Structural Basis for the Autoinhibition and STI-571 Inhibition of c-Kit Tyrosine Kinase*

The activity of the c-Kit receptor protein-tyrosine kinase is tightly regulated in normal cells, whereas deregulated c-Kit kinase activity is implicated in the pathogenesis of human cancers. The c-Kit juxtamembrane region is known to have an autoinhibitory function; however the precise mechanism by which c-Kit is maintained in an autoinhibited state is not known. We report the 1.9-Å resolution crystal structure of native c-Kit kinase in an autoinhibited conformation and compare it with active c-Kit kinase. Autoinhibited c-Kit is stabilized by the juxtamembrane domain, which inserts into the kinase-active site and disrupts formation of the activated structure. A 1.6-Å crystal structure of c-Kit in complex with STI-571 (Imatinib® or Gleevec®) demonstrates that inhibitor binding disrupts this natural mechanism for maintaining c-Kit in an autoinhibited state. Together, these results provide a structural basis for understanding c-Kit kinase autoinhibition and will facilitate the structure-guided design of specific inhibitors that target the activated and autoinhibited conformations of c-Kit kinase.

[1]  D. Heitjan,et al.  Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[2]  T Pawson,et al.  Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[3]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[4]  Marius Sudol,et al.  The WW domain: linking cell signalling to the membrane cytoskeleton. , 2002, Cellular signalling.

[5]  B. Druker,et al.  Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. , 2000, The Journal of pharmacology and experimental therapeutics.

[6]  R. Pearson,et al.  Intrasteric regulation of protein kinases and phosphatases. , 1991, Biochimica et biophysica acta.

[7]  C. Heldin,et al.  Dimerization of cell surface receptors in signal transduction , 1995, Cell.

[8]  T. Schneider.  A genetic algorithm for the identification of conformationally invariant regions in protein molecules. , 2002, Acta crystallographica. Section D, Biological crystallography.

[9]  Duc Hung Le,et al.  SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain , 1998, Molecular and Cellular Biology.

[10]  T. Hunter,et al.  NeW Wrinkles for an Old Domain , 2000, Cell.

[11]  T. Hunter,et al.  Oncogenic kinase signalling , 2001, Nature.

[12]  S. Hubbard,et al.  Structural and Biochemical Evidence for an Autoinhibitory Role for Tyrosine 984 in the Juxtamembrane Region of the Insulin Receptor* , 2003, Journal of Biological Chemistry.

[13]  D E McRee,et al.  XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. , 1999, Journal of structural biology.

[14]  Avijit Chakrabartty,et al.  Autoinhibition of the Kit Receptor Tyrosine Kinase by the Cytosolic Juxtamembrane Region , 2003, Molecular and Cellular Biology.

[15]  D. DiMaio,et al.  A single amino acid substitution in a WW‐like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation , 1998, The EMBO journal.

[16]  Olivier Hermine,et al.  Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms , 2003, Oncogene.

[17]  L. Regan,et al.  Inhibition of Spontaneous Receptor Phosphorylation by Residues in a Putative α-Helix in the KIT Intracellular Juxtamembrane Region* , 1999, The Journal of Biological Chemistry.

[18]  Arthur Weiss,et al.  Switching Signals On or Off by Receptor Dimerization , 1998, Cell.

[19]  S. Hirota,et al.  Familial gastrointestinal stromal tumours with germline mutation of the KIT gene , 1998, Nature Genetics.

[20]  S. Hubbard,et al.  Crystal structure of the MuSK tyrosine kinase: insights into receptor autoregulation. , 2002, Structure.

[21]  T. Pawson,et al.  Structural Basis for Autoinhibition of the EphB2 Receptor Tyrosine Kinase by the Unphosphorylated Juxtamembrane Region , 2001, Cell.

[22]  D. Linnekin,et al.  Early signaling pathways activated by c-Kit in hematopoietic cells. , 1999, The international journal of biochemistry & cell biology.

[23]  C. Moskaluk,et al.  Activating c-kit gene mutations in human germ cell tumors. , 1999, The American journal of pathology.

[24]  D. Birnbaum,et al.  Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells. , 1993, Blood.

[25]  Inder M. Verma,et al.  Structural alteration of viral homologue of receptor proto-oncogene fms at carboxyl terminus , 1986, Nature.

[26]  J Navaza,et al.  Implementation of molecular replacement in AMoRe. , 2001, Acta crystallographica. Section D, Biological crystallography.

[27]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[28]  Shan Zeng,et al.  The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations. , 2002, Blood.

[29]  S. Hirota,et al.  Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. , 1998, Science.

[30]  A. Ullrich,et al.  Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors , 1986, Nature.

[31]  A. Ullrich,et al.  Human proto‐oncogene c‐kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. , 1987, The EMBO journal.

[32]  S. Hubbard,et al.  Autoregulatory Mechanisms in Protein-tyrosine Kinases* , 1998, The Journal of Biological Chemistry.

[33]  S. Hubbard,et al.  Protein tyrosine kinase structure and function. , 2000, Annual review of biochemistry.

[34]  D. Cruickshank,et al.  Remarks about protein structure precision. , 1999, Acta crystallographica. Section D, Biological crystallography.

[35]  F. Lu,et al.  The structural basis for autoinhibition of FLT3 by the juxtamembrane domain. , 2004, Molecular cell.

[36]  E. Chien,et al.  Structure of a c-Kit Product Complex Reveals the Basis for Kinase Transactivation* , 2003, Journal of Biological Chemistry.

[37]  S. Hubbard.  Protein tyrosine kinases: autoregulation and small-molecule inhibition. , 2002, Current opinion in structural biology.

[38]  I. Matsumura,et al.  Constitutive Activation of C-kit by the Juxtamembrane but Not the Catalytic Domain Mutations Is Inhibited Selectively by Tyrosine Kinase Inhibitors STI571 and AG1296 , 2002, International journal of hematology.

[39]  J. Kuriyan,et al.  The Conformational Plasticity of Protein Kinases , 2002, Cell.

[40]  Joseph Schlessinger,et al.  Signal transduction by receptors with tyrosine kinase activity , 1990, Cell.

[41]  Marius Sudol,et al.  WW and SH3 domains, two different scaffolds to recognize proline‐rich ligands , 2002, FEBS letters.

[42]  D. DiMaio,et al.  Definition of an Inhibitory Juxtamembrane WW-like Domain in the Platelet-derived Growth Factor β Receptor* , 2002, The Journal of Biological Chemistry.

[43]  C. Heldin,et al.  cDNA cloning and expression of the human A-type platelet-derived growth factor (PDGF) receptor establishes structural similarity to the B-type PDGF receptor. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[44]  John Kuriyan,et al.  Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). , 2001, Cancer research.

[45]  P. Seeburg,et al.  Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. , 2000, Science.

[46]  S. Hubbard,et al.  Crystal structure of the tyrosine kinase domain of the human insulin receptor , 1994, Nature.

[47]  S. Hirota,et al.  A loss-of-function mutation of c-kit results in depletion of mast cells and interstitial cells of Cajal, while its gain-of-function mutation results in their oncogenesis. , 2001, Mutation research.

[48]  G. Superti-Furga,et al.  Structural Basis for the Autoinhibition of c-Abl Tyrosine Kinase , 2003, Cell.

[49]  John E. Murphy,et al.  A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family , 1986, Nature.

[50]  Daniel Scheibe,et al.  A fully integrated protein crystallization platform for small-molecule drug discovery. , 2003, Journal of structural biology.