Abstract 1073: PIN1 forms a protein complex with Rb2/p130 and controls its phosphorylation status

Abstract

Abstract Since the majority of somatic cells are quiescent, fine controls of cell cycle re-entry and progression are fundamental for the normal life of the cells. Essential to maintaining the quiescent state is what is termed the “restriction point” (R), the point that divides the early and late G1 phase of cell cycle. The pools of proteins that control this mechanism are still not well-defined. However, one of the major players is the RB gene family (Rb, Rb2/p130 and p107) since ablation of these proteins eliminates R. Because their function, RB proteins are considered tumor suppressor. As common feature, RB proteins share a pocket motif able to bind different E2F transcription factors and then regulating their activity. Differential phosphorylation levels of RB proteins are critical for the binding with their E2F partners and consequently for transcriptional inactivation of target genes during G1 and S phase of cell cycle. Phosphorylation is dependent on cyclin/CDKs complexes: it begins in early-G1 phase where gradual inactivation of RB proteins is required to proceed on S phase of cell cycle. PIN1 belongs to peptidyl-prolyl-isomerase family, which role is to catalyze the cis/trans conformation of Serine/Threonine followed by proline residues and to regulate their activity. PIN1 has been shown to be involved in different signalling pathways, many of which concerning cell cycle progression, cellular proliferation, and neoplastic transformation. In glioblastoma cell lines, Rb2/p130 has been demonstrated to be a major player to control cell cycle progression. Since Rb2/p130 has many Ser-Thr/Pro phospho-sites we hypotesized a possible interaction with PIN1 protein. Co-IP and FAR-western blot experiments demonstrated direct interaction between PIN1 and Rb2/p130. The interaction is phosphorylation dependent, as demonstrated by GST-pulldown experiments on protein lysates treated or not with shrimp alkaline phosphatase. To evaluate the effect of PIN1 on Rb2/p130 protein, scrambled- and Pin1-shRNA were prepared by lentiviral approach. Analysis of asynchronous cells showed not difference in Rb2/p130 protein levels. Since Rb2/p130 is mostly active in G0 phase of cell cycle, cells were synchronized in G0 and G1/S phases. Although the total protein level of Rb2/p130 didn't change, phospho- specific Rb2/p130 antibody showed an increase expression level in asynchronous and G0 phase in PIN1 knockdown cells compare to scramble cells. No protein changes are detected in G1/S phase. As demonstrated in other cellular models, PIN1 may affect the level of CDK/cyclin protein complexes eventually affecting Rb2/p130 phosphorylation. Western blot analysis demonstrated no change in protein level among the major CDK/cyclin proteins that control cell cycle. These results suggest that PIN1 regulates directly the functionality of Rb2/p130 during the G0 and probably the early G1 phase of cell cycle when Rb2/p130 has major activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1073.