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.
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 PCNA-p21(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.
Mistafa
Oras
O
Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Ghalali
Aram
A
Kadekar
Sandeep
S
Högberg
Johan
J
Stenius
Ulla
U
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000069059:Atorvastatin; D019703:Calcineurin / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006538:Heptanoic Acids / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction / Q000187:drug effects; D022021:Tacrolimus Binding Proteins / Q000037:antagonists & inhibitors; D054875:Ubiquitination / Q000187:drug effects; D000099159:Tacrolimus Binding Protein 5
D000255:Adenosine Triphosphate; D000069059:Atorvastatin; D019703:Calcineurin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D006538:Heptanoic Acids; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases; D018809:Proliferating Cell Nuclear Antigen; D054648:Protein Phosphatase 2; D051057:Proto-Oncogene Proteins c-akt; D011758:Pyrroles; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D022021:Tacrolimus Binding Proteins; D000099159:Tacrolimus Binding Protein 5; C546468:P2RX7 protein, human; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester