UC San Diego

The prevailing model of G1 cell cycle progression proposes Cyclin D:Cyclin-dependent kinase 4/6 (Cdk4/6) complexes inactivate the retinoblastoma tumor suppressor protein (Rb) during Early G1 phase by progressive multi- phosphorylation or "hypo-phosphorylation" to release E2F transcription factors, resulting in the gradual activation of Cyclin E:Cdk2 complexes. However, due to the use of supra-physiologic overexpression studies, absence of quantification of how many phosphates are present on "hypo -phosphorylated" Rb, and lack of clarity on what is the active isoform of Rb, this model remains largely unproven. Rb contains 16 Cdk phosphorylation sites and is thought to exist in three general isoforms: 1) un-phosphorylated Rb, 2) "hypo-phosphorylated" Rb, and 3) inactive hyper- phosphorylated Rb; however, the un-phosphorylated Rb and "hypo-phosphorylated" Rb isoforms cannot be resolved by 1D SDS-PAGE. Using highly synchronized primary and tumorigenic cells, performing biochemistry on physiologic levels of activities of proteins, and developing 2D isoelectric focusing (2D IEF) of Rb, I show that Rb is exclusively mono-phosphorylated on 14 different sites during Early G1 phase and that this phosphorylation is mediated by Cyclin D:Cdk4/6 complexes. Mono-phosphorylated Rb functioned to induce a G1 arrest, bind E2F transcription factors and regulate the global transcriptional profile, whereas un-phosphorylated Rb was non-functional, showing that mono-phosphorylated Rb is the biologically active isoform of Rb. These observations fundamentally change our understanding of G1 cell cycle regulation and show that Cyclin D:Cdk4/6 complexes activate Rb by mono-phosphorylation during Early G1 phase. Importantly, these observations point to the activation of Cyclin E:Cdk2 complexes as a likely key oncogenic step in the progression of cancer