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[摘要]：P19(INK4d) consists of five ankyrin repeats and controls the human cell cycle by inhibiting the cyclin D-dependent kinases 4 and 6. Posttranslational phosphorylation of p19(INK4d) has been described for Ser66 and Ser76. In the present study we show that mimicking the phosphorylation site of p19(INK4d) by a glutamate substitution at position 76 dramatically decreases the stability of the native but not an intermediate state. At body temperature the native conformation is completely lost and p19(INK4d) molecules exhibit the intermediate state as judged by kinetic and equilibrium analysis. High resolution NMR spectroscopy verified that the three C-terminal repeats remained folded in the intermediate state, whereas all cross-peaks of the two N-terminal repeats lost their native chemical shift. Molecular dynamic simulations of p19(INK4d) in different phosphorylation states revealed large-scale motions in phosphorylated p19(INK4d), which cause destabilization of the interface between the second and third ankyrin repeat. Only doubly phosphorylated p19(INK4d) mimic mutants showed in vitro an increased accessibility for ubiquitination, which might be the signal for degradation in vivo. |
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