Overexpression of a stabilized mutant form of the cyclin-dependent kinase inhibitor p27(Kip1) inhibits cell growth.
OBJECTIVE
The purpose of this study was to test the hypothesis that the expression of the mutant p27(Kip1) protein enhances cell growth inhibition and is more stable than that of the wild-type p27(Kip1).


METHODS
Site-directed mutagenesis was used to mutate threonine 187 to an alanine residue, generating the mutant p27(Kip1). To study the effects of the p27(Kip1) mutant on cell growth, luciferase assays were performed. Cells were transiently transfected with the Renilla luciferase reporter construct and empty vector, wild-type p27(Kip1), or mutant p27(Kip1) using Fugene 6. The transfected cells were lysed and assayed for luciferase activity 24 h later with a dual-luciferase reporter assay system. To further assess the effects of the p27(Kip1) mutant on cell growth, colony count assays were performed. The experiments were repeated in duplicate and a standard two-tailed Student t test was use to analyze the data.


RESULTS
Wild-type p27(Kip1) protein has a half-life of approximately 2 h while the p27(Kip1) mutant has a half-life of greater than 12 h. Furthermore, the p27(Kip1) mutant retained the ability to inhibit CDK2-associated H1 kinase activity. Cells expressing the p27(Kip1) mutant had an 88% reduction in luciferase activity compared to cells expressing the wild-type p27(Kip1) (P = 0.001). Colony assays revealed that cells expressing the p27(Kip1) mutant had fewer colonies compared to cells expressing the wild-type p27(Kip1) (P = 0.04).


CONCLUSIONS
These data are consistent with the hypothesis that the mutated form of p27(Kip1) is more effective in cell growth inhibition than the wild-type p27(Kip1) protein.
Hurteau
Jean A
JA
Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois 60612-7313, USA.
Brutkiewicz
Susan A
SA
Wang
Qi
Q
Allison
Bernadette M
BM
Goebl
Mark G
MG
Harrington
Maureen A
MA
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006131:Growth Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011494:Protein Kinases / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
D018797:Cell Cycle Proteins; D006131:Growth Inhibitors; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D008156:Luciferases; D011494:Protein Kinases; C102333:histone H1 kinase