Yamaguchi M, Oishi H, Araki S, Saeki S, Yamane H, Okamura N, Ishibashi S
Department of Physiological Chemistry, Hiroshima University School of Medicine, Japan.
Arch Biochem Biophys. 1995 Nov 10;323(2):382-6. doi: 10.1006/abbi.1995.0058.
We studied involvement of tyrosine-phosphorylated proteins in activation of NADPH oxidase in guinea pig neutrophils. Pervanadate, which is the oxidized form of orthovanadate, induced O2- production and protein tyrosine phosphorylation in neutrophils. O2- production induced by pervanadate was more sensitive to the tyrosine kinase-specific inhibitor, ST-638, as compared with the production induced by PMA. On the other hand, staurosporine more selectively inhibited PMA-induced O2- production than pervanadate-induced production. These results indicate that tyrosine kinase, not protein kinase C, is involved in pervanadate-induced O2- production. The tyrosine-phosphorylated proteins were detected in both the cytosol and membrane fractions prepared from pervanadate-induced neutrophils. In order to examine if tyrosine residues of some components of NADPH oxidase were directly phosphorylated, tyrosine-phosphorylated proteins were removed from solubilized membranes prepared from the pervanadate-stimulated neutrophils by immunoprecipitation with an anti-phosphotyrosine anti-body. NADPH oxidase activity in the solubilized membranes was not decreased by the treatment. These findings suggest that the components of NADPH oxidase are not tyrosine-phosphorylated by pervanadate treatment, that tyrosine phosphorylation may be involved in the signal transduction pathway of NADPH oxidase activation by pervanadate, and that this pathway is independent of the activation by protein kinase C.
我们研究了酪氨酸磷酸化蛋白在豚鼠中性粒细胞中烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶激活过程中的作用。过氧钒酸盐是原钒酸盐的氧化形式，可诱导中性粒细胞产生超氧阴离子（O₂⁻）并使蛋白酪氨酸磷酸化。与佛波酯（PMA）诱导产生的O₂⁻相比，过氧钒酸盐诱导产生的O₂⁻对酪氨酸激酶特异性抑制剂ST - 638更为敏感。另一方面，星形孢菌素对PMA诱导的O₂⁻产生的抑制作用比过氧钒酸盐诱导的更为选择性。这些结果表明，参与过氧钒酸盐诱导O₂⁻产生的是酪氨酸激酶，而非蛋白激酶C。在从用过氧钒酸盐处理的中性粒细胞制备的胞质溶胶和膜组分中均检测到了酪氨酸磷酸化蛋白。为了检测NADPH氧化酶某些组分的酪氨酸残基是否直接被磷酸化，通过用抗磷酸酪氨酸抗体进行免疫沉淀，从用过氧钒酸盐刺激的中性粒细胞制备的溶解膜中去除酪氨酸磷酸化蛋白。处理后，溶解膜中的NADPH氧化酶活性并未降低。这些发现表明，过氧钒酸盐处理不会使NADPH氧化酶的组分发生酪氨酸磷酸化，酪氨酸磷酸化可能参与过氧钒酸盐激活NADPH氧化酶的信号转导途径，且该途径独立于蛋白激酶C的激活。
