p21

p21 is a potent cyclin-dependent kinase inhibitor (CKI). The p21 (CIP1/WAF1) protein binds to and inhibits the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes, and thus functions as a regulator of cell cycle progression at G₁ and S phase.[12][13] The binding of p21 to CDK complexes occurs through p21's N-terminal domain, which is homologous to the other CIP/KIP CDK inhibitors p27 and p57.[6] Specifically it contains a Cy1 motif in the N-terminal half, and weaker Cy2 motif in the C-terminal domain that allow it to bind CDK in a region that blocks its ability to complex with cyclins and thus prevent CDK activation.[14]

Experiments looking at CDK2 activity within single cells have also shown p21 to be responsible for a bifurcation in CDK2 activity following mitosis, cells with high p21 enter a G₀/quiescent state, whilst those with low p21 continue to proliferate.[15] Follow up work, found evidence that this bistability is underpinned by double negative feedback between p21 and CDK2, where CDK2 inhibits p21 activity via ubiquitin ligase activity.[16]

p21 interacts with proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair.[17][18][19] Specifically, p21 has a high affinity for the PIP-box binding region on PCNA,[20] binding of p21 to this region is proposed to block the binding of processivity factors necessary for PCNA dependent S-phase DNA synthesis, but not PCNA dependent nucleotide excision repair (NER).[21] As such, p21 acts as an effective inhibitor of DNA S-phase DNA synthesis though permits NER, leading to the proposal that p21 acts to preferentially select polymerase processivity factors depending on the context of DNA synthesis.[22]

This protein was reported to be specifically cleaved by CASP3-like caspases, which thus leads to a dramatic activation of CDK2, and may be instrumental in the execution of apoptosis following caspase activation. However p21 may inhibit apoptosis and does not induce cell death on its own.[23] The ability of p21 to inhibit apoptosis in response to replication fork stress has also been reported.[24]

Studies of p53 dependent cell cycle arrest in response to DNA damage identified p21 as the primary mediator of downstream cell cycle arrest. Notably, El-Deiry et al. identified a protein p21 (WAF1) which was present in cells expressing wild type p53 but not those with mutant p53, moreover constitutive expression of p21 led to cell cycle arrest in a number of cell types.[25] Dulcic et al. also found that γ-irradiation of fibroblasts induced a p53 and p21 dependent cell cycle arrest, here p21 was found bound to inactive cyclin E/CDK2 complexes.[26] Working in mouse models, it was also shown that whilst mice lacking p21 were healthy, spontaneous tumours developed and G1 checkpoint control was compromised in cells derived from these mice.[27][13] Taken together, these studies thus defined p21 as the primary mediator of p53-dependent cell cycle arrest in response to DNA damage.

Recent work exploring p21 activation in response to DNA damage at a single-cell level have demonstrated that pulsatile p53 activity leads to subsequent pulses of p21, and that the strength of p21 activation is cell cycle phase dependent.[28] Moreover, studies of p21-levels in populations of cycling cells, not exposed to DNA damaging agents, have shown that DNA damage occurring in mother cell S-phase can induce p21 accumulation over both mother G2 and daughter G1 phases which subsequently induces cell cycle arrest;[29] this responsible for the bifurcation in CDK2 activity observed in Spencer et al..[15]

p21 is negatively regulated by ubiquitin ligases both over the course of the cell cycle and in response to DNA damage. Specifically, over the G1/S transition it has been demonstrated that the E3 ubiquitin ligase complex SCF^Skp2 induces degradation of p21.[30][31] Studies have also demonstrated that the E3 ubiquitin ligase complex CRL4^Cdt2 degrades p21 in a PCNA dependent manner over S-phase, necessary to prevent p21 dependent re-replication,[32] as well as in response to UV irradiation.[33] Recent work has now found that in human cell lines SCF^Skp2 degrades p21 towards the end of G1 phase, allowing cells to exit a quiescent state, whilst CRL4^Cdt2 acts to degrade p21 at a much higher rate than SCF^Skp2 over the G1/S transition and subsequently maintain low levels of p21 throughout S-phase.[29]