Subcellular Localization of Prohibitin 1 is Mediated by Akt Phosphorylation at Thr258

Abstract

The overexpression of intratumoral Prohibitin 1 (PHB1), an evolutionarily conserved gene, has been associated with a 5‐year survival rate of less than 35% in muscle‐invasive bladder cancer patients. When localized to the mitochondria, PHB1 promotes tumor cell proliferation by supporting mitochondrial integrity, whereas nuclear PHB1 assists in inducing cell cycle arrest. As the subcellular localization of PHB1 dictates its function, the mechanisms governing the change in PHB1’s subcellular localization could be exploited to develop novel cancer therapeutics. PHB1 has a mitochondrial signal sequence, but previous work suggested that phosphorylation of PHB1 by Akt at Thr258 is required for mitochondrial localization. Our study confirms and expands this work by using immunofluorescence microscopy to visualize the subcellular location of PHB1 in a muscle‐invasive bladder cancer cell line (5637) after the use of Akt inhibitors. Akt inhibition liberated PHB1 from the mitochondria causing a significant increase in nuclear localization. However, blocking Akt disrupts multiple cellular processes, including those involving the function of PHB1, presenting a potentially confounding explanation for these results. To negate the off‐target effects of Akt inhibition, site‐directed mutagenesis was used to create two mutant PHB1 cDNA plasmids. In the first plasmid, Thr258 was mutated into an aspartate, T258D, which mimics Akt phosphorylation. The second mutation created an alanine, T258A, non‐phosphorylatable. Transfection of T258D showed elevated mitochondrial localization of PHB1, while non‐phosphorylatable T258A pooled in the cytoplasm and failed to enter the mitochondria. Therefore, we concluded that phosphorylation of Thr258 by Akt increases the mitochondrial localization of PHB1.Support or Funding InformationR.J. McElroy Trust Student/Faculty Research FundWartburg College Undergraduate Research FundWartburg College Biology DepartmentMany generous donors to the Group Alpha Research Fund