In the last funding period we obtained important findings suggesting that each of the four important proteins gankyrin, IkappaBalpha, Tax, and SEI-1 interacts with cyclin-dependent kinase 4 (CDK4), with potentially different mechanisms. The main thrust of the next granting period is to understand the structural and biochemical mechanisms and biological functions of the interactions between CDK4 and three constitutively expressed human proteins, gankyrin, IkappaBalpha, and SEI-1, while the viral protein Tax is not included in this revised proposal. Three major approaches will be used: (a) use of NMR to determine solution structures; (b) use of rational mutagenesis combined with activity assays to dissect the structural basis of specificity; (c) use of fluorescence-based binding assay to determine the Kd of gankyrin, IkappaBalpha, and SEI-1 with CDK4.
In Specific Aim 1, we plan to use site-directed mutagenesis to identify key residues of gankyrin important for its binding to CDK4, and the Kd values of gankyrin and mutants will be evaluated using a fluorescence-based assay. Subsequently, a set of CDK4 mutants will be generated through rational design and will be used to map out the residues of CDK4 involved in the binding of gankyrin. Furthermore, a gankyrin/CDK4 structural model will be generated using both NMR and mutagenesis data, hi collaboration with C. Weghorst, we will characterize gankyrin mutants identified from cancer tissues.
In Specific Aim 2, we plan to solve the solution structure of the CDK4-binding domain of IkappaBalpha (residues 1-206), which is also an ankyrin-repeat protein. The same mutagenesis studies as described for gankyrin-CDK4 interactions will be used to map out the interaction between IkappaBalpha and CDK4. These results will allow comparison between gankyrin, IkappaBalpha, and the INK4 family of tumor suppressors p16 and p18.
In Specific Aim 3, we plan to determine the structures of SEI-1 and/or its truncated forms possessing different activities, and use site-directed mutagenesis to dissect the residues important for different activities, and to identify CDK4 residues important for binding of SEI-1 and different functional domains of SEI-1.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA069472-09A1
Application #
6927651
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Knowlton, John R
Project Start
1996-03-01
Project End
2010-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
9
Fiscal Year
2005
Total Cost
$285,919
Indirect Cost
Name
Ohio State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Poi, Ming J; Knobloch, Thomas J; Li, Junan (2017) Deletion of RDINK4/ARF enhancer: A novel mutation to ""inactivate"" the INK4-ARF locus. DNA Repair (Amst) 57:50-55
Poi, Ming J; Knobloch, Thomas J; Sears, Marta T et al. (2015) Alterations in RD(INK4/ARF) -mediated en bloc regulation of the INK4-ARF locus in human squamous cell carcinoma of the head and neck. Mol Carcinog 54:532-42
Li, Junan; Knobloch, Thomas J; Poi, Ming J et al. (2014) Genetic alterations of RD(INK4/ARF) enhancer in human cancer cells. Mol Carcinog 53:211-8
Yuan, Chunhua; Guo, Yi; Zhu, Lu et al. (2013) The study of pH-dependent stability shows that the TPLH-mediated hydrogen-bonding network is important for the conformation and stability of human gankyrin. Biochemistry 52:4848-57
Poi, Ming J; Knobloch, Thomas J; Yuan, Chunhua et al. (2013) Evidence that P12, a specific variant of P16(INK4A), plays a suppressive role in human pancreatic carcinogenesis. Biochem Biophys Res Commun 436:217-22
Li, Junan; Knobloch, Thomas J; Kresty, Laura A et al. (2011) Gankyrin, a biomarker for epithelial carcinogenesis, is overexpressed in human oral cancer. Anticancer Res 31:2683-92
Li, Junan; Poi, Ming Jye; Tsai, Ming-Daw (2011) Regulatory mechanisms of tumor suppressor P16(INK4A) and their relevance to cancer. Biochemistry 50:5566-82
Guo, Yi; Yuan, Chunhua; Weghorst, Christopher M et al. (2010) IKKbeta specifically binds to P16 and phosphorylates Ser8 of P16. Biochem Biophys Res Commun 393:504-8
Guo, Yi; Yuan, Chunhua; Tian, Feng et al. (2010) Contributions of conserved TPLH tetrapeptides to the conformational stability of ankyrin repeat proteins. J Mol Biol 399:168-81
Guo, Yi; Mahajan, Anjali; Yuan, Chunhua et al. (2009) Comparisons of the conformational stability of cyclin-dependent kinase (CDK) 4-interacting ankyrin repeat (AR) proteins. Biochemistry 48:4050-62

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