Use of the two-hybrid system to identify protein-protein interaction temperature-sensitive mutants: application to the CDK2/p21Cip1 interaction.
We describe the application of the two-hybrid system to the identification of protein-protein interaction temperature-sensitive mutants. We applied this strategy to the interaction between the human CDK2 cell cycle regulator and the p21Cip1 regulatory subunit. A library of randomly generated CDK2 mutant proteins was screened for interaction with p21Cip1 at different temperatures. This approach resulted in the isolation of single point mutations in CDK2 causing temperature-sensitive interaction with p21Cip1. Our results demonstrate that the two-temperature two-hybrid screen is an efficient approach for the rational design and screening of protein-protein interaction conditional mutations.
Cayrol
C
C
Institut de Pharmacologie et de Biologie Structurale du CNRS, UPR 9062, Université Paul Sabatier, 205 route de Narbonne, 31077 Toulouse, France.
Cabrolier
G
G
Ducommun
B
B
D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000235:genetics* / Q000378:metabolism; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases