Figure 4: Identification of an LxxLL-like motif in p85α and its interaction with the coactivator-binding groove in tRXRα. | Nature Communications

Figure 4: Identification of an LxxLL-like motif in p85α and its interaction with the coactivator-binding groove in tRXRα.

From: Modulation of nongenomic activation of PI3K signalling by tetramerization of N-terminally-cleaved RXRα

Figure 4

(a) Schematic representations of p85α and mutants. SH3, BCR, nSH2, iSH2, cSH2 domains in p85α and the location of the BCR-LxxLL motif are indicated. BCR and mutant peptides are shown. (b) Interaction of tRXRα with p85α mutants. Myc-tRXRα expression vector was transfected into HEK293T cells along or together with p85α mutant tagged with HA epitope, and their interaction was analysed by coIP using anti-Myc antibody. (c) BCR domain interaction with RXRα mutants. HA-p85α-BCR was cotransfected with Myc-tagged RXRα mutants into HEK293T cells, and their interaction was analysed by coIP using anti-HA antibody. (d) The BCR-LxxLL peptide (structure was extracted from the BCR crystal structure of PDB code 1PBW) docks to the coactivator-binding groove on RXRα (PDB code 3FUG). (e) SPR analysis of BCR-peptide binding to RXRα-LBD in the presence of 9-cis-RA. (f) Enhancing effect of 9-cis-RA on p85α-BCR interaction with RXRα-LBD and tRXRα. Cells transfected with Myc-tRXRα or Myc-RXRα-LBD together with HA-p85α-BCR were treated with 9-cis-RA (10−7M), and analysed by coIP. One of two similar experiments is shown. (g) Inhibitory effect of LxxLL-containing BCR peptide on RXRα-LBD interaction with p85α-BCR. HEK293T cells transfected with HA-p85α-BCR and Myc-RXRα-LBD were exposed to the indicated peptide and compound for 12 h. Cell lysates were prepared and analysed by coIP. (h) Mutation of the LxxLL motif in BCR impairs its interaction with RXRα-LBD. BCR and BCR-LxxLL mutant were tagged with Flag epitope and transfected into A549 cells with Myc-RXRα-LBD. Cell lysates were prepared and analysed for interaction of BCR and its mutant with RXRα-LBD by coIP. (i) Trp305 is essential for RXRα-LBD binding to p85α-BCR. Myc-tagged RXRα-LBD or RXRα-LBD/W305Q was transfected together with HA-p85α-BCR into HEK293T cells in the presence or absence of 9-cis-RA (10−7M). Cell lysates were prepared and analysed by coIP. For western blotting, one of three or four similar experiments is shown.

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