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An absolute requirement for Fyn in T cell receptor-induced caspase activation and apoptosis ¹

JEAN-EHRLAND RICCI, VALÉRIE LANG^*, FRÉDERIC LUCIANO, NATHALIE BELHACENE, VALÉRIE GIORDANENGO, FRÉDÉRIQUE MICHEL, GEORGES BISMUTH^* and PATRICK AUBERGER²

INSERM U526 Activation des Cellules Hématopoïétiques, Physiopathologie de la Survie et de la Mort Cellulaire et Infections Virales, Equipe Labellisée Ligue Nationale contre le Cancer, IFR 50, 06107 Nice Cédex 2, France;
^* Laboratoire d’Immunologie Cellulaire, CNRS UMR 7627, Centre Hospitalier Pitié-Salpètrière, 75013 Paris, France; and
Laboratoire d’Immunologie Moléculaire, Département d’Immunologie, Institut Pasteur, Paris, France

²Correspondence: INSERM U526 Activation des Cellules Hématopoïétiques, Physiopathologie de la Survie et de la Mort Cellulaire et Infections Virales, Equipe Labellisée Ligue Nationale contre le Cancer, IFR 50, 28 Ave. de Valombrose, 06107 Nice Cédex 2, France. E-mail: auberger{at}unice.fr

SPECIFIC AIM

We investigated the possible implication of the tyrosine kinase p59Fyn in TCR-mediated caspase activation and apoptosis in a T cell hybridoma (T8.1 cells) overexpressing different Fyn constructs and in mouse embryo fibroblasts (MEFs) and thymocytes from control or Fyn knockout mice.

PRINCIPAL FINDINGS

1. Cleavage of Fyn upon TCR triggering in T lymphocytes
We first analyzed the cleavage of different molecular forms of Fyn stably expressed in a T cell hybridoma, T8.1. Soluble anti-CD3 mAb failed to induce the cleavage of WT Fyn, but immobilized anti-CD3 was found to generate the cleaved p57 form of the kinase (Fig. 1A ). Cleavage of Fyn was clearly detectable within 4 h of anti-CD3 treatment and was found to be maximal at 8 h (Fig. 1B ). Conversely, a kinase-dead mutant (Fyn KD) and a soluble form of Fyn (Fyn N-ter) were not cleaved after CD3 triggering. Cleavage of Fyn upon TCR engagement, which is reminiscent of our previous observation that Fas induces Fyn cleavage in T lymphocytes, prompted us to investigate the implication of caspases in this process. Intact T8.1 cells overexpressing the WT Fyn kinase were preincubated for 24 h with either Ac-DEVD-CHO or Z-VAD-fmk and then treated for 8 h with coated anti-CD3 mAb. Ac-DEVD-CHO and Z-VAD-fmk were both found to abrogate anti-CD3-induced Fyn cleavage, indicating the involvement of caspases in this process (Fig. 1C ). Consistently coated anti-CD3 mAb induced caspase activity in T8.1 cells overexpressing the WT kinase an effect, prevented completely by preincubation with the caspase inhibitors (Fig. 1D ).

View larger version (19K): [in this window] [in a new window]   Figure 1. Cleavage of p59Fyn upon TCR triggering requires activation of caspases. A) Murine T cell hybridoma stably transfected with Fyn WT or Fyn KD (kinase-dead) were incubated for 4 h with soluble anti-CD3 mAb (2 µg/ml, S) or coated anti-CD3 (cross-linked CL, 50 µg/ml, 2 h, 37°C). Proteins from cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to immobilon membranes for Western blotting with specific anti-Fyn antibody. B) T8.1 cells stably transfected with Fyn WT, Fyn KD, or Fyn N-ter (a soluble form of Fyn lacking the 6 amino-terminal amino acids) were incubated with cross-linked anti-CD3 for various times at 37°C. Western blotting conditions were those of panels A, C, D. T8.1 cells expressing Fyn WT were preincubated for 24 h with 200 µM Ac-DEVD-CHO or 100 µM z-VAD-fmk and incubated for 4 h with cross-linked anti-CD3. Fyn cleavage was detected by Western blot. Caspase activity was assessed using Ac-DEVD-pNA (0.2 mM) as substrate. Caspase activity is expressed in nmol · min-1 · mg-1 of protein.

2. Fyn is required for TCR-induced caspase activation
T cell hybridomas were stimulated with either soluble anti-CD3 mAb or coated anti-CD3 mAb. Soluble anti-CD3 mAb failed to stimulate caspase activity whatever the T8.1 clone tested. However, immobilized anti-CD3 mAb was found to induce caspase activation in T8.1 parental cells. A 100% increase of anti-CD3-induced caspase activation was found in T8.1 cells overexpressing WT Fyn. Caspase activity was virtually undetectable in anti-CD3-treated T8.1 cells overexpressing a kinase-dead or an unanchored (soluble) form of Fyn (Fyn N-ter). These results strongly suggest that Fyn is involved in caspase activation upon TCR triggering in T8.1 cells. To better define the involvement of Fyn in this process, we performed detailed kinetics of caspase activation after TCR triggering. In parental T8.1 cells and cells overexpressing Fyn WT, an increase in caspase activity was detected 4 h after anti-CD3 stimulation that peaked at 8 h, then declined to the basal level after 24 h. However, again the rise in caspase activity was significantly higher in Fyn WT transfected cells compared with their parental counterpart. Long-term kinetics also evidenced a drastic inhibition of anti-CD3 mAb-induced caspase activity in T8.1 cells overexpressing Fyn KD or Fyn N-ter.

3. Lack of apoptosis in Fyn KD and Fyn-ter transfected T cell clones
We sought to determine whether anti-CD3 mAb-induced caspase activation could lead to DNA fragmentation in T8.1 cells. DNA fragmentation was detected 6 to 8 h after anti-CD3 mAb stimulation of parental or Fyn-WT T8.1 clones. There was a significant increase in DNA fragmentation in T8.1 cells overexpressing Fyn WT compared with the parental cell line. Very weak DNA fragmentation was observed in T8.1 cells overexpressing Fyn N-ter after 8 h stimulation with immobilized anti-CD3 mAb whereas T8.1 cells overexpressing Fyn KD exhibited no trace of internucleosomal DNA degradation for the same period.

4. Impaired apoptosis in mouse embryo fibroblasts and thymocytes from Fyn KO mice
We then evaluated the rate of apoptosis in thymocytes prepared from Fyn^+/+ or Fyn^-/- mice. As shown in Fig. 2A , spontaneous DNA fragmentation that normally occurs upon incubation of thymocytes at 37°C (i.e., in the absence of anti-CD3 mAb) was reduced at 6 h in thymocytes from Fyn knockout mice compared with thymocytes from control mice (Fig. 2A ). Anti-CD3 mAb increased significantly DNA fragmentation in thymocytes from control mice. Thymocytes from Fyn^-/- mice were less susceptible to anti-CD3-mediated DNA fragmentation than thymocytes from the control littermate. This was particularly visible at 2 h (Fig. 2A ). Finally, quantitative analysis of DNA fragmentation shown in Fig. 2B clearly indicates that anti-CD3-mediated apoptosis was drastically inhibited in thymocytes from Fyn knockout mice. Consistently, we observed a 50–80% diminution in basal and anti-CD3-mediated caspase activity in thymocytes from Fyn^-/- mice compared with thymocytes from the control littermate (Fig. 2C ). These data support an important role for p59Fyn in the induction of caspase activation and apoptosis in T lymphocytes.

View larger version (42K): [in this window] [in a new window]   Figure 2. Impaired caspase activation and apoptosis in thymocytes from Fyn KO mice. Thymocytes from control (CTL) or Fyn-/- mice were incubated or not for various times at 37°C with coated anti-CD3 mAb. A) T8.1 cells overexpressing different form of Fyn were incubated for various times at 37°C with coated anti-CD3 mAb. Internucleosomal DNA fragmentation was visualized after agarose gel electrophoresis. B) Quantitative analysis of DNA fragmentation is expressed as a percent of the maximal response. C) Under each condition, caspase activity was determined as described previously. The results of two independent experiments are shown for Fyn knockout mice.

CONCLUSIONS

The mechanisms by which TCR cross-linking leads to caspase activation in T lymphocytes remain unknown. In the present study, we addressed the possible involvement of the tyrosine kinase Fyn in TCR-mediated activation of caspases. Using stable transfectants expressing different constructs of Fyn, we show that 1) Fyn is selectively cleaved by caspases after T cell receptor triggering and 2) a Fyn that is both active and membrane-anchored is absolutely required for TCR-induced caspase activation and apoptosis in a murine T cell hybridoma. Evidence that anti-CD3-mAb-induced Fyn cleavage is executed by caspase was further supported by its inhibition by Z-VAD-fmk and Ac-DEVD-CHO, two potent and selective caspase inhibitors.

T cells express two members of the Src kinase family, Lck and Fyn, both of which have been implicated in TCR signaling. p56Lck is critical for T lymphocyte development whereas Fyn^-/- thymocytes appear normal even though they have a diminished response to TCR stimulation. Accordingly, we found that basal and anti-CD3 stimulated caspase activation was significantly reduced in thymocytes from Fyn knockout mice.

Here we show biochemical and cellular evidence that Fyn is capable of mediating caspase activation and apoptosis in a murine T cell hybridoma. This effect required both an active and a plasma membrane-anchored Fyn upon TCR triggering, Fyn is activated and could target to the plasma membrane one or more substrates implicated in the regulation of caspase activation. Once activated, caspases could then cleave Fyn after D19, a mechanism that leads to its relocation from the membrane to the cytosol. The inhibitory effect of a soluble and active form of Fyn (Fyn N) upon TCR-induced caspase activation and apoptosis could be explained by the titration in the cytosol of the substrate(s) implicated in this process (Fig. 3 ). The nature of the potential Fyn interactor(s) is unknown at this time. However, Fyn has been shown to interact with a wide range of proteins involved in T cell receptor signaling including ZAP-70, Cbl, Shc, FYB through SH2 and/or SH3 domain interactions, and with Fas and PKC.

View larger version (16K): [in this window] [in a new window]   Figure 3. A schematic model of the potential implication of Fyn in TCR-induced caspase activation and apoptosis. Fyn is activated upon TCR triggering and could target to the plasma membrane one or many substrates implicated in the regulation of caspase activation. Once activated, caspases could then cleave Fyn by a mechanism that leads to its relocation from the membrane to the cytoplasm. The inhibitory effect of a soluble and active form of Fyn (Fyn N, which is equivalent to the cleaved form of Fyn) upon TCR-induced caspase activation and apoptosis, could be explained by the titration and sequestration in the cytoplasm of the substrates involved in this process.

In conclusion, we have shown that p59Fyn activity and localization are both required for caspase activation and apoptosis after TCR engagement. Caspase activation then induces Fyn cleavage and relocation of the active p57 cleaved form of Fyn into the cytoplasm, where it could bind molecule(s) important for T cell receptor signaling, thus initiating a feedback mechanism that helps to block the TCR-induced apoptotic signal. Finally, this study highlights the role of Fyn in TCR-mediated caspase activation and, more generally, in apoptosis in T cells.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0665fje ; to cite this article, use FASEB J. (June 27, 2001) 10.1096/fj.00-0665fje

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