Figure 5

Akt-dependent oligomerization of TopBP1. (A) HEK293 cells were transfected with FLAG-TopBP1 along with Myc-TopBP1 or Myc-TopBP1(S1159A) (Myc-S1159A) as well as Akt plasmids. The lysates were then immunoprecipitated with anti-FLAG beads followed by immunoblotting as indicated. The overexpression of TopBP1 and dn-Akt in the input lysates is shown in lower panels. The ca-Akt, HA-myr(4–129) Akt, cannot be detected by this Akt antibody, which recognizes the N-terminus of Akt, but can be detected by HA antibody (Figure 2C and data not shown). Transfection of ca-Akt may lead to increasing expression of endogenous Akt in some experiments. (B) HEK293 cells were transfected with FLAG-BRCT678 along with Myc-TopBP1 or Myc-TopBP1(S1159A) (Myc-S1159A) as well as Akt plasmids. The immunoprecipitation by anti-FLAG beads was then carried out as in panel A. (C) HEK293 cells were transfected with FLAG-BRCT78 and Myc-BRCT78 or their S1159A mutant counterparts, or with dn-Akt. The lysates were then immunoprecipitated with anti-FLAG beads followed by immunoblotting. The expression of Myc-BRCT78 or S1159A mutant was confirmed by immunoprecipitation and immunoblotting with anti-Myc (middle panels). Phosphorylation of Ser¹¹⁵⁹ in Myc-BRCT78 was detected by phospho-Akt substrate antibody. Total lysates were also immunoblotted directly to detect the expression of HA-Akt (lower panel). (D) HEK293 cells were transfected with FLAG-TopBP1 and Myc-TopBP1 or FLAG-TopBP178 (FLAG-78) and Myc-TopBP178 (Myc-78). The lysates were then immunoprecipitated with anti-FLAG beads followed by immunoblotting with Myc antibody (upper panel) and FLAG antibody (middle panel) or immunoprecipitated with anti-Myc beads followed by immunoblotting with Myc antibody (lower panel). (E) Akt phosphorylation induces self-association of TopBP1 in vitro. Purified TopBP1 was incubated with GST-BRCT78. Some TopBP1 or GST-BRCT78 was phosphorylated with recombinant Akt kniase as indicated before incubation. S1159A mutant (A) of TopBP1 or BRCT78 serves as a control for its wild-type (wt) counterpart. GST pulldown with glutathione-Sepharose was then performed and probed with TopBP1 antibody to detect the interaction between full-length TopBP1 and BRCT78. GST-BRCT78 was visualized by Ponceau S staining.