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. 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. The stem cell factor receptor c-Kit is a receptor protein-tyrosine kinase (RPTK) 1The abbreviations used are: RPTK, receptor protein-tyrosine kinase; KID, kinase insertion domain; MuSK, autoinhibited muscle-specific kinase; MES, 4-morpholineethanesulfonic acid; PTR, phosphotyrosine; SH, Src homolgy; RMS, root mean square. that initiates cell growth and proliferation signal transduction cascades in response to stem cell factor binding (1Linnekin D. Int. J. of Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar). c-Kit, named after its viral homolog v-Kit (2Besmer P. Murphy J.E. George P.C. Qiu F.H. Bergold P.J. Lederman L. Snyder Jr., H.W. Broudeur D. Zuckerman E.E. Hardy W.D. Nature. 1986; 320: 415-421Google Scholar), is a member of the Type III transmembrane RPTK subfamily, which includes the colony-stimulating factor-1 receptor (3Coussens L. Van Beveren C. Smith D. Chen E. Mitchell R.L. Isacke C.M. Verma I.M. Ullrich A. Nature. 1986; 320: 277-280Google Scholar), also known as the FMS receptor, the related Flt-3 receptor (4Rosnet O. Schiff C. Pebusque M.-J. Marchetto S. Tonnelle C. Toiron Y. Birg F. Birnbaum D. Blood. 1993; 82: 1110-1119Google Scholar), and the platelet-derived growth factor α- and β-receptors (5Claesson-Welsh L. Eriksson A. Westermark B. Heldin C.-H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4917-4921Google Scholar, 6Yarden Y. Escobedo J.A. Kuang W.-J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Ullrich A. Williams L.T. Nature. 1986; 323: 226-232Google Scholar), as well as c-Kit (7Yarden Y. Kuang W.-J. Yang-Feng T. Coussens L. Munemitsu S. Dull T.J. Chen E. Schlessinger J. Francke U. Ullrich A. EMBO J. 1987; 6: 3341-3351Google Scholar). The Type III RPTK family is characterized by five extracellular immunoglobulin (Ig) domains, a single transmembrane helix, an autoinhibitory juxtamembrane domain, and a cytoplasmic kinase domain that is split by a kinase insertion domain (KID) (see Fig. 1A) (6Yarden Y. Escobedo J.A. Kuang W.-J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Ullrich A. Williams L.T. Nature. 1986; 323: 226-232Google Scholar, 8Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Google Scholar). The binding of a stem cell factor dimer to the extracellular Ig domains of c-Kit causes two c-Kit RPTKs to dimerize and permits the kinase domains to act in trans as a substrate and enzyme for one another. The result of stem cell factor binding is the phosphorylation of specific tyrosine residues located in c-Kit juxtamembrane regions (9Heldin C.-H. Cell. 1995; 80: 213-223Google Scholar, 10Hubbard S.R. Mohammadi M. Schlessinger J. J. Biol. Chem. 1998; 273: 11987-11990Google Scholar, 11Weiss A. Schlessinger J. Cell. 1998; 94: 277-280Google Scholar, 12Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Google Scholar). Tyrosine residue 568 is the primary site of in vivo autophosphorylation (see Fig. 1B). Phosphorylation of the tyrosine initiates a cytoplasmic serine/threonine phosphorylation cascade that promotes cell growth and proliferation (12Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Google Scholar). Mutations that cause constitutive activation of c-Kit kinase activity in the absence of stem cell factor binding are implicated in highly malignant human cancers, including gastrointestinal stromal tumors (13Hirota S. Isozaki K. Moriyama Y. Hashimoto K. Nishida T. Ishiguro S. Kawano K. Hanada M. Kurata A. Takeda M. Tunio G.M. Matsuzawa Y. Kanakura Y. Sinomura Y. Kitamura Y. Science. 1998; 279: 577-580Google Scholar, 14Nishida T. Hirota S. Taniguchi M. Hashimoto K. Isozaki K. Nakamura H. Kanakura Y. Tanaka T. Takabayashi A. Matsuda H. Kitamura Y. Nat. Genet. 1998; 19: 323-324Google Scholar), germ cell tumors (15Tian Q. Frierson Jr., H.F. Krystal G.W. Moskaluk C.A. Am. J. Pathol. 1999; 154: 1643-1647Google Scholar), mast cell and myeloid leukemias (16Kitamura Y. Hirota S. Nishida T. Mutat. Res. 2001; 477: 165-171Google Scholar), and in mastocytosis (17Longley Jr., B.J. Metcalfe D.D. Tharp M. Wang X. Tyrrell L. Lu S.-Z. Heitjan D. Ma Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1609-1614Google Scholar). Moreover, activating c-Kit mutations that occur in the kinase domain are resistant to many kinase inhibitors currently in use as chemotherapy treatments (18Frost M.J. Ferrao P.T. Hughes T.P. Ashman L.K. Mol. Cancer Ther. 2002; 12: 1115-1124Google Scholar, 19Ma Y. Zeng S. Metcalfe D.D. Akin C. Dimitrijevic S. Butterfield J.H. McMahon G. Longley B.J. Blood. 2002; 99: 1741-1744Google Scholar, 20Ueda S. Ikeda H. Mizuki M. Ishiko J. Matsumura I. Tanaka H. Shibayama H. Sugahara H. Takai E. Zhang X. Machii T. Kanakura Y. Int. J. Hematol. 2002; 76: 427-435Google Scholar, 21Zermati Y. De Sepulveda P. Feger F. Letard S. Kersual J. Casteran N. Gorochev G. Dy M. Ribadeau Dumas A. Dorgham K. Parizot C. Bieche Y. Vidaud M. Lortholary O. Arock M. Hermine O. Dubreuil P. Oncogene. 2003; 22: 660-664Google Scholar). The kinase activity of c-Kit is tightly regulated throughout its signaling cycle. Binding of the protein-tyrosine phosphatase SHP-1 to the phosphorylated c-Kit juxtamembrane region results in dephosphorylation of the tyrosine residues and termination of the intracellular signal (22Kozlowski M. Larose L. Lee F. Le D.M. Rottapel R. Siminovitch K.A. Mol. Cell. Biol. 1998; 18: 2089-2099Google Scholar). The dual role of the juxtamembrane region as an unphosphorylated autoinhibitory domain and as a phosphorylated intracellular signal has made it difficult to dissect the structural and mechanistic functions of the juxtamembrane region in the signaling cascade. Specific site-directed mutations introduced into the juxtamembrane domains of c-Kit (23Ma Y. Cunningham M.E. Wang X. Ghosh I. Regan L. Longley B.J. J. Biol. Chem. 1999; 274: 13399-13402Google Scholar) and platelet-derived growth factor (24Irusta P.M. DiMaio D. EMBO J. 1998; 17: 6912-6923Google Scholar, 25Irusta P.M. Luo Y. Bakht O. Lai C.-C. Smith S.O. DiMaio D. J. Biol. Chem. 2002; 277: 38627-38634Google Scholar) indicate that several residues are necessary to maintain the kinase in an autoinhibited state (see Fig. 1B). Based on amino acid sequence analysis the Type III RPTK juxtamembrane domains are proposed to adopt a conformation similar to WW domains, which are implicated in regulating cellular processes (26Ilsley J.L. Sudol M. Winder S.J. Cell. Signal. 2002; 14: 183-189Google Scholar, 27Macias M.J. Wiesner S. Sudol M. FEBS Lett. 2002; 513: 30-37Google Scholar, 28Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Google Scholar). Similarly, the c-Kit autoinhibitory juxtamembrane region has been proposed to form a putative α-helix that exerts negative control over uninduced receptor (23Ma Y. Cunningham M.E. Wang X. Ghosh I. Regan L. Longley B.J. J. Biol. Chem. 1999; 274: 13399-13402Google Scholar). Studies with a synthetic peptide of the c-Kit juxtamembrane region suggest that it folds as an autonomous domain and directly interacts with the amino-terminal lobe of the kinase domain (29Chan P.M. Ilangumaran S. La Rose J. Chakrabartty A. Rottapel R. Mol. Cell. Biol. 2003; 23: 3067-3078Google Scholar). These models for the autoinhibition of Type III RPTKs are derived from autoregulation mechanisms inferred from the crystal structures of other protein-tyrosine kinases (reviewed in Refs. 30Hubbard S.R. Hill J.H. Annu. Rev. Biochem. 2000; 69: 373-398Google Scholar and 31Hubbard S.R. Curr. Opin. Struct. Biol. 2002; 12: 735-741Google Scholar). Autoinhibition in cis is seen in the crystal structure of the autoinhibited form of the EphB2 receptor tyrosine kinase domain (32Wybenga-Groot L.E. B. J. T. F. Cell. 2001; Scholar). structure a juxtamembrane domain with mutations of signaling tyrosine residues similar to in the juxtamembrane regions of c-Kit and the Type III The of the EphB2 juxtamembrane domain a α-helix that the amino-terminal lobe of the kinase and disrupts the of the or with residues that form an activated kinase Similarly, the crystal structure of the autoinhibited muscle-specific kinase a of the juxtamembrane region that a which the of the to to binding of J.H. M. A. Lu Y. Ma Y. S.J. S.R. 2002; Scholar). autoinhibitory structural role for a juxtamembrane region tyrosine residue also inferred from the crystal structure of the receptor in which a similar of the conformation of the S. J.H. S.R. J. Biol. Chem. 2003; Scholar). these kinase conformations for a design for the design of binding and specific also known as or is one that to the conformation of the kinase. kinase is directly implicated in the pathogenesis of M.E. B.J. Cancer 2003; Scholar). STI-571 not many other it the two related Type III platelet-derived growth factor and c-Kit E. N. D. S. B.J. J. Ther. 2000; Scholar). H. E.Y. J. J. Biol. Chem. 2003; Scholar) the resolution crystal structure of an activated c-Kit kinase complex in trans with juxtamembrane residues and and that structure an We also the 1.9-Å resolution crystal structure of unphosphorylated c-Kit kinase the autoinhibitory juxtamembrane The juxtamembrane domain inserts directly into the the kinase and the c-Kit control helix, and the kinase from a The kinase activation folds over the and interacts with the kinase-active as a We also a 1.6-Å resolution structure of a complex that that of the inhibitor with regions of the juxtamembrane domain that maintain c-Kit in the autoinhibited These results provide the basis for understanding the mechanism of c-Kit kinase autoinhibition and will facilitate the structure-guided design of specific and inhibitors that target the activated and autoinhibited conformations of c-Kit kinase. and of the human c-Kit that the kinase domains and the juxtamembrane domain (3Coussens L. Van Beveren C. Smith D. Chen E. Mitchell R.L. Isacke C.M. Verma I.M. Ullrich A. Nature. 1986; 320: 277-280Google with the residues and as H. E.Y. J. J. Biol. Chem. 2003; Scholar). c-Kit in in with and the after The and the an amino-terminal by an by the with and the and by a over a c-Kit to by and to in and and c-Kit in by in and and of activated c-Kit kinase domain are as H. E.Y. J. J. Biol. Chem. 2003; Scholar) by enzyme with and to of and of MES, of autoinhibited c-Kit kinase are from the D. J. M. D. J. Struct. Biol. 2003; Scholar) with of and of of the with STI-571 of and of and the of enzyme in with and by in the with an and the and the Scholar). The activated c-Kit kinase to the with cell a and and two enzyme in the The autoinhibited c-Kit kinase to the with cell a and and one enzyme in the The complex are in the with cell a and and one in the and structure of activated c-Kit kinase by J. D. 2001; Scholar) and as H. E.Y. J. J. Biol. Chem. 2003; Scholar). structure two c-Kit enzyme in the and for residues of with the of amino-terminal amino acid residues as well as residues the KID, which are The structure of autoinhibited c-Kit kinase one enzyme in the and also by with the activated c-Kit kinase structure as a with the a of and an of The with D. Scholar), and the models and and the J. Struct. Biol. 1999; Scholar). The structure of autoinhibited c-Kit kinase is with for residues including the the structure of the complex two the activated structure and the autoinhibited structure The autoinhibited structure the and a of this for the The and for these structures are in I. The have been in the and and is the mean of of of of for of from in are for the resolution is the mean of for of from in are for the resolution in a the of D. 1999; Scholar), which is on including the and D. 2002; Scholar). The for the activated conformation from to with a mean of The for the autoinhibited conformation the resolution of the used to the from to with a mean of of c-Kit activated and autoinhibited c-Kit kinase structures from an juxtamembrane region residues from to by the kinase The is a region of and that the sequence for the of many and is in these of the active c-Kit kinase are by with and to the trans autophosphorylation of the by H. E.Y. J. J. Biol. Chem. 2003; Scholar) that the primary of phosphorylation are and which are located the of the juxtamembrane domain and the of the kinase 1B). The and resolution for activated c-Kit are in I. structure two c-Kit enzyme in the with from the amino of in the crystal binding in trans the kinase-active the active c-Kit kinase the amino acid residues of the juxtamembrane domain of in the are as are the residues of the The structure of active c-Kit kinase domain is with the kinase The is of and a single the that activity with the binding site and the of the activation A and of the of active c-Kit a to the of which the α- and of the The c-Kit is also in an active with the the α- and and to binding of the of the and conformations of the c-Kit kinase of the c-Kit kinase-active The is in a conformation that binding with the this complex the with the active and the of has been to the target tyrosine residue of the autoinhibited c-Kit kinase-active The of the autoinhibitory juxtamembrane region inserts into the that in the activated structure. The is in the autoinhibited state with over and the binding this The is and the is from its The active c-Kit kinase enzyme structure is a with a and the of the kinase-active is to enzyme in the substrate peptide binding in trans is by crystal the of binding the active is in of the the of the substrate peptide binding We have on substrate peptide binding to as in this the peptide and the active conformation of the H. E.Y. J. J. Biol. Chem. 2003; Scholar). The activation is in the active the that the target is not The of is a region by the of and A residue this the kinase in an active of Autoinhibited c-Kit kinases are maintained in an state in the absence of activating a for the kinases Scholar), and for the protein-tyrosine kinases M. J. Cell. 2002; Scholar). many tyrosine as the the state of the kinase is maintained and domains that with and the kinase domain B. O. K. D. B. G. J. Cell. 2003; Scholar). The as c-Kit, these domains, and the kinase is activated receptor to its role as a substrate for trans autophosphorylation in is that the juxtamembrane domain of the Type III RPTKs also functions as an autoinhibitory of this region is in the active structure of The structure of autoinhibited c-Kit which has an juxtamembrane domain, permits the dual of the juxtamembrane region to The structure of c-Kit in an autoinhibited conformation the active c-Kit kinase domain structure as a the of the that a and of the structural in the kinase The resolution for the autoinhibited structure for the to including the residues for the and the residues that not in the activated kinase structure. to the that the Type III RPTK juxtamembrane regions adopt a domain (24Irusta P.M. DiMaio D. EMBO J. 1998; 17: 6912-6923Google Scholar) or an structure (23Ma Y. Cunningham M.E. Wang X. Ghosh I. Regan L. Longley B.J. J. Biol. Chem. 1999; 274: 13399-13402Google Scholar), the juxtamembrane domain a that inserts directly into the domain the kinase and The amino-terminal residues of the juxtamembrane domain, from to the of this and form specific that the conformation of the kinase and (see The region that the target tyrosine residues of the autophosphorylation the of the region residues by site-directed as in maintaining the autoinhibited state in the the amino-terminal autoinhibitory domain and the kinase and The of and a by and whereas into the and its and the of and residues of the and the active c-Kit kinase the conformation of the is that of the not the site in the autoinhibited kinase is from this by and the active The kinase the and by the of which the in the in the two enzyme active c-Kit kinase the the whereas in the autoinhibited state the of is the of the active a with the which is the residue the that phosphorylated in and this the active conformation of the kinase. The and activating X. R. P. T. A. Proc. Natl. Acad. Sci. U. S. A. Scholar, Q. Frierson Jr., H.F. Krystal G.W. Moskaluk C.A. Am. J. Pathol. 1999; 154: 1643-1647Google Scholar) the conformation of the that the of residue in the of from its in the autoinhibited structure. the of from the of this residue to a by of the negative of its as the amino-terminal residue for one of α-helix including residues and and a of to a or residue this and the activation its activated structure is in a A similar also for the an activating of an acid residue in the Flt-3 kinase. The as a in the Autoinhibited structural of the kinase the of the c-Kit activation which folds over the to the conformation of the seen in the active c-Kit structure Moreover, in the autoinhibited the amino-terminal of the juxtamembrane domain the region in the active and the from an active the autoinhibited the target inserts into the kinase-active and as a the seen in the complex with the residue A and this the of and and its and and and are the Type III the activated in with from the to and the the inserts and from the kinase and The of the kinase and structural of the autoinhibited the from its in the active structure to a in the autoinhibited state as from the to in the two The of the autoinhibited is similar to the in the receptor S.R. L. L. Nature. Scholar), and also in the structures of to STI-571 T. P. B. J. Science. 2000; Scholar) and other B. O. K. D. B. G. J. Cell. 2003; Scholar, B. P. T. B. J. Cancer Res. 2002; Scholar), the muscle-specific tyrosine J.H. M. A. Lu Y. Ma Y. S.J. S.R. 2002; Scholar), as well as the crystal structure of the Flt-3 kinase J. J. C. L. M. Lu F. J. K. Mol. Cell. Scholar). mechanism of kinase autoinhibition by the juxtamembrane domain seen in c-Kit, as well as in the structure of the related Flt-3 kinase J. J. C. L. M. Lu F. J. K. Mol. Cell. Scholar), is from that seen in other autoinhibited receptor kinase the juxtamembrane regions of these Type III RPTKs with kinase domains not the conformation of the control also the kinase and from an active conformation and with kinase structural are not in other autoinhibited kinase as the EphB2 RPTK the unphosphorylated juxtamembrane region a conformation that interacts with and disrupts the of the kinase (32Wybenga-Groot L.E. B. J. T. F. Cell. 2001; Scholar). A similar specific of the is by an juxtamembrane region of the RPTK J.H. M. A. Lu Y. Ma Y. S.J. S.R. 2002; Scholar), whereas the tyrosine kinase is maintained in an autoinhibited state by an amino-terminal and and These autoinhibitory mechanisms of receptor tyrosine kinases the and of kinases M. J. Cell. 2002; Scholar). and of the Autoinhibited to the the and activated c-Kit kinase the of the kinase that are and the regions that are by the analysis of D. 2002; Scholar). The of the kinase and the as two that with to other in the structural the residues form an domain in which root mean in the two whereas for the an domain is by residues that the a These are the of the for the resolution activated structure and compare with an of the two structures are The of c-Kit the the autoinhibited and activated These residues the amino-terminal autoinhibitory domain the a in the the a that the in the autoinhibited form and a of the that the the these regions the region the a in with the and a in the that the target region of the autoinhibitory domain The conformation the substrate of the juxtamembrane domain is structural The conformation of this region is similar to the peptide conformation in trans in the activated structure that structural of the target region to occur for the trans autophosphorylation to in J. J. C. L. M. Lu F. J. K. Mol. Cell. Scholar) the structure of the Flt-3 kinase domain and proposed a mechanism for the in which the juxtamembrane domain kinase activity the role for the region from to is to maintain and the substrate in the and after the from autoinhibited to activated structure J. J. C. L. M. Lu F. J. K. Mol. Cell. Scholar). c-Kit structures a of this region and indicate that it the substrate in a conformation for binding to kinase in whereas the of this region is the Type III RPTKs to that cis autophosphorylation STI-571 and for c-Kit a the of cellular growth and proliferation signal transduction and activation is implicated in human RPTKs are for by of these inhibitors are with binding the kinase and are to the active kinase for kinases by sequence and the that target kinase are to a of is which is an for by the kinase. STI-571 not the many of the tyrosine including it is an inhibitor of platelet-derived growth factor and c-Kit E. N. D. S. B.J. J. Ther. 2000; Scholar), the two related Type III crystal structures of the autoinhibited c-Kit kinase structure is in the absence of inhibitor and is that this conformation and is not by STI-571 autoinhibited structure that STI-571 is not an for maintaining c-Kit in the autoinhibited state. STI-571 of a a a peptide to a and a c-Kit kinase autoinhibited structure that STI-571 is to the the and of the autoinhibited structure this with and The is resistant to in the absence of STI-571 is STI-571 is in a not that STI-571 binding to c-Kit disrupts the autoinhibitory c-Kit and the structure of a complex and to a 1.6-Å resolution STI-571 to autoinhibited c-Kit in a similar to that in the complex with the kinase B. P. T. B. J. Cancer Res. 2002; however is an of the of a with the of inhibitors of kinases the made with the of into the the kinase and and to the of region is a of the and of STI-571 to c-Kit as with the are with a the and the of c-Kit residue and a the of STI-571 and the of the residue kinase structures are characterized by a a acid residue and a residue that the for with the and is maintained in autoinhibited c-Kit, with the of also a with the of the STI-571 peptide The of the STI-571 peptide is of the of in the c-Kit The of STI-571 the of the of and the of whereas the of the inhibitor specific with the in a by and c-Kit autoinhibited structure on the STI-571 complex structure that these two of the and are with the autoinhibited structure of c-Kit kinase. STI-571 binding not the autoinhibited it with and the mechanism that has to maintain c-Kit in its state. these of STI-571 with that the natural autoinhibited with and for c-Kit inhibitors also the other Type III including platelet-derived growth factor and crystal structures of c-Kit kinase a and in which the juxtamembrane domain functions to maintain the kinase in an autoinhibited state. of this autoinhibitory domain into the the kinase and the into its the activation to over from its conformation in the active kinase it as a the kinase-active A structure of c-Kit kinase in complex with the inhibitor STI-571 that this to the kinase that STI-571 binding in disrupts the mechanism that has to maintain this state an of the autoinhibitory juxtamembrane domain with the kinase These results that inhibitors of the Type III protein-tyrosine kinases that the autoinhibited conformations of these We N. J. D. J. N. and R. A. for and for for and the and of the which is by the of of of the of the