Fig. 1. p85β disassociates from the p110α helical domain-mutant protein.

a Schematic of DLD1 isogenic cell lines. WT-FLAG: DLD1 cells with the endogenous wild-type p110α tagged with 3×FLAG; E545K-FLAG: DLD1 cells with the endogenous p110α E545K mutant protein tagged with 3×FLAG; WT-only: DLD1 cells with the PIK3CA E545K allele knocked out; E545K-only: DLD1 cells with the PIK3CA WT allele knocked out. ABD: adaptor-binding domain; RBD: Ras-binding domain; C2: C2 domain; helical: helical domain; kinase: kinase domain. b–d p85β, but not p85α, disassociates from p110α E545K mutant protein. Cell lysates from the p110α E545K or WT FLAG-tagged cells were immunoprecipitated with anti-FLAG antibody-conjugated beads and blotted with indicated antibodies (b). Cell lysates from the indicated cell lines were immunoprecipitated with either an anti-p85β antibody (c) or an anti-p85α antibody (d) and blotted with indicated antibodies. e A schematic diagram of tumor-derived PIK3CA mutations tested for interaction with p85β. f p85β, but not p85α, disassociates from p110α helical domain mutant proteins. The indicated FLAG-tagged p110α constructs were transfected into 293 T cells. Cell lysates were immunoprecipitated by anti-FLAG agarose and then blotted with the indicated antibodies. g, h p85β disassociates from PI3K complexes in PIK3CA helical domain mutant cells. Cell lysates from indicated cell lines were immunoprecipitated with either an anti-p85β antibody (g) or an anti-p110α antibody (h) and blotted with indicated antibodies. i, j The N-terminal domains of p85β cause disassociation from p110α E545K mutant protein. Schematics of p85α, p85β, and two chimeric p85 constructs (i). The indicated HA-tagged p85 constructs were co-transfected with a Flag-tagged construct expressing either WT or E545K mutant p110α. Cell lysates were immunoprecipitated by anti-FLAG agarose and then blotted with an anti-HA antibody. Similar results were reproduced three times.