Abstract
In a Jurkat cell model of T-cell activation an interleukin-2 promoter/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1. Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C (PKC) with phorbol esters in combination with calcium mobilization by an ionophore. In cotransfection experiments, oncogenic rats obviated the need for PKC stimulation but did not replace either the calcium signal or interleukin-1. Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT. A dominant inhibitory ras mutant specifically blocked antigen receptor agonism, indicating that ras activity is required for antigen receptor signaling. In addition, an inhibitor of PKC blocked both activated ras and phorbol ester stimulation, suggesting a role for ras upstream of PKC.
Highlights
MATERIALS AND METHODSReagents-Recombinant human IL-10 from Escherichia coli (Casagli et al, 1989)was used at 1 ng/ml
From the $.Department of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, Italy andthe §Zmmunobiological Research Institute, Siena, Via Fiorentina I, 53100 Siena, Italy
In a Jurkat cell model of T-cell activation an inter- 1989) delivers an important accessory signal capable of conleukin-2 promoter/reportergeneconstruct was acti- tributing to IL-2 activation (Abrahaemt al., 1987; Macchia et vated by antigen receptor agonism in combinwaittihon al., 1990)
Summary
Reagents-Recombinant human IL-10 from Escherichia coli (Casagli et al, 1989)was used at 1 ng/ml. Panel R, CAT assays of Jurkat cells cotranssequence identified by DNA sequencing was used for a second fectedwith IL"L/CAT (4 pg/sample), pSVT/hILlR (3 pg/sample), round of site-directed mutagenesisof G to A at position 17 (serine to andeitherthe plasmid T24-ras (8 pg/sample, right) orthesame asparagine). T-cell type IL-1 receptors (Baldari et al, 1991; Heguy et al, 1991) In this case, the IL-2/CAT construct responds to a aliquoted, and the test plasmid or an appropriate quanotiftycontrol combination of TCR stimulation by PHA and IL-1R stimuplasmidwasadded.Cellswere allowed to recoverfor 24 hhefore lation by IL-1 (Fig. 1A). Of PKC associated with the membrane (data not showpnl)u,s the calcium ionophore A23187 This combination, like PHA alone, had very little effect on IL-2/CAT activity; it synergized effectively with IL-1 (Fig. lA, right panel).
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