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

Abstract

Human neutrophil protein kinase C (PKC) activity is inhibited by an endogenous protein found primarily in the pellet fraction from homogenized specific granules, which was both heat- and proteinase-sensitive [Balazovich, Smolen & Boxer (1986) J. Immunol. 137, 1665-1673]. We now report that two PKC isoenzymes and the endogenous PKC inhibitor, which we named PKC-I, were purified from human neutrophils. A neutrophil soluble fraction that was subjected to DEAE-Sephacel chromatography yielded highly enriched PKC because, by definition, enzymic activity was strictly dependent on Ca2+ and phosphatidylserine. Hydroxyapatite chromatography resolved two peaks of PKC activity. Type II and Type III PKC isoenzymes were each identified on Western blots by using isoenzyme-specific monoclonal antibodies. Unlike rat brain, from which PKC isoenzymes were also purified, Type I PKC was not detected in human neutrophils. Western blots indicated that both Type II and Type III PKC isoenzymes had molecular masses near 80 kDa. In agreement with other reports, PKC was autophosphorylated in vitro. PKC-I, an endogenous neutrophil inhibitor of PKC, was purified to apparent homogeneity by DEAE-Sephacel and S-400 Sephacel chromatography. PKC-I had a molecular mass of 41 kDa. PKC-I inhibited purified PKC activity stimulated by 1,2-diacylglycerols in a concentration-dependent manner, and inhibited PKC-dependent phosphorylation of proteins present in neutrophil cytosol.

Annexin V and Phospholipid Metabolism

“…Endogenous inhibitors for PKCs have been isolated from rat (18) and sheep brains (19), and from neutrophils (20). An inhibitory activity has also been found in Jurkat cells (21).…”

The inhibitory effect of BSP‐A1/‐A2 on protein kinase C and tyrosine protein kinase

“…PKC inhibitors reported so far in the literature include synthetic compounds and inhibitors of biological origin. [18][19][20][21][22][23] In this paper, we showed for the first time that BSP-A1/A2 inhibits PKC activity, and the inhibition is dose dependent. Since BSP-A1/-A2, BSP-A3 and BSP-30-kDa are homologous proteins, it is reasonable to conjecture that BSP-A3 and BSP-30-kDa can also inhibit the PKC activity and further study is under way.…”

Evidence that the Modulation of Membrane‐Associated Protein Kinase C Activity by an Endogenous Inhibitor Plays a Role in N1E‐115 Murine Neuroblastoma Cell Differentiation

“…The presence of endogenous PKC inhibitors has been reported in a variety of cell types and tissues (Schwantke and Le Peuch, 1984;McDonald and Walsh, 1985;Balazovich et al ., 1986;Eyster, 1990;Toker et al, 1990;Gandini et al, 1993) . Three of these inhibitors, a 17-kDa protein (McDonald and Walsh, 1985;Pearson et al ., 1990) from bovine brain called PKC inhibitor-1 (PKCI-1), a 30-kDa protein Joker et al, 1990Joker et al, , 1992 from sheep brain termed kinase C inhibitory protein-1 (KCIP-1 ), and a 41-kDa PKC inhibitor (PKC-I) from human neutrophils (Balazovich et al, 1986(Balazovich et al, , 1992 have been well characterized . Preliminary characterization of the neuroblastoma PKC inhibitor (N-KCIF) suggested that it may be different from the brain inhibitors PKCI-1 and KCIP-1 .…”

“…However, what role, if any, KCIP-1 plays in these or any other cellular processes remains unknown . The presence of PKC-I in specific granule-containing human neutrophils and not in "resting," specific granule-deficient neutrophils has led to the speculation that PKC-I may play a role in normal neutrophil functions (Balazovich et al, 1986(Balazovich et al, , 1992 . A unique feature of the present study is the demonstration that a correlation exists between PKC and N-KCIF activities in differentiating neuroblastoma cells .…”