title: The 28-kDa/32-kDa activation antigen EA 1. Further characterization and signal requirements for its expression.
abstract: The tumor promoter PMA has been shown to induce the expression of a 28-kDa/32-kDa early activation Ag, termed EA 1, on resting T cells. Under nonreducing conditions, EA 1 was detected by SDS-PAGE as a diffuse band in the 60-kDa region. In this study, this diffuse band was resolved into 56-kDa and 60-kDa bands. Endoglycosidase F treatment of EA 1 resulted in the appearance of a single band with a Mr of 48 kDa. Upon reduction, the 48-kDa band was shown to be composed of 24-kDa peptides. Diagonal gel electrophoresis showed that the major band of EA 1 was composed of a series of disulfide-linked homodimers with subunits of the same 24-kDa core protein that were differentially glycosylated. This analysis also revealed in a minor population of the EA 1 molecules, the presence of proteins of different Mr associated with the core protein. The signal requirements for the induction of EA 1 were investigated. The putative cellular action of PMA is the activation of protein kinase C (PKC). To further investigate the role of PKC activation in the expression of EA 1, the synthetic diacylglycerol, 1,2-sn-dioctanoylglycerol (diOG) was examined for its ability to substitute for PMA. DiOG induced EA 1 expression in a dose dependent manner. H-7, a relatively selective inhibitor of PKC, blocked diOG and PMA induced EA 1 expression. HA1004, a selective inhibitor of cAMP- and cGMP-dependent protein kinases, had no effect. In kinetic studies, EA 1 expression was seen as early as 1 h in diOG- and PMA-activated T cells. However, diOG did not completely mimic PMA-induced EA 1 expression. By 18 h, diOG-induced EA 1 expression was markedly reduced, whereas PMA-induced EA 1 expression was persistent. The role of calcium in EA 1 expression was investigated. mAb against CD3 potentiated diOG-induced EA 1 expression. This potentiation appeared to correlate with the ability of the anti-CD3 mAb to induce rises in intracellular calcium. Addition of EGTA to the media blocked the potentiation of diOG induced EA 1 expression by these mAb. The role of calcium in EA 1 expression was further demonstrated by the ability of ionomycin to potentiate EA 1 expression. These results demonstrate that PKC activation is the primary pathway for the induction of EA 1. However, calcium-dependent pathways appear to have a secondary role.
authors.affiliation: Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
authors.forename: J M
authors.initials: JM
authors.lastname: Bjorndahl
authors.forename: S
authors.initials: S
authors.lastname: Nakamura
authors.forename: T
authors.initials: T
authors.lastname: Hara
authors.forename: L K
authors.initials: LK
authors.lastname: Jung
authors.forename: S M
authors.initials: SM
authors.lastname: Fu
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis / Q000302:isolation & purification*; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D004987:Ethers / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D015759:Ionomycin; D037181:Lectins, C-Type; D008213:Lymphocyte Activation / Q000187:drug effects; D046911:Macromolecular Substances; D008970:Molecular Weight; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D000099192:CD69 Antigens
chemical_list: D000276:Adjuvants, Immunologic; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D004075:Diglycerides; D004987:Ethers; D015759:Ionomycin; D037181:Lectins, C-Type; D046911:Macromolecular Substances; D011493:Protein Kinase C; D000099192:CD69 Antigens; C044295:1,2-dioctanoylglycerol