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. 2014 Apr 16;5(10):3197–3209. doi: 10.18632/oncotarget.1900

Figure 1. HIP-55 interacts with 14-3-3 through S269 and T291.

Figure 1

(A) Interaction of HIP-55 with isoforms of 14-3-3 proteins. HEK293 cells were co-transfected with each of the seven different 14-3-3 isoforms and HIP-55, lysed after 48 h, and subjected to His pull-down assays. Bound proteins were examined by Western blot analysis. (B) The HIP-55/14-3-3 association is mediated by the amphipathic groove of 14-3-3 (R18 competition). HEK293 cells were co-transfected with HIP-55 and 14-3-3τ. After 36 h, the cells were harvested and incubated for 30 min at 4°C with DMSO alone (−) or R18 (+). Cell lysates were subjected to His pull-down, followed by Western blot analysis with the indicated antibodies. (C) The presence of general phosphatase inhibitors preserves the HIP-55/14-3-3 association. Lysates from HEK293 cells co-expressing His-14-3-3τ and HIP-55 were incubated with or without phosphatase inhibitors for 0.5 h at room temperature, followed by Western blot analysis to detect GST-HIP-55 in the His-14-3-3τ complexes. (D) Inhibition of Ser/Thr phosphatases, PP1 and PP2A, by Calyculin A enhances HIP-55/14-3-3 binding. HEK293 cell lysates co-expressing His-14-3-3τ and HIP-55 were incubated with or without Calyculin A for 0.5 h at room temperature, followed by Western blot analysis with the indicated antibodies. (E) Ser269 and Thr291 of HIP-55 are required for 14-3-3 binding. HEK293 cells were co-transfected with His-14-3-3τ and the indicated GST-HIP-55 vectors. His-14-3-3τ complexes were pulled-down from cell lysates and GST-HIP-55 variants were detected by Western blot. Mutation of either S269 or T291 of HIP-55 decreased 14-3-3 binding. (F) Double mutants S269/T291AA and DD of HIP-55 completely prevent the binding of 14-3-3.