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. Author manuscript; available in PMC: 2023 Jan 11.
Published in final edited form as: Nat Cell Biol. 2022 Jul 7;24(7):1099–1113. doi: 10.1038/s41556-022-00949-1

Figure 7. The interaction of p53 with Akt is PIPn-dependent and mediated by the C-terminus of p53.

Figure 7.

a, p53 co-IP-ed with all three Akt isoforms (Akt1, Akt2, and Akt3). FLAG-tagged wild-type p53 and HA-tagged Akt1/2/3 were co-transfected into HEK293FT cells for 48 h. The ectopically-expressed Akt1/2/3 were IP-ed with HA antibody, and analyzed by WB. The co-IP-ed p53 level was normalized to the level obtained by Akt1 IP. N=3.

b, The interaction of the Akt PH with p53 is regulated by PIPn binding to the Akt PH. FLAG-tagged wild-type p53 and GFP-tagged Akt PH domain (WT or PIPn binding-defective R25C mutant) were co-transfected into HEK293FT cells for 48 h. The ectopically expressed WT or R25C mutant Akt PH domain were IP-ed with GFP antibody, and then analyzed by WB. N=3.

c, The interaction of full-length Akt with p53 is regulated by PIPn-binding to the Akt PH. FLAG-tagged p53 and GFP-tagged full-length Akt (WT or R25C mutant) were co-transfected into HEK293FT cells for 48 h. The ectopically expressed WT or R25C mutant Akt were IP-ed with GFP antibody, and then analyzed by WB. N=3.

d, A schematic representation of p53 truncation mutants used in the study. DB, DNA-binding domain; FL, full length; PRD, proline-rich domain; TAD, transactivation domain; TD, tetramerization domain; CTD, C-terminal regulatory domain. The Akt and Sin1 binding domains identified in (e) are indicated.

e, Flag-tagged p53 truncation mutants were transiently transfected into HEK293FT cells for 48 h. The ectopically expressed p53 was IP-ed with FLAG antibody, and the associated endogenous Sin1 and Akt were analyzed by WB. N=3. Binding was normalized to that of FL p53.

f-g, Fluorescent IP-WB detects on-site PI4,5P2 and PI3,4,5P3 association with p53 that is dependent on its PIPn-binding motif. FLAG-tagged p53 (WT or PIPn-binding defective 6Q or 379Q mutants) were transiently transfected into HEK293FT cells for 48 h. The ectopically expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P2, PI3,4,5P3, and p53 simultaneously. N=3.

h-i, The interaction of Akt with p53 requires the PIPn-binding motif of p53. HA-tagged Akt1 and FLAG-tagged WT p53 or mutant p53 (6Q or 379Q) were co-transfected into HEK293FT cells for 48 h. The ectopically expressed Akt1 was IP-ed with HA antibody, and analyzed by WB. N=3.

For all panels, data are represented as mean ± SD, p value denotes t-test.