Interaction studies between the p21Cip1/Waf1 cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen (PCNA) by surface plasmon resonance.
The cyclin-dependent kinase (CDK) inhibitor p21Cip1 consists of two domains that interact with CDKs and proliferating cell nuclear antigen (PCNA), respectively. We have investigated the interaction between p21Cip1 and PCNA using surface plasmon resonance (SPR) technology and compared the results with those obtained from other sources such as the yeast two-hybrid system. Whilst other methods are only semi-quantitative, the SPR technique allowed us to determine the kinetic parameters of the interaction. The apparent equilibrium constant KD calculated for these kinetic parameters was 3.2 x 10(-7) M. We further demonstrate the use of SPR to study the interaction between mutant proteins and to determine their actual KD. The interaction between p21Cip1/PCNA is shown to be dependent upon the trimeric conformation of PCNA since a point mutant that abolishes PCNA-PCNA interaction also abolishes PCNA's interaction with p21Cip1. Finally, we demonstrate that SPR can be used to characterise the interaction of p21Cip1 and PCNA in the presence of short competitive peptides.
Knibiehler
M
M
Institut de Pharmacologie et de Biologie Structurale, Université, P. Sabatier, Toulouse, France.
Goubin
F
F
Escalas
N
N
Jónsson
Z O
ZO
Mazarguil
H
H
Hübscher
U
U
Ducommun
B
B
D000595:Amino Acid Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D005982:Glutathione Transferase; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D018844:Cyclin-Dependent Kinases