The 28-kDa/32-kDa activation antigen EA 1. Further characterization and signal requirements for its expression

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.