Hypo-Phosphorylation of the Retinoblastoma Protein (pRb) by Cyclin D:Cdk4/6 Complexes Results in Active pRb

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G₁ and becomes hyper-phosphorylated in late G₁. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15^INK4b by transforming growth factor β treatment of keratinocytes results in G₁ arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15^INK4b-independent transforming growth factor β-mediated G₁ arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16^INK4a protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G₁ allowing continued E2F binding.