Reactivation of tumor suppressor p16INK4a
Date of Completion
In mammalian cells, non-proliferative signals (i.e. replicative senescence) upregulate expression of proteins including cyclin-dependent kinase inhibitor p16INK4a. Expression of p16 competes with D-type cyclins for cyclin-dependent kinase 4/6 binding to inhibit kinase activity, which prevents hyperphosphorylation of pRb, reactivates the Rb pathway and induces G1-phase cell cycle arrest. Over 300 missense mutations in p16 are associated with inherited and sporadic tumors and most disrupt the protein's structure. We hypothesized that this susceptibility to genetic lesions is due to the proteins low thermodynamic stability ( DGH2On→u is 3.1 kcal/mol) and high backbone flexibility. Furthermore, we predicted that enhancing p16 thermodynamic stability could reactivate cell-cycle inhibitory function of oncogenic variants. To test this hypothesis, we identified stabilizing substitutions using bioinformatics and stability-modeling algorithms. These substitutions were combined to create a hyperstable variant that remains monomeric in solution with wild-type-like structure and Cdk4 binding but is 1.4 kcal/mol more stable than wild-type p16. More importantly, hyperstable substitutions in combination with oncogenic mutations R24P, P81L or V126D significantly restore Cdk4 binding in a yeast two-hybrid assay. Biophysical studies indicate multiple mechanisms are involved in restoring binding activity. In general, helical content ([&thetas;]222) and thermal stability (Tm ) increased while hydrophobic surface area and aggregation decreased in the background of the hyperstable variant compared to wild type p16. We then reported on the intracellular effects of hyperstable amino acid substitutions using a human cell-based system. Each oncogenic variant was expressed in U2OS, a human osteosarcoma cell line deficient in endogenous p16. Western blot, immunofluorescence and flow cytometry analysis showed that in the background of hyperstable substitutions, oncogenic p16 mutants display decreased aggregation with increased nuclear localization, kinase assembly and percentage of cells in the G0/G1 phase of the cell cycle. Taken together, these results provide mechanistic evidence for the repair process aimed at restoring native-like intra and intermolecular interactions to each oncogenic p16 variants. Furthermore, these results suggest that increasing the thermodynamic stability of p16 can be used as a general strategy to restore biological activity to defective variants of this important tumor suppressor protein and provides new insight into correcting protein folding defects in vivo. ^
Cammett, Tobin Jay, "Reactivation of tumor suppressor p16INK4a" (2005). Doctoral Dissertations. AAI3180189.