Type and Size of Fluorescent Proteins Modulate the Localization of PTEN and Its Fragments, but Have Little or No Effect on Mutant PTEN or Stressed Cells

The functions of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor, depend on its subcellular localization. At the plasma membrane, PTEN dephosphorylates phosphatidylinositol-3,4,5-triphosphate to inhibit AKT signaling, whereas nuclear PTEN contributes to the maintenance of genomic stability. Fluorescent proteins (FPs) are widely used to assess PTEN's subcellular localization; however, both the intrinsic properties of FPs (e.g., molecular size) and the choice of FP can influence subcellular localization. This study aimed to determine whether FP fusion affects the subcellular localization of PTEN and its mutant forms under conditions involving DNA damage. mCherry typically promotes cytosolic localization of FP-fused PTEN, indicating that FP selection may affect the interpretation of localization data. Furthermore, FP fusion increases the molecular size of the truncated PTEN fragment, which may impede its nuclear import. In comparison, PTEN mutants such as PTEN_K13R or PTEN_A4, which predominantly localize to the cytoplasm or nucleus, respectively, show a minimal dependence on the type of FP. Similarly, DNA damage-induced nuclear accumulation of PTEN appears to be independent of the FP type. These findings underscore the importance of carefully considering the effects of FP fusion when investigating the mechanisms regulating the nuclear translocation of PTEN.