Mutations in the acetylation hotspots of Rbl2 are associated with increased risk of breast cancer.

Retinoblastoma like protein-2 (Rbl2) is functionally regulated by phosphorylation and acetylation. Previously, we demonstrated that lysine K1083 (K1079 in human Rbl2) is a potential target for acetylation but its functional role remains elusive. We investigated alterations in human Rbl2 gene specifically targeting exons 19-22 harbouring acetylatable residues i.e. K1072, K1083 and K1115 through single stranded conformation polymorphism (SSCP) in breast cancer patients. The K1083 was found altered into arginine (R) in 51% of the cases but K1072 and K1115 remained conserved. The 'K1083R' mutation impairs the acetylation potential of this motif that may result in functional inactivation of Rbl2. These patients also showed poor survival outcome that highlights prognostic relevance of this residue. NIH3T3 cells expressing glutamine (K1083Q) mutated Rbl2 could not be arrested in G1 by serum starvation, whereas cells expressing Rbl2 with K1083R showed prolonged G1 arrest in fluorescence activated cell sorting (FACS) analysis. This suggests that K1083 acetylation is important for G1/S transition. Further, we performed molecular dynamic simulations (MDS) to analyse kinetics of residue K1083 with Cyc-D1/CDK4. Mutations at K1083 impaired this binding exposing neighbouring residues S1080, P1081, S1082 and R1084, hence enhancing the possibility of accelerated phosphorylation. S1080 has previously been reported as a promising candidate of cell cycle dependent phosphorylation in Rbl2. This highlights significance of mutations in the pocket domain of Rbl2 gene in breast cancer, and also strengthen the supposition that K1083 acetylation is pre-requisite for its phosphorylation.