Mao C, Merlin G, Ballotti R, Metzler M, Aguet M
Unité 196 Institut de la Santé et de la Recherche Medicale, Institut Curie, Paris, France.
J Immunol. 1990 Dec 15;145(12):4257-64.
Various cell surface receptors are phosphorylated upon binding of their ligand, and this phosphorylation seems to be involved in the signal transduction or in the feedback regulation of this signal. The possibility of a phosphorylation of the human IFN-gamma receptor (hu-IFN-gamma-R) has been investigated with 32P-labeled whole Raji cells and receptor purification either by immunoprecipitation with an anti-hu-IFN-gamma-R polyclonal antiserum or by affinity chromatography. The hu-IFN-gamma-R was found to be phosphorylated at a basal level. Upon incubation of the cells with recombinant hu-IFN-gamma, a dose-dependent two-fold increase of this phosphorylation was observed. Phosphoamino acid analysis by TLC showed that the same amino acids, serine and threonine, are phosphorylated at a basal level and after incubation with hu-IFN-gamma. Protein kinase C and Ca2+/calmodulin-dependent kinase pathways have been reported in some cases to be involved in the signal transduction pathway of hu-IFN-gamma. Both pathways involved the activation of a serine/threonine kinase and therefore we have investigated the possibility of hu-IFN-gamma-R phosphorylation by these kinases. PMA, an activator of protein kinase C, induced a rapid increase of the receptor phosphorylation in Raji cells, whereas the Ca2+ ionophore A23187 did not. PMA-induced hu-IFN-gamma-R phosphorylation was not associated with any effect on expression or inactivation of the receptor. PMA alone did not mimic the hu-IFN-gamma effect in Raji cells as measured by induction of IP-10 gene expression, a high specific marker of hu-IFN-gamma response. But the protein kinase C inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine, reduced this IFN-gamma-induced expression. However, H7 and staurosporine treatment as well as protein kinase C depletion suppressed PMA-induced receptor phosphorylation, whereas constitutive and hu-IFN-gamma-induced phosphorylation remained unchanged. Our results suggest that the serine/threonine kinase involved in the hu-IFN-gamma-R phosphorylation induced by IFN-gamma is different from protein kinase C.
多种细胞表面受体在与配体结合后会发生磷酸化，这种磷酸化似乎参与了信号转导或该信号的反馈调节。人们利用经32P标记的完整拉吉细胞以及通过用抗人γ干扰素受体（hu-IFN-γ-R）多克隆抗血清进行免疫沉淀或亲和层析来纯化受体，对人γ干扰素受体（hu-IFN-γ-R）发生磷酸化的可能性进行了研究。结果发现，hu-IFN-γ-R在基础水平上就发生了磷酸化。在用重组人γ干扰素孵育细胞后，观察到这种磷酸化呈剂量依赖性地增加了两倍。通过薄层层析进行的磷酸氨基酸分析表明，在基础水平以及与hu-IFN-γ孵育后，相同的氨基酸，即丝氨酸和苏氨酸，会发生磷酸化。在某些情况下，已报道蛋白激酶C和Ca2+/钙调蛋白依赖性激酶途径参与了hu-IFN-γ的信号转导途径。这两条途径都涉及丝氨酸/苏氨酸激酶的激活，因此我们研究了这些激酶使hu-IFN-γ-R发生磷酸化的可能性。蛋白激酶C的激活剂佛波酯（PMA）可诱导拉吉细胞中受体磷酸化迅速增加，而Ca2+离子载体A23187则不会。PMA诱导的hu-IFN-γ-R磷酸化与对受体的表达或失活没有任何影响相关。单独的PMA在通过诱导IP-10基因表达（hu-IFN-γ反应的一种高度特异性标志物）来衡量时，并不会模拟拉吉细胞中的hu-IFN-γ效应。但是蛋白激酶C抑制剂1-(5-异喹啉磺酰基)-2-甲基哌嗪（H7）和星形孢菌素可降低这种γ干扰素诱导的表达。然而，H7和星形孢菌素处理以及蛋白激酶C的缺失会抑制PMA诱导的受体磷酸化，而组成型的以及γ干扰素诱导的磷酸化则保持不变。我们的结果表明，参与γ干扰素诱导的hu-IFN-γ-R磷酸化的丝氨酸/苏氨酸激酶与蛋白激酶C不同。
