Inhibition of protein phosphatases by okadaic acid induces AP1 in human T cells
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C Thévenin1, S J Kim, J H Kehrl
1Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
To examine the role of protein phosphatases in T cell activation, Jurkat cells were treated with okadaic acid, an inhibitor of type 1 and 2A phosphatases, and nuclear extracts were examined for the presence of AP1 as a measure of early T cell activation. Okadaic acid was found to be a potent inducer of AP1. In contrast to phorbol esters such as phorbol myristate acetate (PMA), the induction of AP1 by okadaic acid occurs predominantly by transcriptional activation of the jun and fos family of proto-oncogenes. Surprisingly, while the addition of phytohemagglutinin further enhanced the induction of AP1, the addition of PMA inhibited it. Okadaic acid treatment was found to dramatically increase mRNA transcripts of the jun family of proto-oncogenes including c-jun, junD, and junB and to a lesser extent the fos family including c-fos and fra-1. By comparison, PMA is a very inefficient inducer of the jun gene family in Jurkat cells. Similar to its effect on the induction of AP1 by okadaic acid, PMA inhibits the induction of c-jun mRNA by okadaic acid. Transfection of c-jun promoter constructs confirmed the marked difference between PMA and okadaic acid in inducing c-jun transcription. The induction of AP1 by okadaic acid suggests that protein phosphatases 1 and 2A (PP1 and PP2A) may be involved in T cell activation as important negative regulators of the transcription factor AP1.
Insights
Protein phosphatases 1 and 2A (PP1 and PP2A) act as negative regulators in T cell activation. Inhibiting these phosphatases with okadaic acid strongly induces the transcription factor AP1 in Jurkat cells.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Signaling
Background:
- T cell activation is a complex process involving numerous signaling pathways.
- Protein phosphatases play critical roles in regulating cellular responses, including immune cell activation.
- The transcription factor AP1 is a key regulator of genes involved in T cell activation.
Purpose of the Study:
- To investigate the role of protein phosphatases 1 and 2A (PP1 and PP2A) in T cell activation.
- To determine the effect of PP1 and PP2A inhibition on the early activation marker AP1 in Jurkat cells.
Main Methods:
- Jurkat cells were treated with okadaic acid, a specific inhibitor of PP1 and PP2A.
- Nuclear extracts were analyzed for the presence of AP1.
- mRNA transcripts of jun and fos proto-oncogenes were quantified.
- c-jun promoter constructs were used to assess transcriptional activity.
Main Results:
- Okadaic acid potently induced AP1 in Jurkat cells, primarily through transcriptional activation of jun and fos proto-oncogenes.
- mRNA levels of c-jun, junD, and junB were significantly increased by okadaic acid.
- Phytohemagglutinin enhanced okadaic acid-induced AP1, while phorbol myristate acetate (PMA) inhibited it.
- PMA was inefficient at inducing the jun gene family and inhibited okadaic acid-induced c-jun mRNA expression.
Conclusions:
- Protein phosphatases 1 and 2A (PP1 and PP2A) are likely negative regulators of T cell activation.
- Inhibition of PP1 and PP2A leads to increased AP1 activity via transcriptional upregulation of jun and fos genes.
- Distinct signaling pathways are involved in AP1 induction by okadaic acid and PMA.