Figure 9.
BDCPs bind to the cytosolic tail of CTLA4. (A) Schematic structure of CTLA4 with the amino acid sequence of the cytosolic tail indicated. Bold residues indicate the YxxM AP1-binding motif with underlined Y201 previously reported as being essential for binding to LRBA. Amino acids shown in red were identified as important for binding to ALFY in the current study. (B) ALFY interacts with aa 201–215 in the cytosolic tail of CTLA4. GST-tagged fragments of CTLA4 immobilized on glutathione-sepharose beads were incubated with lysate of HeLa cells stably transfected with 3xFlag-EGFP-ALFY. Bound 3xFlag-EGFP-ALFY were detected by staining with an anti-flag antibody, while GST-tagged proteins were detected by staining with ponceau S. (C) Purified NBEAL2 and LRBA (right panels) can bind to the cytosolic tail of CTLA4. Purified full-length untagged NBEAL2 or LRBA were incubated with GST-tagged CTLA4 (201–215) immobilized on glutathione-sepharose beads. Bound NBEAL2 or LRBA were detected by staining with anti-NBEAL2 or anti-LRBA antibodies, while GST-tagged proteins were detected by staining with an anti-GST antibody. (D) The C-terminal fragment of NBEAL2 interacts with the cytosolic tail of CTLA4. Purified N- (aa 1–1792) or C- (aa 1792–2794) terminal fragments of NBEAL2 fused to 3xFlagEGFP were incubated with GST-tagged CTLA4 (201–215) immobilized on glutathione-sepharose beads. Bound 3xFlag-EGFP-NBEAL2 was detected with an anti-Flag antibody, while GST-tagged proteins were detected by staining with an anti-GST antibody. (E) The PH-BEACH domains of NBEAL1, NBEAL2, LRBA, and ALFY bind to the cytosolic tail of CTLA4. Purified PH-BEACH domains of NBEAL1, NBEAL2, LRBA, and ALFY fused to MBP were incubated with GST-tagged CTLA4 (201–215) immobilized on glutathione-sepharose beads. Bound MBP-fusion proteins were detected with an anti-MBP antibody, while GST-tagged proteins were detected by staining with an anti-GST antibody. (F) Alanine scan mutagenesis of the ALFY-binding region of CTLA4 (aa 201–215). GST-CTLA4 (201–215) point mutants were immobilized on glutathione-sepharose beads and incubated with HeLa 3xFlag-EGFP-ALFY cell lysate. Bound 3xFlag-EGFP-ALFY were detected by staining with an anti-flag antibody, while GST-tagged proteins were detected by staining with an anti-GST antibody. (G) Quantification of data in F from three independent experiments. Values shown are mean ± SD ***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA. (H) Binding of several BDCPs to the cytosolic tail of CTLA4. Wild type or mutant GST-CTLA4(201–215) were immobilized on glutathione-sepharose beads and incubated with lysate from HeLa cells overexpressing 3xFlag-EGFP-NBEAL2, - LYST or -LRBA. Bound proteins were detected by staining with an anti-flag antibody, while GST-tagged proteins were detected by staining with an anti-GST antibody. Source data are available for this figure: SourceData F9.

BDCPs bind to the cytosolic tail of CTLA4. (A) Schematic structure of CTLA4 with the amino acid sequence of the cytosolic tail indicated. Bold residues indicate the YxxM AP1-binding motif with underlined Y201 previously reported as being essential for binding to LRBA. Amino acids shown in red were identified as important for binding to ALFY in the current study. (B) ALFY interacts with aa 201–215 in the cytosolic tail of CTLA4. GST-tagged fragments of CTLA4 immobilized on glutathione-sepharose beads were incubated with lysate of HeLa cells stably transfected with 3xFlag-EGFP-ALFY. Bound 3xFlag-EGFP-ALFY were detected by staining with an anti-flag antibody, while GST-tagged proteins were detected by staining with ponceau S. (C) Purified NBEAL2 and LRBA (right panels) can bind to the cytosolic tail of CTLA4. Purified full-length untagged NBEAL2 or LRBA were incubated with GST-tagged CTLA4 (201–215) immobilized on glutathione-sepharose beads. Bound NBEAL2 or LRBA were detected by staining with anti-NBEAL2 or anti-LRBA antibodies, while GST-tagged proteins were detected by staining with an anti-GST antibody. (D) The C-terminal fragment of NBEAL2 interacts with the cytosolic tail of CTLA4. Purified N- (aa 1–1792) or C- (aa 1792–2794) terminal fragments of NBEAL2 fused to 3xFlagEGFP were incubated with GST-tagged CTLA4 (201–215) immobilized on glutathione-sepharose beads. Bound 3xFlag-EGFP-NBEAL2 was detected with an anti-Flag antibody, while GST-tagged proteins were detected by staining with an anti-GST antibody. (E) The PH-BEACH domains of NBEAL1, NBEAL2, LRBA, and ALFY bind to the cytosolic tail of CTLA4. Purified PH-BEACH domains of NBEAL1, NBEAL2, LRBA, and ALFY fused to MBP were incubated with GST-tagged CTLA4 (201–215) immobilized on glutathione-sepharose beads. Bound MBP-fusion proteins were detected with an anti-MBP antibody, while GST-tagged proteins were detected by staining with an anti-GST antibody. (F) Alanine scan mutagenesis of the ALFY-binding region of CTLA4 (aa 201–215). GST-CTLA4 (201–215) point mutants were immobilized on glutathione-sepharose beads and incubated with HeLa 3xFlag-EGFP-ALFY cell lysate. Bound 3xFlag-EGFP-ALFY were detected by staining with an anti-flag antibody, while GST-tagged proteins were detected by staining with an anti-GST antibody. (G) Quantification of data in F from three independent experiments. Values shown are mean ± SD ***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA. (H) Binding of several BDCPs to the cytosolic tail of CTLA4. Wild type or mutant GST-CTLA4(201–215) were immobilized on glutathione-sepharose beads and incubated with lysate from HeLa cells overexpressing 3xFlag-EGFP-NBEAL2, - LYST or -LRBA. Bound proteins were detected by staining with an anti-flag antibody, while GST-tagged proteins were detected by staining with an anti-GST antibody. Source data are available for this figure: SourceData F9.

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