TY - JOUR
T1 - Phosphatidylinositol 3-kinase-dependent activation of protein kinase C- ζ in bacterial lipopolysaccharide-treated human monocytes
AU - Herrera-Velit, Patricia
AU - Knutson, Keith L.
AU - Reiner, Neil E.
PY - 1997/6/27
Y1 - 1997/6/27
N2 - 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-ζ. In addition to primary monocytes, activation of PKC-ζ in response to LPS was also observed in the human promonocytic cell lines, U937 and THP- 1. Consistent with its identity as PKC-ζ, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide ε and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-ζ 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-ζ. Furthermore, activation of PKC-ζ failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-ζ 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-ζ is activated downstream of PI 3-kinase in monocytes in response to LPS.
AB - 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-ζ. In addition to primary monocytes, activation of PKC-ζ in response to LPS was also observed in the human promonocytic cell lines, U937 and THP- 1. Consistent with its identity as PKC-ζ, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide ε and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-ζ 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-ζ. Furthermore, activation of PKC-ζ failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-ζ 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-ζ is activated downstream of PI 3-kinase in monocytes in response to LPS.
UR - http://www.scopus.com/inward/record.url?scp=0030985901&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030985901&partnerID=8YFLogxK
U2 - 10.1074/jbc.272.26.16445
DO - 10.1074/jbc.272.26.16445
M3 - Article
C2 - 9195953
AN - SCOPUS:0030985901
SN - 0021-9258
VL - 272
SP - 16445
EP - 16452
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -