Cell death, cell growth and cell cycle regulation by the Retinoblastoma family

Resumen

The Retinoblastoma gene (RB1) was the first tumor suppressor gene to be cloned. Its encoded protein, the Retinoblastoma protein (pRB) is a member of the Retinoblastoma family, which is constituted also by p107 and p130. This family plays a critical role in the control of the progression through the different phases of the cell cycle. pRB is particularly involved in the G1-to-S transition and, although its role in cell cycle regulation is well defined, its role in the regulation of cell death remains controversial. In this work, we have performed a systematic and global study of the Retinoblastoma family members and of the various functional domains of pRB in the regulation of E2F activity, cell cycle progression, DNA replication, and in the regulation of cell death triggered by ionizing radiation (IR). While all family members inhibited E2F activity and DNA replication, and accumulated cells in G0/G1 in the short-term, pRB was the only member of the family that inhibited IR-induced cell death and arrested cells in the G0/G1 cell cycle phase in the long-term. We have found a strong correlation between the capacity of pRB to induce a long-term arrest of the cell cycle and its capacity to inhibit cell death, suggesting that a sustained arrest of the cell cycle in G0/G1 is necessary and sufficient to inhibit cell death caused by DNA damage. These data suggest that pRB may not play a direct role in the regulation of cell death induced by DNA damage and that cell survival might be the consequence of its main function, the arrest of the cell cycle until DNA damage has been eliminated. We have forced the expression of various domains of pRB and the results obtained strongly suggest that a correct folding of pRB, integrating its small pocket and carboxyl terminus into the large pocket, is critically required to efficiently inhibit IR-induced cell death and to block cell cycle progression in the long-term. Finally, we have created in-silico a 3-dimensional model of full-length pRB that shows the presence of an amino acid from the carboxyl terminus (R798) in the soil of the pocket, next to an amino acid from the A-domain (K530). The analysis of the conformational changes induced by inactivating mutations in the A- or in the B-domain showed that the carboxyl terminus was more severely affected than the small pocket. The in-silico analysis therefore supports the notion that the functional pRB large pocket is the result of the structural integration of the small pocket and the carboxyl terminus.