Transforming growth factor beta targeted inactivation of cyclin E:cyclin-dependent kinase 2 (Cdk2) complexes by inhibition of Cdk2 activating kinase activity.
Transforming growth factor beta (TGF-beta)-mediated G(1) arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. We report here that TGF-beta-treated human HepG2 hepatocellular carcinoma cells arrest in G(1), but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-beta-treated cells failed to induce p15(INK4b), down-regulate CDC25A, or increase levels of p21(CIP1), p27(KIP1), and p57(KIP2). However, TGF-beta treatment resulted in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr(160) phosphorylation on Cdk2. Whole-cell lysates from TGF-beta-treated cells showed inhibition of Cdk2 Thr(160) Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, was not altered. Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7. Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regulated in response to TGF-beta treatment. Taken together, these observations demonstrate that TGF-beta signaling mediates a G(1) arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.
Nagahara
H
H
Howard Hughes Medical Institute, Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Ezhevsky
S A
SA
Vocero-Akbani
A M
AM
Kaldis
P
P
Solomon
M J
MJ
Dowdy
S F
SF
D042846:CDC2-CDC28 Kinases; D019927:Cyclin E / Q000378:metabolism*; D056748:Cyclin H; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation; D016193:G1 Phase / Q000187:drug effects*; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C534751:CCNH protein, human; D019927:Cyclin E; D056748:Cyclin H; D016213:Cyclins; D016212:Transforming Growth Factor beta; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human