Figure 4. Akt interacts with and phosphorylates CSN6. (A) Interaction of Akt with CSN6. Equal amounts of 293T cell lysates transfected with indicated plasmids were immunoprecipitated (IP) with anti-Flag and immunoblotted (IB) with anti-HA. Equal amounts of U2OS cell lysates were also immunoprecipitated with anti-CSN6 and immunoblotted with anti-Akt. (B) Consensus phosphorylation sequence for Akt phosphorylation sites in CSN6. (C) CSN6 is a phosphorylated target of Akt. 293T cells were transfected with the indicated expression vectors in the presence or absence of LY294002. Equal amounts of cell lysates were immunoprecipitated (IP) with anti-Flag antibody and immunoblotted with anti-phosphorylated Akt substrates antibody. (D) Akt phosporylates CSN6 in vitro. Baculovirus-produced active recombinant Akt1 was incubated with indicated equal amounts of bacterially produced and purified recombinant CSN6 and mutant CSN6 (S60A) protein for kinase activity. ³²P autoradiography demonstrated the phosphorylated CSN6 and Coomassie blue-stained SDS-PAGE gel of CSN6 proteins showed comparable amounts of wild-type CSN6 and mutant CSN6 (S60A).