Tyrosine Protein Kinase ABL1

1. Tyrosine Protein Kinase ABL1 Mira Patel H Molecular Human Anatomy

2. Kinases • Attach a phosphate group to an amino acid residue of a protein • Result is usually a conformational change that activates the substrate protein • Activation allows proteins to take part in chemical reactions • Over 500 different types of kinases • Function is shared, so it can safely be assumed that structure will also be highly conserved

3. c-abl Protein • Named as a homolog of the v-abl oncogene, found in the Abelson murine leukemia virus • Human c-abl tyrosine kinase is a product of the Abelson (abl) gene, located on the 9th chromosome • Non-receptor tyrosine kinase • Transfers a phosphate group from ATP to a tyrosine residue in a protein • Located in both cell cytoplasm and the nucleus • Functions are altered depending on its location in the cell • protein is thought to move from one location to the other, depending on which of its functions are needed in the cell

4. ribbon view of abl tyrosine kinase (PDB 1opk X-RAY)

5. Structure: N-terminal half • N terminal cap ~80 amino acid residues • Src Homology 2 (SH2) domain ~100 amino acid residues • Src Homology 3 (SH3) domain ~50 amino acid residues • Kinase domain encloses the ATP-binding site

6. ATP Binding Pocket of Kinase Domain Kinase domain of c-abl (PDB 2v7a X-Ray) ribbon and surface views of hydrophobic ATP-binding cleft (PDB 1opk X-RAY)

7. Structure: C-terminal half • Binding elements for the SH3 domain • Three nuclear localization signals • Export signal • DNA binding domain • Actin-binding domain

8. Cellular Function: Cytoplasm • cytoskeleton remodeling • actin binding domain suggests that c-abl can anchor actin filaments • a similar domain is seen in proteins with cytoskeletal associations f-actin binding domain of c-abl (PBD 1zzp NMR)

9. Cellular Function: Nucleus • Cell is exposed to ionizing radiation • c-abl phosphorylates p73 on Y99  • Activation of p73 • Induction of p21 gene • p21 inhibits Cdk kinase activity • Conclusion: c-abl can be involved in G1/S checkpoint, in a pathway independent of p53

10. ABL 1a and 1b • Two variants of the abl protein—abl 1a and abl 1b • Only difference is that form 1b has a post-translational modification that form 1a lacks • abl 1b is myristoylated • For reasons that are not known, abl 1b is found in larger amounts in cells than abl 1a

11. Abl and Src Kinases • Many structural similarities between abl and src family kinases • N-terminal half, minus the cap, is almost identical to an Src family kinase protein • However, abl kinases differ in their C-terminal half, the domains of which are absent in Src family proteins

12. Ribbon view ofc-abl , c-src overlap Human tyrosine kinase c-abl (PDB 1opk X-RAY) Human tyrosine kinase c-src (PDB 2src X-RAY) http://jkweb.berkeley.edu/external/pdb/2003/hlabl/C0301076_Nagar_etal_fig2.jpg

13. ClustalW between c-abl and c-src kinases (SH2, SH3, and kinase domains) * sequence identity : strong conservation . weaker conservation 35.93% identity http://align.genome.jp/sit-bin/clustalw

14. Regulation and Activity • Pathways used to regulate abl activity are not clear • Key structural differences between abl and src kinases lead to different regulatory mechanisms for the two proteins • Activation of src kinase controlled by phosphotyrosine residue, Y527 • This residue is absent in abl kinase • Both kinases exist in an autoinhibited form • Kinase domains are controlled negatively by other domains on the protein • Mutations in or deletion of SH3 domain result in an active abl kinase that cannot otherwise be inactivated • This suggests that the SH3 domain regulates the activity of the kinase domain in c-abl

15. c-abl Proto-oncogene • Translocation, t(9;22)(q34;q11), between abl (Abelson) and bcr (breakpoint cluster region) genes results in Philadelphia chromosome • Fusion bcr/abl gene encodes altered kinase (bcr/abl kinase) that allows cell proliferation without regulation • Philadelphia chromosome is directly responsible for chronic myeloid leukemia (CML), although individuals with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) can also have this chromosome

16. Chronic Myelogenous Leukemia • Unnecessary and dangerous increase in the production of immature granulocytes • Blood tissue becomes so packed with non-functional white blood cells that healthy blood cells can no longer be produced • Leads to anemia, a dysfunctional immune system, and dangerous infections

17. CML Treatment • Kinase inhibitors designed bind to hydrophobic ATP-binding pocket of kinase • ATP cleft is small and requires a small inhibitor molecule • FDA approved kinase inhibitors to fight CML: • Imatinib, marketed as Gleevec in 2001 by Novartis Pharmaceuticals Corporation • Dasatinib, marketed as Sprycel in 2006 by Bristol-Myers Squibb Company • Nilotinib, marketed as Tasigna in 2007 by Novartis Pharmaceuticals Corporation • Patients diagnosed with CML must immediately begin taking kinase inhibitors in addition to chemotherapy treatment

18. Gleevec 1st generation inhibitor Binds to only inactive conformations of bcr/abl Many mutation sites cause resistance A surface view of Gleevec in the hydrophobic ATP-binding pocket of the abl kinase domain (PDB 2hyy X-RAY)

19. Sprycel 2nd generation inhibitor Binds to both active and inactive conformations of bcr/abl Overcomes many imatinib mutation sites A surface view of Sprycel in the hydrophobic ATP-binding pocket of the abl kinase domain (PDB 2gqg X-RAY)

20. Gleevec in the hydrophobic ATP-binding pocket of the abl kinase domain (PDB 1iep X-RAY) Gleevec: Mutation Sites

21. Sprycel in the hydrophobic ATP-binding pocket of the abl kinase domain (PDB 2gqg X-RAY) Sprycel: Mutation Sites

22. Overlap of Gleevec and Sprycel in the hydrophobic ATP-binding pocket of the abl kinase domain (PDB 1iep X-RAY, 2gqg X-RAY)

23. Drug Resistance • Mutation in T315, the “gatekeeper residue,” leads to resistance to Gleevec, Sprycel, and Tasigna • To combat this problem, another class of inhibitors must be used • SGX Pharmaceuticals is currently working with Novartis to produce a generation of abl-bcr kinase inhibitors that can overcome T315 mutation • Testing of this new drug, SGX393, is expected to be complete by June 2008, after which the developers may seek FDA approval for the drug

24. T315I Mutation (c-abl in complex with imatinib PDB 1iep X-RAY) (c-abl in complex with dasatinib PDB 2gqg X-RAY) (mutated c-abl kinase domain PDB 2v7a X-Ray)

25. Conclusion • Understanding structure of a protein can lead to structure-based drug designs, which have proven to be very effective • Individuals afflicted with cancer are able to live normal and productive lives

26. References • Nagar, et al. Structural Basis for the Autoinhibition of c-Abl Tyrosine Kinase. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-486WNCN-D&_user=526750&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000023759&_version=1&_urlVersion=0&_userid=526750&md5=3c430835fc17373b68b770c1da093d1f • Shaul, Y. c-Abl: activation and nuclear targets. http://www.nature.com/cdd/journal/v7/n1/full/4400626a.html • Pendergast, Anne Marie. Nuclear tyrosine kinases: from Abl to WEE1. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VRW-4547875-C7&_user=6824479&_coverDate=04%2F30%2F1996&_rdoc=1&_fmt=summary&_orig=article&_cdi=6245&_sort=v&_docanchor=&view=c&_ct=29845&_acct=C000023759&_version=1&_urlVersion=0&_userid=6824479&md5=4feed9094a9b76b1ac3d8372c4e9c766 • Hantschel, et al. Structural Basis for the Cytoskeletal Association of Bcr-Abl/c-Abl. http://www.cemm.oeaw.ac.at/downloads/MolecularCell19p461to473.pdf • Shah, et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WWK-46KR83D-7&_user=6824479&_origUdi=B6WWK-4D4KPHG-4&_fmt=high&_coverDate=08%2F31%2F2002&_rdoc=1&_orig=article&_acct=C000023759&_version=1&_urlVersion=0&_userid=6824479&md5=72adfb4aa21d767908ef62e79d2ac3ab

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