Abstract
Signaling via the phosphoinositide 3-kinase (PI3K)/AKT pathway is crucial for the regulation of endothelial cell (EC) proliferation and survival, which involves the AKT-dependent phosphorylation of the DNA repair protein p21(Cip1) at Thr-145. Because p21(Cip1) is a short-lived protein with a high proteasomal degradation rate, we investigated the regulation of p21(Cip1) protein levels by PI3K/AKT-dependent signaling. The PI3K inhibitors Ly294002 and wortmannin reduced p21(Cip1) protein abundance in human umbilical vein EC. However, mutation of the AKT site Thr-145 into aspartate (T145D) did not increase its protein half-life. We therefore investigated whether a kinase downstream of AKT regulates p21(Cip1) protein levels. In various cell types, AKT phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). Upon serum stimulation of EC, GSK-3beta was phosphorylated at Ser-9. Site-directed mutagenesis revealed that GSK-3 in vitro phosphorylated p21(Cip1) specifically at Thr-57 within the Cdk binding domain. Overexpression of GSK-3beta decreased p21(Cip1) protein levels in EC, whereas the specific inhibition of GSK-3 with lithium chloride interfered with p21(Cip1) degradation and increased p21(Cip1) protein about 10-fold in EC and cardiac myocytes (30 mm, p < 0.001). These data indicate that GSK-3 triggers p21(Cip1) degradation. In contrast, stimulation of AKT increases p21(Cip1) via inhibitory phosphorylation of GSK-3.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adjuvants, Immunologic / pharmacology
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Androstadienes / pharmacology
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Animals
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Blotting, Western
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Bromodeoxyuridine / pharmacology
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COS Cells
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
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Calcium-Calmodulin-Dependent Protein Kinases / physiology*
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Cell Cycle
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Cell Division
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Cell Survival
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Cells, Cultured
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Chromones / pharmacology
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins / metabolism*
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Dose-Response Relationship, Drug
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Endothelium, Vascular / cytology
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Endothelium, Vascular / enzymology*
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Enzyme Inhibitors / pharmacology
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Glycogen Synthase Kinase 3
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Glycogen Synthase Kinases
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Humans
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Immunoblotting
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Lithium Chloride / pharmacology
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Morpholines / pharmacology
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Mutagenesis, Site-Directed
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Phosphorylation
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Protein Binding
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Protein Serine-Threonine Kinases*
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-akt
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Serine / chemistry
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Signal Transduction
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Threonine / chemistry
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Time Factors
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Transfection
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Umbilical Veins / cytology
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Wortmannin
Substances
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Adjuvants, Immunologic
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Androstadienes
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CDKN1A protein, human
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Chromones
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins
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Enzyme Inhibitors
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Morpholines
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Proto-Oncogene Proteins
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Threonine
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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Serine
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Glycogen Synthase Kinases
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AKT1 protein, human
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Calcium-Calmodulin-Dependent Protein Kinases
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Glycogen Synthase Kinase 3
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Bromodeoxyuridine
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Lithium Chloride
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Wortmannin