to novel lipids such as PIP3 and ceramides [9, 15, 16]

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