PROTEIN-KINASE-C ISOTYPES AND SIGNAL-TRANSDUCTION IN HUMAN NEUTROPHILS - SELECTIVE SUBSTRATE-SPECIFICITY OF CALCIUM-DEPENDENT BETA-PKC AND NOVEL CALCIUM-INDEPENDENT NPKC
PROTEIN-KINASE-C ISOTYPES AND SIGNAL-TRANSDUCTION IN HUMAN NEUTROPHILS - SELECTIVE SUBSTRATE-SPECIFICITY OF CALCIUM-DEPENDENT BETA-PKC AND NOVEL CALCIUM-INDEPENDENT NPKC
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DOI：
10.1016/0167-4889(93)90056-u
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发表时间：
1993-04-16
期刊：
影响因子：
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通讯作者：
KORCHAK, HM
中科院分区：
文献类型：
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作者：
MAJUMDAR, S;KANE, LH;KORCHAK, HM
中性粒细胞具有至少两种磷脂依赖性形式的蛋白激酶C，一种经典的Ca/PS/DG依赖性β-同种型蛋白激酶C和一种Ca非依赖性但PS/DG依赖性的新型蛋白激酶C（nPKC），我们现在证明其具有不同的底物特异性。人类中性粒细胞的激活触发膜中NADPH氧化酶的组装和O2-的产生。中性粒细胞中主要钙依赖性同种型β-PKC的作用被认为是刺激诱导的磷酸化和细胞溶质47 kDa蛋白（p47-phox）与膜NADPH氧化酶的结合。在这项研究中，我们证明，纯化的β-PKC和nPKC有非常不同的底物特异性，β-PKC，但不nPKC磷酸化内源性和重组p47-phox。此外，β-PKC而非nPKC磷酸化[ser 25]PKC（19-31），底物肽基于Ca依赖性α、β和γ同种型中的序列。来源于Ca依赖性PKC同种型C-末端的假底物（19-36）抑制β-PKC活性，但不抑制使用组蛋白III S或肽（19-31）作为底物的nPKC活性。假底物（19-36）也抑制内源性和重组p47-phox的β-PKC催化的磷酸化。假底物（19-36）抑制电穿孔的中性粒细胞中由GTP γ S触发的O2产生50%。在GTP γ S存在下电穿孔的P-32标记的中性粒细胞显示出多种胞质蛋白的磷酸化，包括47 kDa条带，以及膜相关的34 kDa、47 kDa和54 kDa蛋白的磷酸化。假底物（19-36）抑制膜中p47-phox的磷酸化，但不抑制胞质溶胶中的磷酸化。这些发现表明，可转位的，钙依赖性的PKC亚型，如β-PKC可能在膜相关p47-phox的磷酸化和组装或维持活性NADPH氧化酶中发挥作用。
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) inhibited the O2- generation triggered by GTPgammaS in electroporated neutrophils by 50%. P-32-labelled neutrophils electroporated in the presence of GTPgammaS 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.