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Inhibition of Cdk2 activation by selected tyrphostins causes cell cycle arrest at late G1 and S phase
N Kleinberger-Doron1, N Shelah, R Capone
1Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel.
Abstract:
We have previously reported that certain tyrphostins which block EGF-R phosphorylation in cell-free systems fail to do so in intact cells. Nevertheless, we found that this family of tyrphostins inhibits both EGF- and calf serum-induced cell growth and DNA synthesis [Osherov, N.A., Gazit, C., Gilon, and Levitzki, A. (1993). Selective inhibition of the EGF and HER2/Neu receptors by Tyrphostins. J. Biol. Chem. 268, 11134-11142.] Now we show that these tyrphostins exert their inhibitory activity even when added at a time when the cells have already passed their restriction point and receptor activation is no longer necessary. AG555 and AG556 arrest 85% of the cells at late G1, whereas AG490 and AG494 cause cells to arrest at late G1 and during S phase. No arrest occurs during G2 or M phase. Further analysis revealed that these tyrphostins act by inhibiting the activation of the enzyme Cdk2 without affecting its levels or its intrinsic kinase activity. Furthermore, they do not alter the association of Cdk2 to cyclin E or cyclin A or to the inhibitory proteins p21 and p27. These compounds also have no effect on the activating phosphorylation of Cdk2 by Cdk2 activating kinase (CAK) and no effect on the catalytic domain of cdc25 phosphatase. These compounds lead to the accumulation of phosphorylated Cdk2 on tyrosine 15 which is most probably the cause for its inhibition leading to cell cycle arrest at G1/S. A structure-activity relationship study defines a very precise pharmacophore, suggesting a unique molecular target not yet identified and which is most probably involved in the regulation of the tyrosine-phosphorylated state of Cdk2. These compounds represent a new class of cell proliferation blockers whose target is Cdk2 activation.
Insights
Certain tyrphostins block cell proliferation by inhibiting cyclin-dependent kinase 2 (Cdk2) activation. These compounds arrest cells in the G1/S phase, representing a new class of cell proliferation blockers targeting Cdk2 activation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Tyrphostins previously shown to inhibit epidermal growth factor receptor (EGF-R) phosphorylation in cell-free systems were found to inhibit EGF- and calf serum-induced cell growth and DNA synthesis in intact cells.
- These tyrphostins retain inhibitory activity even when added after the restriction point, when receptor activation is no longer required for cell cycle progression.
Purpose of the Study:
- To investigate the mechanism by which specific tyrphostins inhibit cell growth and DNA synthesis.
- To identify the molecular target and understand the structure-activity relationship of these novel cell proliferation blockers.
Main Methods:
- Cell cycle analysis using flow cytometry to determine cell cycle arrest points.
- Biochemical assays to assess enzyme activity, protein levels, and protein-protein interactions.
- Structure-activity relationship (SAR) studies to define the pharmacophore.
Main Results:
- Tyrphostins AG555 and AG556 arrested 85% of cells at late G1, while AG490 and AG494 caused arrest at late G1 and during S phase. No G2 or M phase arrest was observed.
- These tyrphostins inhibit the activation of cyclin-dependent kinase 2 (Cdk2) without affecting its levels, intrinsic kinase activity, or association with cyclins (E, A) or inhibitors (p21, p27).
- The compounds did not affect Cdk2 activating kinase (CAK) or cdc25 phosphatase activity, but led to the accumulation of tyrosine 15-phosphorylated Cdk2, indicating inhibition of Cdk2 activation and subsequent G1/S cell cycle arrest.
Conclusions:
- The studied tyrphostins represent a new class of cell proliferation blockers targeting Cdk2 activation.
- The precise pharmacophore identified suggests a novel molecular target involved in regulating the tyrosine-phosphorylated state of Cdk2.
- These findings provide a basis for developing new therapeutic strategies targeting cell cycle regulation in proliferative diseases.