Abstract 1072: PIN1 controls cell cycle progression through its interaction with pRb protein
Authors: Flavio Rizzolio , Lucchetti Chiara , Marchesi Irene , Bagella Luigi , Giordano Antonio 
Published: February 2011

Normal cells become tumor cells through the deregulation of multiple pathways. Nevertheless, there are some pathways that are altered in many tumors and the pRb pathway is one of the most important. pRb controls the cell cycle through its interaction with E2F transcription factors. These interactions are regulated during the cell cycle by a phosphorylation mechanism. In early and mid-G1, the protein complex D type cyclins/CDK4(6) and in late G1, cyclins E(A)/CDK2, gradually phosporylate pRb. Hyper-phosphorylated pRb releases E2Fs transcription factors and allows for the expression of genes that mediate S phase entry.

Ser or Thr followed by Pro are major phosphorylation motifs in the proteins but their significance was obscured until the discovery of the PIN1 protein (protein interacting with NIMA (never in mitosis A)-1). PIN1 has a two-domain structures that consist of an N-terminal WW and the C-terminal PPIase domain. The WW domain binds only to specific phosphorylated Ser/Thr-Pro-motifs and the PPIase isomerase domain catalyzes the conformational switch of target proteins from cis to trans. This reaction is especially important because Pro-directed kinases and phosphatases are conformation-specific and act only on the trans isoform. Many PIN1 protein targets have now been discovered, most of which are involved in the control of G0, G1/S and M cell cycle phases. For instance, PIN1 controls Cyclin D1 mRNA levels and it is involved in the regulation of CyclinD1, c-MYC and Cyclin E protein stability.

In order to clarify the role of PIN1 in the RB/E2F pathway, we generated a PIN1 knock down (kd) glioblastoma cell line. PIN1 kd cells proliferate at 25% less compared to normal cells as determined by XTT assay. PIN1 kd cells have 7.1% more cells in the G0/G1 cell cycle phase than normal cells. Since siRNA targeting PIN1 in LNCaP and PC3 prostate cancer cells increased cellular senescence and apoptosis, we tested if it is the case in our cell line. Caspase and β-galactosidase assays exclude the involvement of these pathways. In line with previously published data, pRb is hypo-phosphorylated in PIN1 kd cells and mRNA and total protein level did not change. To clarify if the CDK/cyclin protein complexes were altered we analyzed the protein levels by western blot and found that CDK2, CDK4, cyclin A, E, D1 levels are unchanged. Based on these results, we hypothesized that PIN1 and pRb directly interact. GST pull-down, Co-IP and FAR-western blot experiments confirmed that the two proteins interact and treatment of total protein lysate with CIP alkaline phosphatase demonstrated that the interaction is phosphorylation dependent. Finally, PIN1/pRb double knockdown cells rescued the cell proliferation defects observed in PIN1 kd cells. In addition, some E2F target genes are downregulated in PIN1 kd cells suggesting that pRb is the major PIN1 target to control transition from the G1 to S phase of the cell cycle.

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 1072.