Cyclin-binding motifs are essential for the function of p21CIP1.
The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.
Chen
J
J
Department of Pathology, Division of Molecular Oncology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Saha
P
P
Kornbluth
S
S
Dynlacht
B D
BD
Dutta
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D029867:Xenopus Proteins; D014982:Xenopus laevis
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases