The C-terminal region of p21SDI1/WAF1/CIP1 is involved in proliferating cell nuclear antigen binding but does not appear to be required for growth inhibition.
The cyclin-dependent kinase (Cdk) inhibitor p21SDI1/WAF1/CIP1 has been found to be involved in cell senescence, cell cycle arrest, and differentiation. p21SDI1 inhibits the activity of several Cdks, in contrast to other inhibitors such as p15INK4B and p16INK4A, which act on specific cyclin-Cdk complexes. Of interest were reports that p21SDI1 also bound proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase delta, and inhibited DNA replication but not DNA repair in vitro. To better understand the function of this interaction in vivo, we first determined the region of p21SDI1 that was needed for PCNA binding. Analysis of deletion mutants of p21SDI1, which covered the majority of the protein, revealed that deletion of either amino acids 142-147 or 149-154 resulted in loss of ability to bind a glutathione S-transferase-PCNA fusion protein. Site-directed mutagenesis in this region led to the identification of the PCNA binding motif RQXXMTXFYXXXR and demonstrated that mutation of either amino acid Met-147 or Phe-150 resulted in almost complete ablation of PCNA binding. Interestingly, when we determined DNA synthesis inhibitory activity of deletion mutants or point mutants that were unable to bind Cdk2 and/or PCNA, we found that loss of binding to PCNA did not affect inhibitory activity, whereas lack of Cdk2 binding greatly reduced the same. This result suggests that the primary mechanism for inhibition of DNA synthesis by p21SDI1 occurs via inhibition of Cdk activity.
Nakanishi
M
M
Roy M. and Phyllis Gough Huffington Center on Aging, Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030-3498, USA.
Robetorye
R S
RS
Pereira-Smith
O M
OM
Smith
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016213:Cyclins / Q000737:chemistry / Q000502:physiology*; D004247:DNA / Q000096:biosynthesis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013329:Structure-Activity Relationship
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases