Control ofp63 protein stability and prolyl isomerase Pin1-mediated regulation of retinoblastoma protein

This thesis investigated novel regulation of two cell cycle regulators, p53-related p63 and retinoblastoma protein (Rb).; The p63 gene encodes six isoforms. The TAp63 isoforms possess a N-terminal domain capable of transactivating a set of genes including some p53 downstream targets. Accumulating evidence indicates that TAp63 plays an important role in the regulation of cell proliferation, differentiation and apoptosis. The DeltaNp63 isoforms lack the N-terminal transactivation domain and function to inhibit p63 and other p53 family members. Mutations in the p63 gene have been linked to human diseases including ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndromes. In this study, we show that mutant p63 proteins with a single amino acid substitution found in EEC syndromes are transcriptionally inert and highly stable. We demonstrate that TAp63 protein expression is tightly controlled by its specific DNA-binding and transactivation activity and that p63 is degraded in a proteasome-dependent, murine double minute-2 (MDM2)-independent pathway. In addition, the N-terminal transactivation domain of p63 is critical for its protein degradation. Furthermore, the wild type TAp63gamma acts in trans to promote degradation of mutant TAp63gamma defective in DNA-binding. Moreover, DeltaTAp63gamma inhibits transactivation activity of TAp63 and stabilizes TAp63 protein. Taken together, these data suggest a feedback loop for p63 regulation, analogous to the p53-MDM2 feedback loop.; The tumor suppressor retinoblastoma protein is frequently inactivated in a variety of human cancers. While Rb plays a pivotal role in cell cycle G1-S transition, emerging evidence indicates that Rb is also critical for S-phase checkpoint control in cellular response to DNA damage. Here we show that CDK-mediated phosphorylation of Rb facilitates its interaction with Pin1, a phosphorylation-specific peptidyl-prolyl isomerase. Pin1 inhibits protein phosphatase 2A (PP2A)-mediated Rb dephosphorylation in vitro , attenuates Rb dephosphorylation upon S-phase DNA damage, and compromises the gamma-irradiation induced S-phase checkpoint control. Ablation of Pin1 leads to a significant increase in hypophosphorylated Rb, accelerated Rb dephosphorylation and increased cell growth arrest upon S-phase DNA damage. Furthermore, Pin1 inhibits Rb dephosphorylation at mitotic exit. Moreover, Pin1 is overexpressed, concomitant with hyperphosphorylated Rb in human breast cancers. Together, these data reveal a novel regulatory pathway for Rb in which Pin1 is critical for modulation of Rb function in S-phase checkpoint control upon DNA damage.