Immunoglobulin secretion and phosphorylation of common proteins are induced by IL-2, IL-4, and IL-6 in the factor responsive human B cell line, SKW6.4.
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.
Goldstein
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Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY 10461.
Kim
A
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D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D015342:DNA Probes; D019833:Genistein; D006801:Humans; D007075:Immunoglobulin M / Q000096:biosynthesis* / Q000235:genetics; D007376:Interleukin-2 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D015342:DNA Probes; D007075:Immunoglobulin M; D007376:Interleukin-2; D015850:Interleukin-6; D007529:Isoflavones; D007546:Isoquinolines; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011506:Proteins; D012333:RNA, Messenger; D015847:Interleukin-4; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019833:Genistein