Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Abstract

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the “translocation” of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.

Purification of PKC-I, an endogenous protein kinase C inhibitor, and types II and III protein kinase C isoenzymes from human neutrophils

“…In support of its cellular localization, both neutrophil cytoplasts and specific-granuledeficient neutrophils appear to lack PKC-I activity [29]. Functionally, PKC-I may modulate PKC activity in vivo, because resting specific-granule-deficient neutrophils display translocated PKC and increased endogenous protein phosphorylation compared with normal neutrophils [29]. With regard to the other endogenous PKC inhibitors which have been reported, no characteristic appears common to the group; for example, reported molecular masses range from 0.6 to 67 kDa, only about half are heat-sensitive, and some interact with the regulatory fragment of PKC, whereas others interact with the catalytic fragment.…”

“…The Ca2+-and phospholipid-dependent family of enzymes known as protein kinase C (PKC) has been described in several types of blood cells, including lymphocytes [1], platelets [2] and neutrophils [3][4][5]. PKC becomes mostly insoluble (i.e.…”

“…PKC activity has been quantified in vitro in crude fractions of human neutrophils by using an assay that measures histone phosphorylation. Qualitatively, a large number of different endogenous PKC substrates have been described by observing their phosphorylation in situ [5,[10][11][12], some of which may be involved in cellular functional responses. PKC activity assayed by both methods has been correlated with degranulation and superoxideanion production in neutrophils [13][14][15], although recent investigations [16][17][18] suggest that there may not be an absolute requirement for its activity to initiate these functions.…”

“…We [29] and others [30][31][32] have previously described endogenous inhibitors of PKC which may modulate PKC activity and thereby certain cellular functional events in vivo. The neutrophil PKC inhibitor, PKC-I, is heat-and proteinasesensitive, is not itself a proteinase, and appears to be present primarily in the membrane fraction of specific granules and in the plasma-membrane fraction [29]. In support of its cellular localization, both neutrophil cytoplasts and specific-granuledeficient neutrophils appear to lack PKC-I activity [29].…”

“…The neutrophil PKC inhibitor, PKC-I, is heat-and proteinasesensitive, is not itself a proteinase, and appears to be present primarily in the membrane fraction of specific granules and in the plasma-membrane fraction [29]. In support of its cellular localization, both neutrophil cytoplasts and specific-granuledeficient neutrophils appear to lack PKC-I activity [29]. Functionally, PKC-I may modulate PKC activity in vivo, because resting specific-granule-deficient neutrophils display translocated PKC and increased endogenous protein phosphorylation compared with normal neutrophils [29].…”