Isolation of Proteins Interacting With the Cyclin D1-CDK6 Complex From Normal and Tumorigenic Human Breast Cells Using a Novel Yeast Three-Hybrid System
Abstract
The eukaryotic cell division cycle is primary controlled by a family of protein serine/threonine kinases, the cyclin-dependent-kinases (CDKs). Activation of CDKs is dependent upon the interaction with an activating cyclin subunit. Presumably, these kinases progress the cell cycle by phosphorylation of critical cellular proteins. The detection of these transient kinase-substrate protein-protein interactions using conventional methods presents a major technical challenge, and is primarily responsible for our poor understanding of CDK substrates. The yeast two-hybrid system is a method for detecting protein-protein interactions. A major limitation of this method is the fact that only binary interactions can be detected. Using a modified version of the two-hybrid system, I am able to detect the interaction of ternary protein complexes. Using the cyclin D1-CDK6 protein complex as bait, I have identified the interaction of this complex with the tumor suppressor protein tuberin. Tuberin interacts specifically with the D-type cyclins, and this interaction has been found to exist in lysates from whole mouse embryos. Using the yeast three-hybrid system, it may be possible to detect the cellular substrates of cyclin-CDK complexes in human breast cells and further our understanding of the pathogenesis of human breast cancer.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 1999
- Accession Number
- ADA392324
Entities
People
- Michael A. Nichols
- Yue Xiong
Organizations
- University of North Carolina at Chapel Hill