begins to accumulate and completes pRb phosphorylation, as shown in the figure. Cdk inhibitors and regulation of Cyclin D/Cdk complex activity p27 and p21 are stoichiometric inhibitors of G1/S- and S-cyclin-Cdk complexes. While p21 levels increase during cell-cycle entry, p27 is generally inactivated as cells progress to late G1. High cell density, mitogen starvation, and TGF-β result in accumulation of p27 and cell cycle arrest. Similarly, DNA damage and other stressors increase p21 levels, while mitogen-stimulated ERK2 and Akt activity leads to inactivating phosphorylation of p21. Early work on p27 overexpression suggested that it can associate with and inhibit cyclin D-Cdk4/6 complexes and cyclin E/A-Cdk2 complexes in vitro and in select cell types. However, kinetic studies by LaBaer et al. (1997) found that titrating in p21 and p27 promotes assembly of the cyclin d-Cdk complex, increasing overall activity and nuclear localization of the complex. Subsequent studies elucidated that p27 may be required for cyclin D-Cdk complex formation, as p27-/-, p21-/- MEFs showed a decrease in cyclin D-Cdk4 complexation that could be rescued with p27 re-expression. Work by James et al. (2008) further suggests that phosphorylation of tyrosine residues on p27 can switch p27 between an inhibitory and non-inhibitory state while bound to cyclin D-Cdk4/6, offering a model for how p27 is capable of regulating both cyclin-Cdk complex assembly and activity. Association of p27 with cyclin D-Cdk4/6 may further promote cell cycle progression by limiting the pool of p27 available for inactivating cyclin E-Cdk2 complexes. Increasing cyclin E-Cdk2 activity in late G1 (and cyclin A-Cdk2 in early S) leads to p21/p27 phosphorylation that promotes their nuclear export, ubiquitination, and degradation. Dynamics A paper published by the Lingchong You and Joe Nevins groups at Duke University in 2008 demonstrated that the a bistable hysteric E2F switch underlies the restriction point. E2F promotes its own activation, and also promotes the inhibition of its own inhibitor (pRb), forming two feedback loops (among others) that are important in establishing bistable systems. The authors of this study used a destabilized GFP-system under the control of the E2F promoter as a readout of E2F activity. Serum-starved cells were stimulated with varying serum concentrations, and the GFP readout was recorded at a single-cell level. They found that the GFP reporter was either on or off, indicating that E2F was either completely activated or deactivated at all of the different serum levels analyzed. Further experiments, in which they analyzed