The tumor suppressor gene PTEN is a phosphoinositide phosphatase that is inactivated by deletion and/or mutation in diverse human tumors. Wild-type PTEN is expressed both in the cytoplasm and nucleus in normal cells, with a preferential nuclear localization in differentiated or resting cells. To elucidate the relationship between PTENs subcellular localization and its biologic activities, we constructed different PTEN mutants that targeted PTEN protein into different subcellular compartments. Our data show that the subcellular localization patterns of a PTEN (⌬PDZB) mutant versus a G129R phosphatase mutant were indistinguishable from those of wild-type PTEN. In contrast, the Myr-PTEN mutant demonstrated an enhanced association with the cell membrane. We found that nuclear PTEN alone is capable of suppressing anchorage-independent growth and facilitating G 1 arrest in U251MG cells without inhibiting Akt activity. Nuclear compartmentspecific PTEN-induced growth suppression is dependent on possessing a functional lipid phosphatase domain. In addition, the down-regulation of p70S6K could be mediated, at least in part, through activation of AMPactivated protein kinase in an Akt-independent fashion. Introduction of a constitutively active mutant of Akt, Akt-DD, only partially rescues nuclear PTEN-mediated growth suppression. Our collective results provide the first direct evidence that PTEN can contribute to G 1 growth arrest through an Akt-independent signaling pathway.
The tumor suppressor phosphatase and tensin homologue (PTEN) plays distinct growth-regulatory roles in the cytoplasm and nucleus. It has been shown to be preferentially localized to the nucleus in differentiated or resting cells, and to the cytoplasm in advanced tumor cells. Thus, the regulation of PTEN's subcellular localization seems to be critical to its tumor-suppressing functions. In this study, we showed that activation of the phosphoinositide-3-kinase (PI3K) pathway triggers PTEN's cell cycle-dependent chromosome region maintenance 1-mediated nuclear export, as PTEN was predominantly expressed in the cytoplasm of TSC2 À/À mouse embryo fibroblasts or activated Akt mutant-transfected NIH3T3 cells. In contrast, dominant-negative mutants of Akt and pharmacologic inhibitors of PI3K, mTOR, and S6K1, but not of MEK, suppressed the nuclear export of PTEN during the G 1 -S transition. The nuclear-cytoplasmic trafficking of exogenous PTEN is likewise regulated by the PI3K cascade in PTEN-null U251MG cells. The nuclear export of PTEN could also be blocked by short interfering RNA to S6K1/2. In addition, PTEN interacts with both S6K1 and S6K2. Taken together, our findings strongly indicate that activation of the PI3K/Akt/mTOR/S6K cascade, specifically S6K1/2, is pivotal in regulating the subcellular localization of PTEN. This scenario exemplifies a reciprocal regulation between PI3K and PTEN that defines a novel negative-feedback loop in cell cycle progression.
The regulation of the subcellular localization of phosphatase and tensin homologue (PTEN) is critical to its tumor-suppressing functions. Previously, we found that the activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR/S6 protein kinase (S6K) cascade triggers the nuclear export of PTEN during the G1/S transition. Because mTOR can be alternatively downregulated by tuberous sclerosis complex 2 (TSC2) activation mediated by 5' adenosine monophosphate-activated protein kinase (AMPK), we proposed that the activation of AMPK α1/2 by LKB1 and/or by calmodulin-dependent protein kinase kinase (CaMKK) would also block the nuclear export of PTEN in a manner similar to that of inhibitors of PI3K, mTOR, and S6K. We found that in LKB1-null A549 lung adenocarcinoma cells, an AMPK activator, metformin, failed to block the nuclear export of PTEN, and the reintroduction of functional LKB1 into these cells restored the metformin-mediated inhibition of the nuclear export of PTEN. In addition, the nuclear export of PTEN was blocked in cells treated with the CaMKK activator ATP, and this inhibition was reversed by the addition of inhibitors of either AMPK (compound C) or CaMKK (STO-609). Although the nuclear export of PTEN is blocked by metformin in MCF-7 breast cancer cells carrying wild-type LKB1, this inhibition could not be reversed by an AMPK inhibitor, suggesting that LKB1 could regulate the nuclear export of PTEN by bypassing AMPK α1/2. Moreover, ATP could not block the nuclear export of PTEN in AMPK α1/2(-/-) or TSC2(-/-) mouse embryonic fibroblasts. However, metformin was still able to induce the LKB1-mediated inhibition of the nuclear export of PTEN in these cells. Taken together, these findings strongly suggest that although CaMKK mediates the nuclear retention of PTEN mainly through the activation of AMPK, LKB1 can regulate the nuclear-cytoplasmic trafficking of PTEN, with or without the AMPK/TSC2/mTOR/S6K-signaling intermediates.
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