Majumdar S, Kane L H, Rossi M W, Volpp B D, Nauseef W M, Korchak H M
Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia.
Biochim Biophys Acta. 1993 Apr 16;1176(3):276-86. doi: 10.1016/0167-4889(93)90056-u.
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.
中性粒细胞至少拥有两种依赖磷脂的蛋白激酶C形式，一种是经典的依赖钙/磷脂酰丝氨酸/二酰甘油的蛋白激酶Cβ亚型，另一种是不依赖钙但依赖磷脂酰丝氨酸/二酰甘油的新型蛋白激酶C（nPKC），我们现在证明它们具有不同的底物特异性。人中性粒细胞的激活会触发膜中NADPH氧化酶的组装并产生超氧阴离子（O2-）。有人提出，主要的依赖钙的β-PKC亚型在中性粒细胞中，在刺激诱导的磷酸化以及一种胞质47 kDa蛋白（p47-phox）与膜NADPH氧化酶的结合中发挥作用。在本研究中，我们证明纯化的β-PKC和nPKC具有非常不同的底物特异性；β-PKC而非nPKC能使内源性和重组p47-phox磷酸化。此外，β-PKC而非nPKC能使[ser25]PKC(19-31)磷酸化，该底物肽基于依赖钙的α、β和γ亚型中的一个序列。源自依赖钙的PKC亚型C末端的假底物(19-36)，以组蛋白IIIS或肽(19-31)作为底物时，抑制β-PKC的活性，但不抑制nPKC的活性。假底物(19-36)也抑制β-PKC催化的内源性和重组p47-phox的磷酸化。假底物(19-36)还能使电穿孔中性粒细胞中由GTPγS触发的O2-产生减少50%。在GTPγS存在的情况下进行电穿孔的32P标记中性粒细胞显示多种胞质蛋白发生磷酸化，包括一条47 kDa条带，以及膜相关的34 kDa、47 kDa和54 kDa蛋白发生磷酸化。假底物(19-36)抑制膜中p47-phox的磷酸化，但不抑制胞质中的磷酸化。这些发现表明，可转位的、依赖钙的PKC亚型，如β-PKC，可能在膜相关p47-phox的磷酸化以及活性NADPH氧化酶的组装或维持中发挥作用。
