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-depen dent kinase (Cdk) 4/6 complexes. We report here that TGF-beta-treated human HepG2 hepatocellular carcinoma cells arrest in G1, but retain continued cy clin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-beta-treated cell s failed to induce p15(INK4b), down-regulate CDC25A, or increase levels of p21(CIP1), P27(KIP1), and p57(KIP2). However, TGF-beta treatment resulted i n the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr(160) phosphorylation on Cdk2, Whole-cell lysates from T GF-beta-treated cells showed inhibition of Cdk2 Thr(160) Cdk activating kin ase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed m ammalian CAK, was not altered. Saccharomyces cerevisiae contains a genetica lly and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kD a Cak1p protein unrelated to Cdk7, Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regula ted in response to TGF-beta treatment. Taken together, these observations d emonstrate that TGF-beta signaling mediates a G1 arrest in HepG2 cells by t argeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.