Abstract Amino acids (aa) 50-77 of azurin, p28, a 128 aa member of the cupredoxin family of copper containing redox proteins, isolated from the opportunistic pathogen Pseudomonas aeruginosa is a cell penetrating peptide (CPP) that preferentially penetrates a wide variety of human cancer cells. Exposure of p53wt, mut cancer cells to p28 produces a post translational increase in the level of p53, inhibition of the cell cycle at G2/M and apoptosis (Yamada et. al., Mol. Can. Therap., 8:2947-58, 2009). Computer simulation (ClusPro, GROMACS) and domain specific antibodies to p53 suggest p28 binds to motifs that span aa 110–146 in flexible L1 loop and 80 and 276 of the p53 DNA binding domain (DBD), respectively. Western and RT-PCR analyses of human breast cancer, MCF-7 (p53wt), MCF-7 derived MDD2 (p53dom/neg), MDA-MB-231 (p53mut), and melanoma cells, Mel-29 (p53wt), Mel-23 (p53mut) and Mel-6 (p53null) exposed to p28 for 72 hrs showed a significant increase in the levels of p53 and p21 in MCF-7, MDA-MB-231, Mel-29 and Mel-23 cells, while significantly reducing the level of FoxM1 over a 72hr exposure. The level of STMN1 was not altered suggesting the block at G2/M does not involve microtubules. p28 significantly decreased the level of the E3 ligase COP1 in p53wt, mut MCF-7, MDA-MB-231, Mel-29 and Mel-23, but not in p53null Mel-6 cells. In addition, p28 did not significantly decrease the level of E3 ligases TOPORS, Pirh2 or HDM2 which also ubiquitinate p53. The levels of p53, p21, FoxM 1, STMN1, COP1, TOPORS and Pirh2 either remained essentially at control levels (p53, p21) or were elevated (FoxM1, STMN1, COP1, TOPORS, Pirh2) at some point during a 72 hr exposure of p53dom/neg MDD2 cells to p28. In contrast, COP1 has also been reported to negatively regulate cell proliferation in a c-jun-dependent manner and not regulate intracellular levels of p53 (Migliorini et. al., J. Clin. Invest., 121: 1329-43, 2011). The p28 induced decrease in the level of COP1 in p53wt, mut cancer cells was not accompanied by a sustained increase in c-jun, in any cancer cell line, suggesting that COP1 regulates the level of p53 in cancer cells independent of any effect on c-jun. These data also suggest that COP1, but not TOPORS or Pirh2, also binds within this region of p53, and that COP1 is a major regulator of p53 activity. In sum, these data suggest that p28 binds to a specific motif within the p53 DBD where it inhibits COP1 mediated binding to and ubiquitination of p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2870. doi:1538-7445.AM2012-2870