The Tpl-2 protooncoprotein activates the nuclear factor of activated T cells and induces interleukin 2 expression in T cell lines - PubMed (original) (raw)

The Tpl-2 protooncoprotein activates the nuclear factor of activated T cells and induces interleukin 2 expression in T cell lines

C Tsatsanis et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Tpl-2 expression is induced within 30-60 min after ConA stimulation of rat splenocytes, suggesting that it may contribute to the induction of IL-2 during T cell activation. Herein we show that wild-type and carboxyl-terminally truncated (activated) Tpl-2 activate the nuclear factor of activated T cells (NFAT) and induce interleukin 2 (IL-2) expression in EL4 cells. In Jurkat cells the truncated Tpl-2 activates NFAT and induces IL-2, whereas wild-type Tpl-2 activates NFAT only when cotransfected with NFAT expression constructs, suggesting that Tpl-2 may induce NFAT activation signals. Experiments in NIH 3T3 cells revealed that the NFATp isoform, but not the NFATc or NFATx isoform, undergoes nuclear translocation when coexpressed with wild-type Tpl-2 and confirmed this hypothesis. Activation of NFAT by anti-CD3 stimulation but not by phorbol 12-myristate 13-acetate and ionomycin in Jurkat cells was inhibited by the kinase-dead Tpl-2K167M, suggesting that Tpl-2 contributes to the transduction of NFAT activation signals originating in the T cell receptor. The Tpl-2-mediated induction of IL-2 was not observed in T cell lymphoma lines other than EL4 and Jurkat, as well as in normal T cells. NFAT activation by Tpl-2, however, was observed in several cell lines including some of nonhematopoietic origin. The activation of NFAT by Tpl-2 in different cell types defines a molecular mechanism that may contribute to its oncogenic potential.

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Figures

Figure 1

Figure 1

Overexpression of Tpl-2 induces IL-2 expression in EL4 and Jurkat cells. (A) IL-2 was measured by ELISA in culture supernatants of three clones of EL4 cells transfected stably with the carboxyl-terminally truncated _Tpl_-2 construct (Tpl-2tr) and two independent clones of EL4 cells transfected stably with the wild-type Tpl-2 construct (Tpl-2wt). Vector (pBabe) transfected cells secrete no IL-2. (B and C) EL4 and Jurkat cells were transiently transfected with Tpl-2 and carboxyl-terminally truncated Tpl-2 expression constructs. Thirty-six hours after transfection, empty vector-transfected control cells were stimulated with PMA plus ionomycin and cultured for an additional 8 h period. Culture supernatants were harvested and IL-2 was measured by ELISA.

Figure 2

Figure 2

Tpl-2 activates the IL-2 promoter: The role of NFAT. (A) Diagrammatic representation of the IL-2 promoter showing the position of previously mapped transcription factor binding sites. (B) Both wild-type and truncated Tpl-2 activate an IL-2 promoter/CAT reporter in EL4 cells. (C) The same reporter is activated by truncated Tpl-2 but not wild-type Tpl-2 in Jurkat cells. (D) Both wild-type and truncated Tpl-2 activate a CAT reporter construct containing a minimal SV40 promoter with three NFAT binding sites upstream, in EL4 cells. The asterisk indicates that the NFAT binding site is mutated and fails to bind NFAT. (E) The same reporter construct is activated by truncated but not wild-type Tpl-2 in Jurkat cells. Reporter constructs containing a mutant NFAT binding site are inactive in both EL4 and Jurkat cells. All transfections were done in quadruplicate and expression was normalized based on the expression of βgal from a cotransfected CMV-β-Gal construct.

Figure 3

Figure 3

Tpl-2 induces NFAT binding activity. EMSA of a 32P-labeled double-stranded oligonucleotide containing an NFAT binding site after incubation with nuclear extracts of Jurkat cells transiently transfected with the indicated constructs or stimulated with PMA and ionomycin.

Figure 4

Figure 4

Tpl-2 collaborates with NFATp and NFATc but not NFATx to activate IL-2/CAT and NFAT/CAT reporters. Jurkat cells were cotransfected with expression constructs of wild-type Tpl-2 and NFATp, NFATc, or NFATx in combination with IL-2/CAT (A) or NFAT/CAT (B) reporter constructs. All transfections were done in quadruplicate and expression was normalized based on the expression of βgal from a cotransfected CMV-β-Gal construct.

Figure 5

Figure 5

Tpl-2 activates NFATp. (A) Relative CAT activity in freeze–thaw lysates of NIH 3T3 cells stably transfected with an SRα-based Tpl-2 construct or with SRα and cotransfected with expression constructs of NFATp, NFATc, or NFATx and the reporter construct NFAT/CAT. All transfections were performed in triplicate and transfection efficiency was monitored by using βgal. (B–D) Subcellular localization of NFATp, NFATc, and NFATx expressed from transiently transfected expression constructs in the NIH 3T3 shown in A. Localization was determined by immunofluorescence using anti-HA (HA-NFATc) or anti-Flag (Flag-NFATp or Flag-NFATx) mouse monoclonal antibodies and an anti-mouse IgG secondary antibody conjugated with fluorescein isothiocyanate. Stained cells were examined by confocal microscopy. (Left) Entire cell. (Right) Confocal image of a slice through the nucleus.

Figure 6

Figure 6

Dominant negative mutant Tpl-2K167M inhibits the activation of NFAT by anti-CD3 but not by PMA plus ionomycin. Relative CAT activity in lysates of TAg-Jurkat cells transiently transfected with the NFAT/CAT reporter construct and carboxyl-terminally truncated or kinase-dead (K167M) Tpl-2 expression constructs is shown. Selected cultures were stimulated with the human anti-CD3 antibody OKT3 or PMA and ionomycin as indicated. All transfections were done in quadruplicate. Similar results were obtained in two independent experiments.

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