Mutant p53R273H disrupts PDPK1 homodimerization and contributes to PDPK1 activation.

The tumor suppressor p53 protein is frequently mutated in cancer, conferring oncogenic gain-of-function properties. One such mutant, p53R273H, is known to promote the activation of the PI3K/AKT pathway, but the underlying mechanism is unclear. Here, we demonstrated a direct interaction between p53R273H and 3-phosphoinositide-dependent protein kinase 1 (PDPK1), an upstream kinase of AKT. Interestingly, this interaction was unique to p53R273H, as it was not seen with wild-type p53 or other hotspot mutants such as p53R175H and p53R248W. Mechanistically, our in silico analysis revealed that the R273H mutation exposed a hydrophobic region within the p53R273H DNA-binding domain (DBD), facilitating PDPK1 binding. This binding disrupted PDPK1 homodimerization, a known inhibitory mechanism of PDPK1 activity. In contrast, the PDPK1-binding deficient mutant p53F270A/R273H did not disrupt PDPK1 homodimerization. We propose that p53R273H promotes AKT phosphorylation, potentially by enhancing PDPK1 homodimer dissociation. Supporting this, the small molecule PRIMA-1Met disrupted the p53R273H-PDPK1 interaction, resulting in reduced AKT phosphorylation. These findings highlight a new gain-of-function role of p53R273H, suggesting that targeting the p53R273H-PDPK1 interaction with PRIMA-1Met could dampen AKT phosphorylation in p53R273H-expressing cancers.