Modulation of cyclin D1 and its signaling components by the phorbol ester TPA and the tyrosine phosphatase inhibitor vanadate.
The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) triggers cell-cycle progression at G1 phase in mouse embryonic fibroblast C3H 10T1/2 cells was examined. TPA treatment resulted in a temporary induction of cyclin D1 peaking at 9 h post stimulation. PD98059 (10 microM), the specific inhibitor of MAPK kinase, completely blocked TPA-stimulated cyclin D1 induction and DNA synthesis, confirming that MAPK activation plays an essential role in TPA-stimulated cell-cycle progression. Although both PKCalpha and PKCepsilon are expressed in C3H 10T1/2 cells, inhibitor studies suggest that PKCepsilon activation is required for the activation of MEK/MAPK signal transduction cascade. p70s6K, an important kinase involved in the regulation of protein synthesis and cell-cycle progression, has been reported to be activated through a PKC-dependent pathway (TPA-activatable) in addition to a PI3K-dependent pathway. Here, we demonstrate for the first time that TPA-stimulated MAPK activation is essential for the phosphorylation of several key residues involved in the activation of p70s6K, namely, thr389, thr421, and ser424. Vanadate, the tyrosine phosphatase inhibitor, triggered a sustained elevation of the level of active MAPK. However, corresponding to a rapid loss of cyclin D1 protein, vanadate treatment resulted in a significant shut out of 3H-thymidine incorporation into DNA regardless of TPA cotreatment. Vanadate treatment also led to the increase of active MEK, increased phosphorylation of p70s6K at thr389, thr421, and ser424 yet without activation of PKB. These data suggest that vanadate can selectively perturb the activation of signaling components which raises the interesting issue as to how vanadate downregulates the cyclin D1 level.
Yan
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Department of Biochemistry, Roswell Park Cancer Institute, Buffalo, New York 14260, USA.
Wenner
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CE
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D019938:Cyclin D1 / Q000502:physiology*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts; D005419:Flavonoids / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014638:Vanadates / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D010982:Platelet-Derived Growth Factor; D019938:Cyclin D1; D014638:Vanadates; D004815:Epidermal Growth Factor; C497256:Prkce protein, mouse; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D020928:Mitogen-Activated Protein Kinases; D017027:Protein Tyrosine Phosphatases; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one