Goldstein H, Kim A
Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY 10461.
J Immunol. 1993 Dec 15;151(12):6701-11.
After the binding of IL-2, IL-4, or IL-6 to their respective receptors on activated human B cells, a multistep cascade of intracellular events is initiated that results in the secretion of Ig. However, it is not known whether these different cytokine receptors use common or divergent signal transduction pathways to stimulate Ig secretion. Therefore, we examined the signaling mechanisms used by a human lymphoblastoid cell line arrested at a late stage of differentiation, SKW6.4, that secretes IgM following stimulation with IL-2, IL-4, or IL-6 alone. Our study demonstrated that IL-2, IL-4, and IL-6-stimulation of IgM secretion by SKW6.4 cells was inhibited by either the serine/threonine kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H7) or the tyrosine kinase inhibitor, genistein. To investigate the early phosphorylation events initiated by these cytokines, a membrane-enriched preparation from SKW6.4 cells was isolated in a manner that minimized the disruption of membrane protein complexes and then incubated with IL-2, IL-4, or IL-6 in the presence of [gamma-32P]ATP. IL-2, IL-4, and IL-6 stimulated the rapid serine/threonine phosphorylation of 47-, 49-, and 91-kDa proteins. However, in contrast to the 47- and 49-kDa proteins that remained phosphorylated for up to 30 min poststimulation, the 91-kDa protein was rapidly dephosphorylated within 15 min of stimulation. The observation that serine/threonine phosphorylation of the same proteins was stimulated by IL-2, IL-4, and IL-6 suggested that the cytokines activated either different protein kinases with the same substrate specificity or the same protein kinase. In addition, stimulation of intact SKW6.4 cells with either IL-2, IL-4, or IL-6 induced the phosphorylation of two proteins with molecular masses of 45- to 50-kDa and 85 to 90-kDa. Taken together, our data demonstrate that activation of both a serine/threonine kinase and a tyrosine kinase is involved in the IL-2, IL-4, and IL-6-stimulation of IgM secretion by SKW6.4 cells and activation of the same or a similar serine/threonine protein kinase is an early step in the signal transduction pathway used by these cytokines.
白细胞介素-2（IL-2）、白细胞介素-4（IL-4）或白细胞介素-6（IL-6）与活化的人B细胞上各自的受体结合后，会启动一系列细胞内多步骤级联反应，最终导致免疫球蛋白（Ig）的分泌。然而，尚不清楚这些不同的细胞因子受体是通过共同的还是不同的信号转导途径来刺激Ig分泌的。因此，我们研究了一种处于分化后期停滞状态的人淋巴母细胞系SKW6.4所使用的信号传导机制，该细胞系在单独用IL-2、IL-4或IL-6刺激后会分泌IgM。我们的研究表明，SKW6.4细胞中IL-2、IL-4和IL-6刺激IgM分泌的过程可被丝氨酸/苏氨酸激酶抑制剂1-(5-异喹啉磺酰基)-2-甲基哌嗪二盐酸盐（H7）或酪氨酸激酶抑制剂染料木黄酮抑制。为了研究这些细胞因子引发的早期磷酸化事件，我们以尽量减少膜蛋白复合物破坏的方式分离了SKW6.4细胞的富含膜的制剂，然后在[γ-32P]ATP存在的情况下与IL-2、IL-4或IL-6一起孵育。IL-2、IL-4和IL-6刺激了47 kDa、49 kDa和91 kDa蛋白质的快速丝氨酸/苏氨酸磷酸化。然而，与刺激后长达30分钟仍保持磷酸化的47 kDa和49 kDa蛋白质不同，91 kDa蛋白质在刺激后15分钟内迅速去磷酸化。IL-2、IL-4和IL-6刺激相同蛋白质的丝氨酸/苏氨酸磷酸化这一观察结果表明，这些细胞因子激活了具有相同底物特异性的不同蛋白激酶或相同的蛋白激酶。此外，用IL-2、IL-4或IL-6刺激完整的SKW6.4细胞会诱导两种分子量分别为45至50 kDa和85至90 kDa的蛋白质发生磷酸化。综上所述，我们的数据表明，丝氨酸/苏氨酸激酶和酪氨酸激酶的激活都参与了SKW6.4细胞中IL-2、IL-4和IL-6刺激IgM分泌的过程，并且相同或相似的丝氨酸/苏氨酸蛋白激酶的激活是这些细胞因子所使用的信号转导途径中的早期步骤。
