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Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1
1Cell Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10021, USA.
Abstract:
Mammalian cell-cycle control by antimitogenic signals involves p21Cip1/WAF1 (refs 1-4), p27Kip1 (refs 5, 6) and p57Kip2 (refs 7, 8), a family of proteins that bind to and inhibit cyclin-dependent kinases (CDKs) required for initiation of S phase. The protein p21 also binds to the DNA polymerase delta processivity factor, proliferating-cell nuclear antigen (PCNA), and inhibits in vitro PCNA-dependent DNA replication. The CDK and PCNA inhibitory activities of p21 are shown here to be functionally independent and to reside in separate protein domains. The PCNA binding and inhibitory activities, which are not observed with p27 or p57, reside in the C-terminal domain of p21, whereas the CDK inhibitory activity resides in the conserved N-terminal domains of these proteins. When separately overexpressed in mammalian cells, the CDK and PCNA inhibitory domains prevent DNA replication, demonstrating a dual function of p21 as a cell-cycle inhibitor in vivo.
Insights
The protein p21 inhibits cell division by targeting both cyclin-dependent kinases (CDKs) and proliferating cell nuclear antigen (PCNA). These dual inhibitory functions are independent and located in separate domains of p21, controlling DNA replication.
Area of Science:
- Molecular Biology
- Cell Biology
Background:
- Mammalian cell-cycle progression is regulated by antimitogenic signals.
- Key regulators include p21Cip1/WAF1, p27Kip1, and p57Kip2, which inhibit cyclin-dependent kinases (CDKs).
- p21Cip1/WAF1 also interacts with proliferating cell nuclear antigen (PCNA), inhibiting DNA replication.
Purpose of the Study:
- To investigate the functional independence of p21's CDK and PCNA inhibitory activities.
- To determine the specific protein domains responsible for these distinct functions.
- To confirm the role of these domains in cell-cycle inhibition in vivo.
Main Methods:
- Analysis of protein-protein interactions between p21, CDKs, and PCNA.
- Domain mapping to identify regions responsible for CDK and PCNA binding/inhibition.
- Overexpression of separate p21 functional domains in mammalian cells.
Main Results:
- p21's CDK and PCNA inhibitory activities are functionally independent.
- CDK inhibition is mediated by the N-terminal domain of p21.
- PCNA binding and inhibition reside in the C-terminal domain of p21, a function not shared by p27 or p57.
- Separate overexpression of these domains in cells inhibits DNA replication.
Conclusions:
- p21 possesses a dual function in cell-cycle inhibition.
- The N-terminal domain inhibits CDKs, while the C-terminal domain inhibits PCNA and DNA replication.
- These findings elucidate the distinct molecular mechanisms underlying p21's role as a cell-cycle inhibitor.