1. Bub1 & Colorectal Cancer Julie Clarke March 18, 2003

2. Bub1- quick facts 150kD Protein Chromosome 2, 2q14 Serine/Threonine Protein Kinase –Self phosphorilates Highly Conserved First discovered in Budding Yeast

3. Bub1- Localized to Kinetochore 3 Domains Kinetochore Localization Domain Central Domain Kinase Domain Yu Ru et. al.

4. Proof of Kinetochore Localization Blue Stain for Chromosomes Pink Stain for Tubulin Green Stain for Bub1 with antibody Sharp-Baker et. al.

5. Bub1 allows others to bind Kinetochore Sharp-Baker et. al.

6. Bub1 is required for Mad1, Mad2, Bub3 and CENP-E to bind Sharp-Baker et. al.

7. Bub1- Normal vs. Mutant Function a. Wild Type Cells- Bub1 does its job b. Bub1 mutant STOP!! Chromosome loose Spindle Reattached, continue on to anaphase No Stop signal, continue on to anaphase with mis-segregation Causes Chromosome Instability Jallepalli et. al.

8. Bub1- Mitotic Spindle Checkpoint Mutations inactivate mitotic spindle checkpoint –Insert mutant Bub1 into normal cell –Treat Cells with nocodazole to disrupt microtubules –Cells do not arrest and continue into anaphase with out proper segregation –Causes Chromosome Instability –Shows a Dominant Negative Mutation Sharp-Baker et.al.

9. Bub1 checkpoint independent of kinase activity Bub1(K872R) mutant with disrupted kinase domain Insert normal Bub1 and Bub1(K872R) into Bub1 depleted Xenopus egg extracts Both Bub1 and Bub1(K872R) restored mitotic checkpoint and allowed other kinetochore proteins to bind

10. Bub1 is required for Mad1, Mad2, Bub3 and CENP-E to bind Bub1(K872R) also allows Mad1, Mad2, Bub3, and CENP-E to bind Sharp-Baker et. al.

11. Colorectal Cancer? Yes- 2 out of 19 CIN colorectal tumors had Bub1 mutations Shows reduction in in number of cells in mitosis and reduction in BrdU incorporation in mutant cell lines this means less cells in mitosis, more cells in S phase(replicating DNA) Cahill et. al.

12. Bub1 and Cancer The Jury is still out Does Bub1 play a role in other CIN tumors? –Some say yes some say no Bub1 does play a roll in CIN therefore infer a step to cancer