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...

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Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro.

Circulating nucleic acids,s uch as short interfering RNA( siRNA), regulate many biological processes;h owever, the mechanism by whichthese molecules enter the cell is poorly understood. The role of extracellular-matrix-derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilizesiRNAvia hydrogen bonding and Vand er Waals interactions.T his stabilization facilitated HA size-and concentration-dependent gene silencing in aCD44-positive human osteosarcoma cell line (MG-63) and in human mesenchymal stromal cells (hMSCs). This native HA-based siRNAtransfection represents the first report on an anionic,n on-viral delivery method that resulted in approximately 60 %gene knockdowninboth cell types tested, which correlated with ar eduction in translation levels.