Glycogen synthase kinase-3 couples AKT-dependent signaling to the regulation of p21Cip1 degradation.
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.
Rössig
Lothar
L
Division of Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
Badorff
Cornel
C
Holzmann
Yvonne
Y
Zeiher
Andreas M
AM
Dimmeler
Stefanie
S
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism* / Q000502:physiology*; D002453:Cell Cycle; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D015151:Immunoblotting; D018021:Lithium Chloride / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology; D000077191:Wortmannin
D000276:Adjuvants, Immunologic; D000730:Androstadienes; C493956:CDKN1A protein, human; D002867:Chromones; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D009025:Morpholines; D011518:Proto-Oncogene Proteins; D013912:Threonine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012694:Serine; D038341:Glycogen Synthase Kinases; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D001973:Bromodeoxyuridine; D018021:Lithium Chloride; D000077191:Wortmannin