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 G(1) and becomes hyper-phosphorylated in late G(1). The role of hypophosphorylation 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 beta treatment of keratinocytes results in G(1) arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb, Conversely, p15(INK4b)-independent transforming growth factor beta-mediated G(1) 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 it 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 G(1) allowing continued E2F binding.