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Tyrosine Protein Kinase ABL1. Mira Patel H Molecular Human Anatomy. 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
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Tyrosine Protein Kinase ABL1 Mira Patel H Molecular Human Anatomy
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
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
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
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)
Structure: C-terminal half • Binding elements for the SH3 domain • Three nuclear localization signals • Export signal • DNA binding domain • Actin-binding domain
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)
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
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
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
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
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
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
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
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
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
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)
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)
Gleevec in the hydrophobic ATP-binding pocket of the abl kinase domain (PDB 1iep X-RAY) Gleevec: Mutation Sites
Sprycel in the hydrophobic ATP-binding pocket of the abl kinase domain (PDB 2gqg X-RAY) Sprycel: Mutation Sites
Overlap of Gleevec and Sprycel in the hydrophobic ATP-binding pocket of the abl kinase domain (PDB 1iep X-RAY, 2gqg X-RAY)
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
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)
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
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