Complex Regulation of RETINOBLASTOMA-RELATED's Interactions With E2Fs via Phosphorylation

Arabidopsis RETINOBLASTOMA-RELATED (RBR) regulates cell proliferation by interacting with E2F transcription factors and DIMERISATION PARTNER, RB-LIKE, E2F AND MULTI-VULVAL CLASS B COMPLEX (DREAM) components. Although CDK-CYCD phosphorylation is believed to affect RBR's E2F-binding capacity, the precise phosphorylation events inhibiting RBR's cell cycle function remain unclear. This study found RBR phosphorylated at 13 of 16 CDK sites in Arabidopsis, with many phosphorylated forms still binding E2Fs. In contrast, multi-phosphorylated RBR forms with phosphorylated 911S site in Arabidopsis thaliana and corresponding sites in Medicago truncatula or Brassica napus do not co-purify with E2Fs and DREAM components but interact with RNA-binding proteins involved in post-transcriptional regulation through ribosomal biogenesis and protein translation. The 911S phosphorylation is high in proliferating cells and rapidly diminishes under DNA damage conditions, indicating its role in switching from proliferation to quiescence under stress. However, molecular modelling implies that this site is not accessible for phosphorylation if RBR is in complex with E2Fs. These findings suggest that different phosphorylation events inhibit RBR's capacity to form complexes with E2Fs and to release E2Fs from RBR inhibition. We posit that multi-site phosphorylation coupled to 911S impedes free RBR's binding to E2Fs and DREAM components, but this is not the initial inhibitory phosphorylation contributing to the disruption of RBR-E2F-DP complexes. Rather, it facilitates RBR interaction with proteins involved in post-transcriptional cell cycle regulation.