DataSheet_1_Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction.docx

BackgroundPhosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion.ResultsHerein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity.ConclusionWe discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response.</p

DGKα mediates SDF-1α-induced cell invasion by regulating aPKCs recruitment to cell pseudopods.

<p>A) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium, transfected with CTRL or DGKα –specific siRNA and cultured for further 20 hours in serum free medium. Cells were then stimulated for 6 hours with 50 ng/ml SDF-1α, fixed, and stained for actin (red) and aPKCs (green). Arrowhead indicates aPKCs at protrusions. Scale bar 24 µm. B) Histogram reports the percentage of cells displaying aPKCs at protrusions as mean ± SE of 3 independent experiments with **t-test p<0.005, ***t-test p<0.0005. C) MDA-MB-231 cells were transfected with CTRL or DGKα –specific siRNA and lysed. The efficiency of DGKα down–regulation by siRNA was verified at 48 hours after transfection by western blot, tubulin was used as loading control. D) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium and cultured for further 20 hours in serum free medium. Cells were then stimulated for 6 hours with 50 ng/ml SDF-1α, in presence or in absence of 1 µM R59949, fixed and stained for actin (red) and aPKCs (green). Arrowheads indicate aPKCs at protrusions. Scale bar 24 µm. E) Histogram reports the percentage of cells displaying aPKCs at protrusions as mean ± SE of 3 independent experiments with ***t-test p<0.0005. F) MDA-MB-231 cells (10⁶/well) were plated on matrigel invasion chamber and stimulates for 24 hours with SDF-1α (50 ng/ml) in presence or absence of PKCζ pseudosubstrate (PS-PKCζ, 10 µM). Histogram reports mean ± SE of folds over control values from 3 independent experiments with *t-test p<0.05.</p

DGKα and aPKCs mediates MMP-9 secretion and localization at protrusions.

<p>A) MDA-MB-231 cells were transfected with CTRL or DGKα –specific siRNA and shifted to serum free media. After 24 hours cells were treated with 100 ng/ml SDF-1α in serum free medium for further 20 hours. MMP9 content in the supernatants was measured by ELISA assay, histogram reports secreted MMP-9 as mean ± SE of 3 independent experiments normalized for control, with *t-test p<0.05. B) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium, transfected with CTRL or DGKα –specific siRNA and cultured for further 20 hours in serum free medium. Cells were stimulated for 6 hours with 50 ng/ml SDF-1α, fixed and stained for actin (red) and MMP-9 (green). Arrowhead indicates MMP-9 at protrusions. Scale bar 24 µm. C) Histogram reports the percentage of cells displaying MMP-9 at protrusions as mean ± SE of 3 independent experiments with ***t-test p<0.0005. D) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium and cultured for further 20 hours serum free medium. Cells were stimulated for 6 hours with 50 ng/ml SDF-1α, in presence or in absence of 1 µM R59949, fixed and stained for actin (red) and MMP-9 (green). Arrowhead indicates MMP-9 at protrusions. Scale bar 24 µm. E) Histogram reports the percentage of cells displaying MMP-9 at protrusions as mean ± SE of 3 independent experiments with **t-test p<0.005, ***t-test p<0.01. F) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium, transfected with CTRL or PKCζ/ι –specific siRNA and cultured for further 20 hours in serum free medium. Cells were then stimulated for 6 hours with 50 ng/ml SDF-1α, fixed and stained for actin (red) and MMP-9 (green). Arrowhead indicates MMP-9 at protrusions. Scale bar 24 µm. G) Histogram reports the percentage of cells displaying MMP-9 at protrusions as mean ± SE of 3 independent experiments with *t-test p<0.05, **t-test p<0.005.</p

DGKα is necessary for SDF-1α-induced cell invasion.

<p>MDA-MB-231 cells were infected with lentiviral vectors expressing an inducible shRNA against DGKα (shRNA-DGKα1) or an inducible control shRNA (shRNA-CTRL). Parental and infected cells were treated with 1 µg/ml doxycycline for 72 hours to promote shRNA transcription. A) 50,000 cells were plated on matrigel invasion chamber and incubated for 24 hours in presence or in absence of SDF-1α (100 ng/ml). Histogram reports mean ± SE of fold over control values from 3 independent experiments with *t-test p<0.05, **t-test p<0.01. B) The efficiency of DGKα down–regulation by shRNA was verified by quantitative RT-PCR. **t-test p<0.01. A) Cells were lysed and the efficiency of DGKα down–regulation by shRNA was verified by western blot, tubulin was used as a loading control.</p

SDF-1α stimulates DGKα activity and localization at protrusions site.

<p>A) MDA-MB-231 cells, stably expressing myc-DGKα, were plated on matrigel-coated coverslips for 20 hours in FCS containing medium and cultured for further 20 hours in serum free medium. Cells were then stimulated with 50 ng/ml of SDF-1α for the indicated times, fixed and stained for actin (red) and myc-DGKα (green). Representative images at 4 hours after stimulation. Arrowheads indicate DGKα at protrusions. Histogram (B) reports the percentage of cells displaying myc-DGKα at protrusion as mean ± SE of 5 independent experiments, *t-test p<0.05, **t-test p<0.005. Scale bar 24 µm. C) MDA-MB-231 cells were infected with a lentiviral vector expressing inducible OST-tagged DGKα or an empty vector. To induce DGKα expression, cells were treated overnight with doxycycline (1 µg/ml) in serum free medium. Cell were homogenized with buffer B in absence of detergent and analysed for DGK activity (upper panel). Values are mean ± SE of 4 independent experiments with *t-test p<0.05. OST-DGKα and actin protein expression was verified by anti-OST and anti-actin western blot (lower panel).</p

DGKα and aPKCs mediate SDF-1α-induced recruitment of β1 integrin to pseudopods.

<p>A) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium, transfected with CTRL or DGKα–specific siRNA and cultured for further 20 hours in serum free medium. Cells were then stimulated for 6 hours with 50 ng/ml SDF-1α, fixed and stained for actin (red) and β1 integrin (green). Arrows indicate β1 integrin at protrusions. Scale bar 24 µm. B) Histogram reports the percentage of cells displaying β1 integrin at protrusions as mean ± SE values of 3 independent experiments with **t-test p<0.005. C) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium and cultured for further 20 hours in serum free medium. Cells were then stimulated for 6 hours with 50 ng/ml SDF-1α, in presence or in absence of 1 µM R59949, fixed and stained for actin (red) and β1 integrin (green). Arrow indicates β1 integrin at protrusions. Scale bar 24 µm. D) Histogram reports the percentage of cells displaying β1 integrin at protrusions as mean ± SE of 3 independent experiments with *t-test p<0.05, **t-test p<0.005. E) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium, transfected with CTRL or PKCζ/ι –specific siRNA and cultured for further 20 hours in serum free medium. Cells were then stimulated for 6 hours with 50 ng/ml SDF-1α, fixed and stained for actin (red) and β1 integrin (green). Arrowheads indicate β1 integrin at protrusions. Scale bar 24 µm. F) Histogram reports the percentage of cells displaying β1 integrin at protrusions as mean ± SE of 3 independent experiments with **t-test p<0.005. G) MDA-MB-231 cells were transfected with CTRL and PKCζ/ι –specific siRNA and lysed. The efficiency of PKCζ/ι down–regulation by siRNA was verified by western bloting, tubulin was used as a loading control. H) MDA-MB-231 cells were infected with lentiviral vectors expressing a shRNA against β1-integrin (shRNA-β1) or a control sequence (shRNA-CTRL). 50,000 cells were plated on matrigel invasion chamber and incubated for 24 hours in presence or in absence of SDF-1α (100 ng/ml). Histogram reports mean ± SE of fold over control values from 3 independent experiments with *t-test p<0.05, **t-test p<0.01. I) The efficiency of β1-integrin down–regulation by shRNA was verified by quantitative RT-PCR.</p

DGKα overexpression promotes a PKC-dependent cell elongation.

<p>MDA-MB-231 cells were infected with lentiviral vector expressing inducible OST-tagged DGKα or an empty vector. To induce DGKα expression, cells were treated overnight with doxycycline (1 µg/ml) in serum free medium. A) After cell lysis OST-DGKα induction was verified by western blotting with an antibody recognizing the OST-tag, while the extent of overexpression was verified with anti DGKα antibodies. Tubulin was used as loading control. B) Phase contrast images of control and OST-DGKα cells cultured in presence or absence of doxycycline. Arrows indicate cells with long protrusions. Scale bar 50 µm. C) Confocal images of doxycycline induced cells showing OST-DGKα localization, cells were stained for actin (red) and OST (green). Scale bar 24 µm. D) Time course of cell elongation at 2, 10 and 18 hours with or without doxycycline treatment. Time lapse videos were recorded and total cell length measured. Box and whiskers plots (black lines show median, whiskers: 5–95 percentile) of data from 3 independent experiments are shown, ***p<0.0001, 1 way ANOVA. E) MDA-MB-231 cells expressing OST-DGKα were transiently transfected with control or PKCζ/ι-specific siRNA. After 48 hours DGKα expression was induced by overnight treatment with doxycycline (1 µg/ml) in serum free medium. Images were acquired with a phase contrast microscope, representative images are shown. Scale bar 50 µm. Total cell length was measured for at least 100 cells and reported as box and whiskers plot. F) MDA-MB-231 cells expressing OST-DGKα were induced by overnight treatment with doxycycline (1 µg/ml) in serum free medium with or without NSC23766 (100 µM). Images were acquired with a phase contrast microscope, representative images are shown. Scale bar 50 µm. Total cell length was measured for at least 100 cells and reported as box and whiskers plot. MDA-MB-231 cells were transfected with CTRL and PKCζ/ι –specific siRNA and lysed. The efficiency of PKCζ/ι down–regulation by siRNA was verified by western blotting, tubulin was used as a loading control.</p