The Fas/FasL system plays a critical role in death by apoptosis and immune escape of cancer cells. The Fas receptor being ubiquitously expressed in tissues, its apoptotic-inducing function, initiated upon FasL binding, is tightly regulated by several negative regulatory mechanisms to prevent inappropriate cell death. One of them, involving the non-receptor tyrosine kinase Btk, was reported mainly in B cells and only poorly described. We report here that Btk negatively regulates, through its tyrosine kinase activity, the FasL-mediated cell death in epithelial cell lines from colon cancer origin. More importantly, we show that Btk interacts not only with Fas but also with the phosphatidylinositol-4-phosphate 5-kinase, PIP5K1γ, which, upon stimulation by Fas ligand, is responsible of a rapid and transient synthesis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). This production requires both the presence and the tyrosine kinase activity of Btk, and participates in the negative regulation of FasL-mediated cell death since knocking down PIP5K1γ expression significantly strengthens the apoptotic signal upon FasL engagement. Altogether, our data demonstrate the cooperative role of Btk and PIP5K1γ in a FasL-induced PI(4,5)P2 production, both proteins participating to the threshold setting of FasL-induced apoptotic commitment in colorectal cell lines.
Fas Btk PIP5K1γ PI(4,5)P2Cell death
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We are grateful to Bernard Payrastre for discussion and input at the initial phase of this work, to Marcel Deckert for providing us the mouse Btk expression vector and to Sébastien Huault for technical assistance. The authors acknowledge Agnès Loubat (Plateforme de Cytométrie Cyan, iBV, Université Côte d’Azur) for advises and assistance.
This work was supported by institutional funds from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Medicale (Inserm) and by grants from the Institut National du Cancer (INCa) (Grant No. 2009-197) and the Agence Nationale de la Recherche (ANR) (Grant No. ANR-10-BLAN-1226; LABEX SIGNALIFE program Grant No. ANR-11-LABX-0028-01).
Supplementary Figure 1—(a) Efficient Btk knock down was assessed by immunoblot in the indicated cell lines. (b) HCT15, HT29 and SW48 cells transfected with Btk si RNA were exposed to indicated doses of TRAIL crosslinked with M2 (1 ng/ml) and the percentage of cell death was determined by flow cytometry quantification of sub-G1 particles. (c) SW48, HT29 and HCT15 cells pre-treated for 2 hours with DMSO or Ibrutinib (1 and 3 µM) were incubated for 5 hours with indicated doses of FasL or TRAIL crosslinked with M2 (1 µg/ml). The percentage of cell death was assessed as in b. (TIFF 1192 KB)
Supplementary Figure 2—(a) Efficient PIP5K1γ knock down was assessed by immunoblot in the indicated cell lines. (b) HCT15 and HT29 cells transfected with PIP5K1γ si RNA were exposed to indicated doses of TRAIL crosslinked with M2 (1 µg/ml) and the percentage of cell death was determined by flow cytometry quantification of sub-G1 particles. (TIFF 854 KB)
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