(A) HEK293T cells were transfected with plasmids expressing GST-PTEN or individual deletion fragments, as indicated, for 24 h. interacts with Rad52 and colocalizes with Rad52, as well as H2AX, after genotoxic stress. Moreover, BMS-794833 PTEN is involved in regulating Rad52 sumoylation. Combined, our studies strongly suggest that nuclear/chromatin PTEN mediates DNA damage repair through interacting with and modulating the activity of Rad52. is a tumor suppressor gene whose importance in protection of mammalian cells from malignant transformation is second only to that of p53.1,2 The PTEN tumor suppressor is frequently mutated in cancer cells, and inherited PTEN mutations cause cancer-susceptibility conditions.1,2 The PTEN level as well as its activity in an organism profoundly influences tumor susceptibility, because mice also develop tumors in multiple organs.3,4 PTEN functions as a lipid and protein phosphatase.1,2 The PTEN protein consists of an N-terminal phosphatase domain, a lipid-binding C2 domain, and a C-terminal tail domain. The N terminus contains a PIP2-binding motif spanning from residues 6C15; the C tail contains PEST motifs which commonly serve as molecular signals for rapid proteolytic cleavage.5 Biochemically, PTEN dephosphorylates the lipid second-messenger phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) to generate phosphatidylinositol 3, 4-bisphosphate (PIP2) and, by doing so, antagonizes the PI3K/Akt signaling pathway. PTEN also has nuclear functions, since a fraction of PTEN is consistently detected in the nucleus.6,7 Several independent studies have shown that nuclear PTEN plays a significant role in the maintenance of genomic stability through the modulation of DNA repair, chromosomal segregation, and cell cycle arrest.8-12 PTEN is extensively phosphorylated at the C-tail region by several protein kinases. Phosphorylation of several Ser/Thr residues, including S370 in the C-tail region of PTEN by casein kinase 2 (CK2), is essential for the tail-dependent regulation of stability as phospho-defective mutant proteins exhibit decreased stability.13-15 GSK3 phosphorylates PTEN at S362 and T366.13 PTEN can be modified by mono- or polyubiquitination, which regulates its nuclear localization and stability, respectively.5 NEDD4-1 and WWP2 and RFP are 3 reported ubiquitin E3 ligases that mediate PTEN ubiquitination, 16-18 although NEDD4-1 appears to be dispensable for the regulation of its subcellular localization and stability. 19 PTEN also contains atypical nuclear localization sequences.20,21 Rad52 is a key component in DNA double-strand break repair and homologous recombination in yeast.22,23 Rad52 homologs in mammals are also identified and characterized as having similar biochemical activities to those of the yeast counterpart.22,23 Intriguingly, mammalian Rad52 plays a more modest role in vivo as Rad52 knockout in mice results in neither lethality, nor overt adverse phenotypes. However, Rad52 inactivation is synthetically lethal with BRCA2 deficiency,24 strongly suggesting that BRCA2 perform functions similar to those of Rad52 in vivo. Rad52 is regulated by sumoylation, which is induced by DNA damage and involved BMS-794833 in regulating Rad52 stability and activity.25,26 In the current study, we report that a significant fraction of PTEN underwent time-dependent nuclear translocation after DNA damage induced by reactive oxygen species, and that the accumulation of chromatin PTEN was associated with its phosphorylation on S366/T370. Deletional analysis coupled with ectopic expression shows that the C2 domain was responsible for chromatin translocation of PTEN. PTEN specifically interacted with Rad52, and Rabbit Polyclonal to ADORA1 the interaction was enhanced after treatment with H2O2. Our further study revealed that nuclear PTEN was involved in regulating sumoylation of Rad52, a process essential for Rad52 function in DNA homologous recombination. Results PTEN has been implicated in DNA damage responses.27 To understand how PTEN is regulated during DNA damage responses, we first examined PTEN subcellular localization after treatment with adriamycin, etoposide, and H2O2, chemical compounds that cause DNA double-strand breaks. We observed that at high concentrations, both adriamycin and H2O2, but not etoposide, induced BMS-794833 chromatin association of PTEN (Fig.?1). The presence of chromatin-PTEN was inversely correlated with the amount of cytoplasmic PTEN, especially after H2O2 treatment, suggesting its subcellular translocation under severe genotoxic stress. Both adriamycin BMS-794833 and etoposide induced cleavage of PARP-1 at high concentrations, BMS-794833 indicating apoptosis caused by these agents. On the other hand, H2O2-induced chromatin translocation was not.