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 M(r) 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 and 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 M(r) 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. Thes 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.
| Original language | English |
|---|---|
| Pages (from-to) | 4094-4100 |
| Number of pages | 7 |
| Journal | Journal of Immunology |
| Volume | 141 |
| Issue number | 12 |
| Publication status | Published - Dec 1 1988 |
| Externally published | Yes |
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All Science Journal Classification (ASJC) codes
- Immunology and Allergy
- Immunology
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The 28-kDa/32-kDa activation antigen EA 1. Further characterization and signal requirements for its expression. / Bjorndahl, J. M.; Nakamura, Seiji; Hara, T.; Jung, L. K.L.; Man Fu, S.
In: Journal of Immunology, Vol. 141, No. 12, 01.12.1988, p. 4094-4100.Research output: Contribution to journal › Article
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TY - JOUR
T1 - The 28-kDa/32-kDa activation antigen EA 1. Further characterization and signal requirements for its expression
AU - Bjorndahl, J. M.
AU - Nakamura, Seiji
AU - Hara, T.
AU - Jung, L. K.L.
AU - Man Fu, S.
PY - 1988/12/1
Y1 - 1988/12/1
N2 - 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 M(r) 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 and 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 M(r) 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. Thes 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.
AB - 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 M(r) 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 and 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 M(r) 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. Thes 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.
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M3 - Article
C2 - 3264302
AN - SCOPUS:0024209585
VL - 141
SP - 4094
EP - 4100
JO - Journal of Immunology
JF - Journal of Immunology
SN - 0022-1767
IS - 12
ER -