The molecular events governing entry into S phase in mammalian—and in fact all metazoan—cells are remarkably similar to those in budding yeast (Figure 19-12b). G₁ cyclins are present throughout G₁ and are often found to be expressed at increased levels in response to growth factors. In turn, the G₁ CDKs activate members of a small family of related transcription factors, referred to collectively as E2F transcription factors (E2Fs). During G₁, E2Fs are held inactive through their association with the retinoblastoma protein (Rb) until G₁ CDKs activate E2Fs by phosphorylating and inactivating Rb. E2Fs then activate genes encoding many of the proteins involved in DNA synthesis. They also stimulate transcription of genes encoding the G₁/S phase cyclins and the S phase cyclins. Thus the E2Fs have a function in late G₁ that is similar to that of the S. cerevisiae transcription factor complex SBF.

Key to the regulation of E2F function is the Rb (retinoblastoma) protein. When E2Fs are bound to Rb, they function as transcriptional repressors. This is because Rb recruits chromatin-modifying enzymes that promote deacetylation and methylation of specific histone lysines, causing chromatin to assume a condensed, transcriptionally inactive form. RB was initially identified as the gene mutated in retinoblastoma, a childhood cancer of the retina. Subsequent studies found Rb to be inactivated in many cancers, either by mutations in both alleles of RB or by abnormal regulation of Rb phosphorylation.

Rb protein regulation by G₁ CDKs in mammalian cells is analogous to Whi5 regulation by Cln3-CDK in yeast. Phosphorylation on multiple sites by G₁ CDKs prevents Rb from associating with E2F and promotes its export out of the nucleus. This allows E2F to activate the transcription of genes required for entry into S phase. Once the expression of genes coding for the G₁/S cyclins and CDK has been induced by phosphorylation of some of the Rb molecules, the resulting G₁/S phase CDK complexes further phosphorylate Rb in late G₁. This is one of the principal biochemical events responsible for passage through the restriction point. Since E2F stimulates its own expression as well as that of the G₁/S cyclin-CDKs, positive cross-regulation of E2F and G₁/S cyclin-CDKs produces a rapid rise of both activities in late G₁.

As they accumulate, S phase CDKs and mitotic CDKs maintain Rb protein in the phosphorylated state throughout the S, G₂, and early M phases. After cells complete anaphase and enter early G₁ or G₀, a fall in all cyclin-CDK activities leads to dephosphorylation of Rb. As a consequence, hypophosphorylated Rb is available to inhibit E2F activity during early G₁ of the next cycle and in G₀-arrested cells. Thus G₁/S phase CDK activity remains low until cells decide to enter a new cell cycle and G₁ CDKs break the inhibitory grip of Rb on E2F.