Kinetics of CDK4/6 inhibition determine different temporal locations of the restriction point

Mitogenic and stress signaling pathways regulate CDK4/6 activity to control entry into the cell cycle until cells pass the restriction point, after which cells irreversibly commit to the cell cycle. Previous studies have proposed that positive feedback between CDK2 and Rb, and double-negative feedback between APC/CCdh1 and Emi1 cause irreversible Rb hyperphosphorylation and APC/CCdh1 inactivation, respectively, to trigger the restriction point. Here, we propose that CDK2-Rb feedback is the primary signaling network that regulates the restriction point with respect to both mitogenic and stress signaling. We show that CDK4/6 activity is sufficient to initiate Rb hyperphosphorylation and E2F activation, and then high CDK2 activity maintains Rb hyperphosphorylation, triggering the transition into CDK4/6-independent cell-cycle entry. While mitogen removal results in the slow inactivation of CDK4/6, induction of stress rapidly suppresses CDK4/6 activity and can reverse cell-cycle entry even after APC/CCdh1 inactivation. An increase in CDK2 activity triggers CDK2-Rb feedback without involvement of other mechanisms in S phase, indicating that activity-based substrate specificity for CDK2 is the key determinant in initiating CDK2-Rb feedback. Our finding that mitogenic and stress signaling induce different temporal locations of the restriction point reveals how cells utilize a safety mechanism to differentially regulate cell-cycle entry depending on extracellular environment.