Protein kinase C isotypes and signal-transduction in human neutrophils: selective substrate specificity of calcium-dependent beta-PKC and novel calcium-independent nPKC.
Neutrophils possess at least two phospholipid-dependent forms of protein kinase C, a classical Ca/PS/DG-dependent beta-isotype of protein kinase C and a Ca-independent but PS/DG-dependent novel protein kinase C (nPKC) which we now demonstrate to have different substrate specificities. Activation of human neutrophils triggers assembly of an NADPH oxidase in the membrane and generation of O2-. A role for the major Ca-dependent isotype beta-PKC in neutrophils is proposed in stimulus-induced phosphorylation and association of a cytosolic 47 kDa protein (p47-phox) with the membrane NADPH oxidase. In this study we demonstrate that purified beta-PKC and nPKC have very different substrate specificities; beta-PKC but not nPKC phosphorylated both endogenous and recombinant p47-phox. In addition, beta-PKC but not nPKC phosphorylated [ser25]PKC(19-31), the substrate peptide based on a sequence in the Ca-dependent alpha, beta and gamma-isotypes. Pseudosubstrate(19-36), derived from the C-terminus of Ca-dependent PKC isotypes, inhibited beta-PKC but not nPKC activity using either Histone IIIS or peptide(19-31) as substrate. Pseudosubstrate(19-36) also inhibited beta-PKC catalyzed phosphorylation of endogenous and recombinant p47-phox. Pseudosubstrate(19-36) also inhibited the O2- generation triggered by GTP gamma S in electroporated neutrophils by 50%. 32P-Labelled neutrophils electroporated in the presence of GTP gamma S showed phosphorylation of multiple cytosolic proteins including a 47 kDa band, and phosphorylation of membrane-associated 34 kDa, 47 kDa and 54 kDa proteins. Pseudosubstrate(19-36) inhibited phosphorylation of p47-phox in the membrane but not in the cytosol. These findings suggest translocatable, Ca-dependent isotypes of PKC such as beta-PKC may play a role in the phosphorylation of membrane associated p47-phox and the assembly or maintenance of an active NADPH oxidase.
Majumdar
S
S
Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia.
Kane
L H
LH
Rossi
M W
MW
Volpp
B D
BD
Nauseef
W M
WM
Korchak
H M
HM
D002118:Calcium / Q000494:pharmacology*; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity
D007527:Isoenzymes; D010455:Peptides; D010750:Phosphoproteins; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D011493:Protein Kinase C; D002118:Calcium