Construction of a cyclin D1-Cdk2 fusion protein to model the biological functions of cyclin D1-Cdk2 complexes.
Cyclin D1 is frequently overexpressed in human breast cancers, and cyclin D1 overexpression correlates with poor prognosis. Cyclin D1-Cdk2 complexes were previously observed in human breast cancer cell lines, but their role in cell cycle regulation and transformation was not investigated. This report demonstrates that Cdk2 in cyclin D1-Cdk2 complexes from mammary epithelial cells is phosphorylated on the activating phosphorylation site, Thr(160). Furthermore, cyclin D1-Cdk2 complexes catalyze Rb phosphorylation on multiple sites in vitro. As a model to investigate the biological and biochemical functions of cyclin D1-Cdk2 complexes, and the mechanisms by which cyclin D1 activates Cdk2, a cyclin D1-Cdk2 fusion gene was constructed. The cyclin D1-Cdk2 fusion protein expressed in epithelial cells was phosphorylated on Thr(160) and catalyzed the phosphorylation of Rb on multiple sites in vitro and in vivo. Kinase activity was not observed if either the cyclin D1 or Cdk2 domain was mutationally inactivated. Mutational inactivation of the cyclin D1 domain prevented activating phosphorylation of the Cdk2 domain on Thr(160). These results indicate that the cyclin D1 domain of the fusion protein activated the Cdk2 domain through an intramolecular mechanism. Cells stably expressing the cyclin D1-Cdk2 fusion protein exhibited several hallmarks of transformation including hyperphosphorylation of Rb, resistance to TGFbeta-induced growth arrest, and anchorage-independent proliferation in soft agar. We propose that cyclin D1-Cdk2 complexes mediate some of the transforming effects of cyclin D1 and demonstrate that the cyclin D1-Cdk2 fusion protein is a useful model to investigate the biological functions of cyclin D1-Cdk2 complexes.
Chytil
Anna
A
Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37232, USA.
Waltner-Law
Mary
M
West
Robert
R
Friedman
David
D
Aakre
Mary
M
Barker
Dana
D
Law
Brian
B
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism; D042846:CDC2-CDC28 Kinases / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D006801:Humans; D046911:Macromolecular Substances; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism; D020123:Sirolimus / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D019938:Cyclin D1; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D020123:Sirolimus