Phosphorylation of p53 at threonine 155 is required for Jab1-mediated nuclear export of p53

Abstract

The Jun activation-domain binding protein 1 (Jab1) induces p53 nuclear export and cytoplasmic degradation, but the underlying mechanism is poorly understood. Here, we show that phosphorylation at the threonine 155 residue is essential for Jab1-mediated p53 nuclear export. Jab1 stimulated phosphorylation of p53 at T155 was inhibited by curcumin, an inhibitor of COP9 signalosome (CSN)-associated kinases. The T155E mutant, which mimics phosphorylated p53, exhibited spontaneous cytoplasmic localization in the absence of Jab1. This process was prevented by leptinomycin B (LMB), but not by curcumin. The substitution of threonine 155 for valine (T155V) abrogated Jab1-mediated p53 nuclear export, indicating that phosphorylation at this site is essential for Jab1-mediated regulation of p53. Although T155E can be localized in the cytoplasm in the absence of Mdm2, the translocation of T155E was significantly enhanced by ectopic Hdm2 expression. Our data suggests that Jab1-mediated phosphorylation of p53 at Thr155 residue mediates nuclear export of p53. [BMB Reports 2017; 50(7): 373-378].

Fig. 1

Curcumin suppressed Jab1-mediated nuclear export of p53. (A) H1299 cells were transfected with the plasmids expressing HA-p53, Myc-Jab1, or both for 24 h and then treated with 50 μM curcumin for 6 h. The cells were analyzed by fluorescence microscopy using anti-HA and Myc antibodies. The cells were counterstained with DAPI to visualize the nuclei. Representative images are shown in the left panel. A total of 200 cells expressing HA-p53 were counted according to their localization, and the results are presented in the right panel (N: Nucleus, N/C: Nucleus and cytoplasm). (B) Nuclear and cytoplasmic fractions of H1299 cells transfected and treated as described in (A) were prepared using an NE-PER extraction kit. Protein levels were determined by western blot (WB) using anti-phospho-p53 (Thr155), HA, or Myc antibodies. HDAC and tubulin were used as loading controls for the nuclear and cytoplasmic proteins, respectively. (C) U2Os cells transfected with the plasmid expressing HA-Jab1 were treated with DMSO or 50 μM curcumin for 6 h. The cells analyzed by fluorescence microscopy as in (A). (D) Nuclear and cytoplasmic fractions of U2OS cells transfected and treated as described in (C) were prepared using NE-PER extraction kit. Protein levels were determined by WB.

Fig. 2

Phosphomimetic mutant p53 (T155E) is mainly localized in the cytoplasm, in a CRM-dependent manner. (A) H1299 cells transfected with the plasmids expressing HA-p53 WT or T155E were analyzed by fluorescence microscopy. Representative images are shown and summarized in the right panel. (B) Nuclear and cytoplasmic fractions of H1299 cells expressing HA-p53 WT and T155 were subjected to WB. (C, D) H1299 cells expressing HA-p53 T155 were treated with 50 μM curcumin for 6 h or 10 nM leptomycin B (LMB) for 12 h. Immunofluorescence (C) and WB (D) analysis were performed as described above. (E) H1299 cells were transfected with the plasmid expressing Jab1 and p53 wild-type (WT) or mutants. Whole cell extracts (WCE) were immunoprecipitated with an anti-Myc antibody, followed by WB using anti-HA and Myc antibody. (F) H1299 cells were transfected as indicated, then treated with or without 50 μM curcumin for 6 h. The cells analyzed by fluorescence microscopy. Representative images are shown in the supplementary Fig. 2.

Fig. 3

The Thr155 residue on p53 are indispensable for Jab1-mediated cytoplasmic localization and degradation. (A) Extracts from H1299 cells transfected as indicated were immunoprecipitated with an anti-Myc antibody, followed by WB using anti-HA and Myc antibody. (B) H1299 cells transfected with the plasmids expressing HA-p53 WT and T155V in the absence and presence of Myc-Jab1. Protein levels were determined by western blot (WB). (C) H1299 cells transfected with the indicated plasmids were analyzed by fluorescence microscopy. Representative images are shown and summarized in the right panel. (D) Nuclear and cytoplasmic fractions of H1299 cells transfected as indicated were subjected to WB.

Fig. 4

Phosphorylation at the Thr155 residue and ubiquitination at the six lysine residues on p53 cooperatively facilitate cytoplasmic localization. (A) H1299 cells transfected with HA-p53 6KR, T155E, or T155E6KR were analyzed by fluorescence microscopy. Representative images are shown and summarized in the right panel. (B) H1299 cells were transfected with the plasmids expressing HA-p53 and Hdm2, and then treated with 50 μM curcumin for 6 h. The cells were analyzed by fluorescence microscopy. Representative images are shown, and summarized in the right panel.