Tsai Laboratory
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Tsai Laboratory |
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INK4 Tumor Suppressor Proteins in the CDK4/Rb Pathway. Tumor suppressor p16. Discovered in 1995, p16 has been found to be mutated more frequently than the best known tumor suppressor p53 in various cancer cells. Its main function is to serve as a negative regulator of the cell cycle by binding to and inhibiting cyclin-dependent kinase 4 (CDK4). Our lab is the first and only group to solve the tertiary structure of free p16 and identified key binding residues. Several more papers have been published subsequently to address the structural and functional properties of p16 and its homologous proteins p15, p18, and p19. Other labs have solved the crystal structures of p18, p19, as well as p16-CDK6 complexes.
Stereo view of the structure of tumor suppressor p16 (pdbID=1A5E)
Stereo view of the structure of tumor suppressor p18 (pdbID=1BU9)
Gankyrin. Our studies with the INK4 family, which are ankyrin-repeat proteins, led us to study another newly discovered ankyrin-repeat oncogenic protein called gankyrin, which contains six ankyrin repeats. It has been reported to be involved in the phosphorylation and degradation of the retinoblsatoma gene product, Rb. Using in vitro systems, we have identified a peptide fragment of gankyrin responsible for binding of gankyrin to Rb. We further demonstrated a different mechanism for gankyrin to facilitate the phosphorylation of Rb – by binding with CDK4. This binding does not inhibit the Rb-phosphorylating kinase activity of CDK4, but it competes with p16 binding to CDK4 and counteracts the inhibitory function of p16. We then showed that the two mechanisms involve different structural regions of gankyrin: the Rb-binding motif is located at the fifth ankyrin repeat, whereas the CDK4-binding region is located in the first three or four ankyrin repeats. The solution structure of gankyrin is in the final stage of refinement and will be available soon.
Tax Protein. In another study, we showed that the Tax oncoprotein encoded by human T-cell leukemia virus 1 (HTLV-1) binds to and activates CDK4 in vitro, and that such binding counteracts against the inhibition of p16 and p18, and acts as the major path to regulate Tax-mediated activation of CDK4. We have identified the binding region lies in the first 40 residues. also report that Tax40N retains the transactivation ability. These results of in vitro studies demonstrate a potentially novel, p16-independent route to regulate CDK4 activity by the Tax oncoprotein in HTLV-1 infected cells.
IkB. IkBa, a protein composed of 6 ankyrin repeats, is a specific inhibitor of nuclear factor kB (NF-kB) and functions in signal transduction in many different cell types. Using both in vivo yeast two-hybrid assays and in vitro activity and binding assays, we showed that IkBa binds to cyclin-dependent kinase 4 (CDK4) specifically and inhibits the kinase activity. The potency of binding and inhibition of IkBa are comparable to those of INK4 proteins, the specific CDK4 inhibitors that also contain ankyrin repeats. We showed that INK4 proteins and IkBa compete with each other for binding to CDK4. These results led us to propose a hypothesis that there is crossing between the NF-kB /IkBa pathway and the p16/CDK4/Rb pathway in cells, and that IkBa could substitute for the CDK4-inhibiting function of p16, a tumor suppressor frequently inactivated in human tumors. To further understand the structural basis of IkBa-CDK binding, we used different mutants of CDK4 to show that there are notable differences between IkBa and INK4 proteins in CDK4 binding since the inhibition is affected differently by different CDK4 mutations. We also demonstrated that, while most of the contacts contributing to NF-kB binding are located within the last two C-terminal ankyrin repeats and the loop region bridging these, the first four ankyrin repeats at the N-terminus are responsible for CDK4 binding and inhibition.
New publications:
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