Purinergic Receptor-mediated Rapid Depletion of Nuclear Phosphorylated Akt Depends on Pleckstrin Homology Domain Leucine-rich Repeat Phosphatase, Calcineurin, Protein Phosphatase 2A, and PTEN Phosphatases

Abstract

Akt is an important oncoprotein, and data suggest a critical role for nuclear Akt in cancer development. We have previously described a rapid (3-5 min) and P2X7-dependent depletion of nuclear phosphorylated Akt (pAkt) and effects on downstream targets, and here we studied mechanisms behind the pAkt depletion. We show that cholesterol-lowering drugs, statins, or extracellular ATP, induced a complex and coordinated response in insulin-stimulated A549 cells leading to depletion of nuclear pAkt. It involved protein/lipid phosphatases PTEN, pleckstrin homology domain leucine-rich repeat phosphatase (PHLPP1 and -2), protein phosphatase 2A (PP2A), and calcineurin. We employed immunocytology, immunoprecipitation, and proximity ligation assay techniques and show that PHLPP and calcineurin translocated to the nucleus and formed complexes with Akt within 3 min. Also PTEN translocated to the nucleus and then co-localized with pAkt close to the nuclear membrane. An inhibitor of the scaffolding immunophilin FK506-binding protein 51 (FKBP51) and calcineurin, FK506, prevented depletion of nuclear pAkt. Furthermore, okadaic acid, an inhibitor of PP2A, prevented the nuclear pAkt depletion. Chemical inhibition and siRNA indicated that PHLPP, PP2A, and PTEN were required for a robust depletion of nuclear pAkt, and in prostate cancer cells lacking PTEN, transfection of PTEN restored the statin-induced pAkt depletion. The activation of protein and lipid phosphatases was paralleled by a rapid proliferating cell nuclear antigen (PCNA) translocation to the nucleus, a PCNAp21 cip1 complex formation, and cyclin D1 degradation. We conclude that these effects reflect a signaling pathway for rapid depletion of pAkt that may stop the cell cycle.The serine/threonine kinase Akt is a central regulator of apoptosis and cell growth and is activated by insulin, growth factors, and cellular stress. Many studies indicate a key role for Akt in carcinogenesis and as a target for therapeutic agents (1-4). 3-Hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, statins, have anticancer properties (5), and we have shown that statins decrease levels of phosphorylated Akt (pAkt) 3 in lung and pancreatic cancer cells (6 -9). A conspicuous finding was that dephosphorylation of nuclear Akt was induced within minutes and that this effect was associated with inhibited cell proliferation. Extracellular ATP also induced nuclear pAkt depletion, and several lines of evidence indicated that the rapid pAkt depletion was mediated by the purinergic receptor P2X7 (7,8).A family of protein phosphatases, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP), was identified as a phosphatase for Akt (10). Two isoforms, PHLPP1 and PHLPP2, have been shown to dephosphorylate distinct Akt isoforms, at one (Ser 473 ) of two phosphorylation sites required for activation (11). This dephosphorylation is regulated by the immunophilin FKBP51 (FK506-binding protein 51), which acts as scaffolding protein for Akt and PHLPP (12). Other phosphatases shown to dephosphoryl...

Statins exhibit anticancer effects through modifications of the pAkt signaling pathway

“…Different types of statins have been reported to decrease the levels of phosphorylated Akt in several cell lines, and our group has shown that pravastatin, simvastatin and atorvastatin lower the insulininduced nuclear pAkt accumulation without affecting the total Akt levels in A549 cells (15). This effect is mediated by P2X7, and is associated with altered phosphorylations of downstream targets of pAkt, including GSK3β and cyclin D1 (13,14). Here we investigated the cellular effects of the long-term treatment with atorvastatin to gain insight into their mode of action in the prevention of cancer.…”

“…Mutaton/deletion of PTEN has been reported in many cancers (20): therefore, the pAkt/PTEN axis is currently attracting great interest for its role in carcinogenesis. Moreover, we provided evidence for the involvement of PTEN, together with the protein phosphatases PHLPP1, PHLPP2, PP2A and calcineurin, in the rapid statin-induced P2X7-mediated nuclear pAkt depletion (14).…”

“…Here we investigated the cellular effects of the long-term treatment with atorvastatin to gain insight into their mode of action in the prevention of cancer. Atorvastatin-induced selection of A549 cells was studied as the short-term effects on Akt/P2X7 signaling are best characterized in this cell model (13)(14)(15)22).…”

“…We previously demonstrated that statins rapidly activate the purinergic receptor P2X7 and affect Akt signaling, which may have important anti-cancer effects (13)(14)(15): these effects were observed within few minutes, and were not prevented by pre-incubation with mevalonate. The Akt pathway is one of the major anti-apoptotic factors in cells (16): it is activated by growth factors and cellular stress and is commonly overexpressed in different tumors, but is also often induced by cytostatic treatment (17,18).…”

Potenciais mecanismos de regulação da fosfatase PTEN pelas proteínas SET e PP2A e seu envolvimento na predisposição ao carcinoma bucal

“…Alguns estudos têm relacionado a atividade de PTEN e PP2A (Mistafa et al, 2010;Nho e Kahm, 2010;Bononi et al, 2011;Xu et al, 2011) …”