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 prote in kinase C, a classical Ca/PS/DG-dependent beta-isotype of protein ki nase C and a Ca-independent but PS/DG-dependent novel protein kinase C (nPKC) which we now demonstrate to have different substrate specifici ties. Activation of human neutrophils triggers assembly of an NADPH ox idase in the membrane and generation of O2-. A role for the major Ca-d ependent isotype beta-PKC in neutrophils is proposed in stimulus-induc ed phosphorylation and association of a cytosolic 47 kDa protein (p47- phox) with the membrane NADPH oxidase. In this study we demonstrate th at purified beta-PKC and nPKC have very different substrate specificit ies; beta-PKC but not nPKC phosphorylated both endogenous and recombin ant p47-phox. In addition, beta-PKC but not nPKC phosphorylated [ser25 ]PKC(19-31), the substrate peptide based on a sequence in the Ca-depen dent alpha, beta and gamma-isotypes. Pseudosubstrate(19-36), derived f rom the C-terminus of Ca-dependent PKC isotypes, inhibited beta-PKC bu t not nPKC activity using either Histone IIIS or peptide(19-31) as sub strate. Pseudosubstrate(19-36) also inhibited beta-PKC catalyzed phosp horylation of endogenous and recombinant p47-phox. Pseudosubstrate(19- 36) inhibited the O2- generation triggered by GTPgammaS in electropora ted neutrophils by 50%. P-32-labelled neutrophils electroporated in th e presence of GTPgammaS showed phosphorylation of multiple cytosolic p roteins including a 47 kDa band, and phosphorylation of membrane-assoc iated 34 kDa, 47 kDa and 54 kDa proteins. Pseudosubstrate(19-36) inhib ited phosphorylation of p47-phox in the membrane but not in the cytoso l. These findings suggest translocatable, Ca-dependent isotypes of PKC such as beta-PKC may play a role in the phosphorylation of membrane a ssociated p47-phox and the assembly or maintenance of an active NADPH oxidase.