Phospho PTEN mediated dephosphorylation of mitotic kinase PLK1 and Aurora Kinase A prevents aneuploidy and preserves genomic stability.

PTEN, dual phosphatase tumor suppressor protein, is found to be frequently mutated in various cancers. Post-translational modification of PTEN is important for its sub-cellular localization and catalytic functions. But how these modifications affect cytological damage and aneuploidy is not studied in detail. We focus on the role of phosphatase activity along with C-terminal phosphorylation of PTEN in perspective of cytological damage like micronucleus, nuclear bud, and nuclear bridge formation. Our data suggest that wild-type PTEN, but not phospho-mutant PTEN significantly reduces cytological damage in PTEN null PC3 cells. In case of phosphatase-dead PTEN, cytological damage markers are increased during 24 h recovery after DNA damage. When we use phosphorylation and phosphatase-dead dual mutant PTEN, the extent of different cytological DNA damage parameters are similar to phosphatase-dead PTEN. We also find that both of those activities are essential for maintaining chromosome numbers. PTEN null cells exhibit significantly aberrant γ-tubulin pole formation during metaphase. Interestingly, we observed that p-PTEN localized to spindle poles along with PLK1 and Aurora Kinase A. Further depletion of phosphorylation and phosphatase activity of PTEN increases the expression of p-Aurora Kinase A (T288) and p-PLK1 (T210), compared to cells expressing wild-type PTEN. Again, wild-type PTEN but not phosphorylation-dead mutant is able to physically interact with PLK1 and Aurora Kinase A. Thus, our study suggests that the phosphorylation-dependent interaction of PTEN with PLK1 and Aurora Kinase A causes dephosphorylation of those mitotic kinases and by lowering their hyperphosphorylation status, PTEN prevents aberrant chromosome segregation in metaphase.

Both phosphorylation and phosphatase activity of PTEN are required to prevent replication fork progression during stress by inducing heterochromatin.

PTEN is a tumor suppressor protein frequently altered in various cancers. PTEN-null cells have a characteristic of rapid proliferation with an unstable genome. Replication stress is one of the causes of the accumulation of genomic instability if not sensed by the cellular signaling. Though PTEN-null cells have shown to be impaired in replication progression and stalled fork recovery, the association between the catalytic function of PTEN regulated by posttranslational modulation and cellular response to replication stress has not been studied explicitly. To understand molecular mechanism, we find that PTEN-null cells display unrestrained replication fork progression with accumulation of damaged DNA after treatment with aphidicolin which can be rescued by ectopic expression of full-length PTEN, as evident from DNA fiber assay. Moreover, the C-terminal phosphorylation (Ser 380, Thr 382/383) of PTEN is essential for its chromatin association and sensing replication stress that, in response, induce cell cycle arrest. Further, we observed that PTEN induces HP1α expression and H3K9me3 foci formation in a C-terminal phosphorylation-dependent manner. However, phosphatase dead PTEN cannot sense replication stress though it can be associated with chromatin. Together, our results suggest that DNA replication perturbation by aphidicolin enables chromatin association of PTEN through C-terminal phosphorylation, induces heterochromatin formation by stabilizing and up-regulating H3K9me3 foci and augments CHK1 activation. Thereby, PTEN prevents DNA replication fork elongation and simultaneously causes G1-S phase cell cycle arrest to limit cell proliferation in stress conditions. Thus PTEN act as stress sensing protein during replication arrest to maintain genomic stability.

Non-canonical function of nuclear PTEN and its implication on tumorigenesis.

Suppression of genomic instability is the key to prevent tumor development. PTEN is a unique tumor suppressor protein having both lipid and protein phosphatase activities. Interestingly though it is a cytoplasmic protein, but a significant pool of PTEN can also be localized in nucleus. The function of cytoplasmic PTEN is well defined and extensively studied in various literatures focusing mainly on the negative regulation of oncogenic PI-3Kinase-AKT pathway but functional regulation of nuclear PTEN is less defined and therefore it is a fascinating subject of research in cancer biology. Post-translation modulation of PTEN such as phosphorylation, sumorylation, acetylation and methylation also regulates its cellular localization, protein-protein association and catalytic function. Loss or mutation in PTEN is associated with the development of tumors in various tissues from the brain to prostate. Here we have summarized the role of nuclear PTEN and its epigenetic modulation in various DNA metabolic pathways, for example, DNA damage response, DNA repair, DNA replication, DNA segregation etc. Further, pathways involved in nuclear PTEN degradation are also discussed. Additionally, we also emphasize probable potential targets associated with PTEN pathway for chemotherapeutic purpose.

Phosphorylation of PTEN at STT motif is associated with DNA damage response.

Phosphatase and tensin homolog deleted on chromosome Ten (PTEN), a tumor suppressor protein participates in multiple cellular activities including DNA repair. In this work we found a relationship between phosphorylation of carboxy (C)-terminal STT motif of PTEN and DNA damage response. Ectopic expression of C-terminal phospho-mutants of PTEN, in PTEN deficient human glioblastoma cells, U87MG, resulted in reduced viability and DNA repair after etoposide induced DNA damage compared to cells expressing wild type PTEN. Also, after etoposide treatment phosphorylation of PTEN increased at C-terminal serine 380 and threonine 382/383 residues in PTEN positive HEK293T cells and wild type PTEN transfected U87MG cells. One-step further, DNA damage induced phosphorylation of PTEN was confirmed by immunoprecipitation of total PTEN from cellular extract followed by immunobloting with phospho-specific PTEN antibodies. Additionally, phospho-PTEN translocated to nucleus after etoposide treatment as revealed by indirect immunolabeling. Further, phosphorylation dependent nuclear foci formation of PTEN was observed after ionizing radiation or etoposide treatment which colocalized with γH2AX. Additionally, etoposide induced γH2AX, Mre11 and Ku70 foci persisted for a longer period of times in U87MG cells after ectopic expression of PTEN C-terminal phospho-mutant constructs compared to wild type PTEN expressing cells. Thus, our findings strongly suggest that DNA damage induced phosphorylation of C-terminal STT motif of PTEN is necessary for DNA repair.

Multinucleation regulated by the Akt/PTEN signaling pathway is a survival strategy for HepG2 cells.

Hepatocellular carcinoma (HCC) is non-responsive to many chemotherapeutic agents including etoposide. The aim of this study was to examine the survival strategy of the HCC cell line HepG2 after etoposide treatment. Here we analyzed and compared spontaneous and etoposide-induced DNA damage in HepG2 (α-fetoprotein (AFP)-positive) and Chang Liver (AFP-negative) cell lines. Compared to Chang Liver cells, HepG2 cells exhibited a significantly higher degree of micronucleation and a higher nuclear division index, as determined by the cytokinesis-block micronucleus assay, following exposure to etoposide. HepG2 cells were also more resistant to etoposide-induced cytotoxicity compared to Chang Liver cells. We also establish that increased etoposide-induced multinucleation in HepG2 cells is dependent on the catalytic activity of Akt, as phosphatidylinositol-3-kinase inhibitors as well as the overexpression of kinase-defective Akt reversed this phenotype. Moreover, ectopic expression of wild type PTEN reduced the frequency of etoposide-induced multinucleated HepG2 cells, and restored HepG2 etoposide sensitivity. Taken together, these results implicate the Akt/PTEN cellular axis as a major determinant of the etoposide resistance of HCC cells.