Fig. 1
From: Phosphorylation at tyrosine 317 and 508 are crucial for PIK3CA/p110α to promote CRC tumorigenesis
p110α protein undergoes tyrosine phosphorylation. A Schematic of isogenic cell lines in which endogenous p110α was tagged with SBP-3×FLAG-6×HIS. ABD: adaptor-binding domain; RBD: Ras-binding domain; C2: C2 domain; helical: helical domain; kinase: kinase domain; SBP: SBP tag; FLAG: 3×FLAG tag; HIS: 6×HIS tag. B-C DLD1-p110α-SFH cells (B) or HCT116-p110α-SFH cells (C) were serum-starved overnight and then treated with or without pervanadate for 30 min. Cell lysates were immunoprecipitated with FLAG agarose beads and immunocomplex were blotted with indicated antibodies. DLD1-p110α-SFH: DLD1 isogenic cells in which endogenous p110α was tagged with SBP-3×FLAG-6×HIS; HCT116-p110α-SFH: HCT116 isogenic cells in which endogenous p110α was tagged with SBP-3×FLAG-6×HIS. D HCT116-p110α-SFH cells were serum-starved overnight and then treated with growth factors, insulin, or pervanadate for indicated time. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and immunocomplex were blotted with the indicated antibodies. FGF: fibroblast growth factor; PDGF: platelet derived growth factor BB; HGF: hepatocyte growth factor; EGF: epidermal growth factor; Per: pervanadate. E Schematic of p110α tyrosine phosphorylation sites. Tyrosine 317 and 508 located in the linker regions RBD-C2 and C2- Helical of p110α respectively. F Mutation at Y317 and/or Y508 reduced tyrosine phosphorylation of p110α. Wild-type or mutant p110α constructs were transfected into 293T cells. Cells were serum-starved overnight and then treated with or without pervanadate for 30 min. Cell lysates were immunoprecipitated with FLAG agarose beads and immunocomplex were blotted with indicated antibodies