The Strength and Cooperativity of KIT Ectodomain Contacts Determine Normal Ligand-Dependent Stimulation or Oncogenic Activation in Cancer

Abstract

SUMMARY The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoietic cells and interstitial pacemaker cells. Oncogenic KIT mutations play an important `driver' role in gastrointestinal stromal tumors, acute myeloid leukemias and melanoma among other cancers. Here we describe the crystal structure of a recurring, somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain rendering KIT tyrosine kinase activity constitutively activated. The structural analysis together with biochemical and biophysical experiments and detailed analyzes of the activities of a variety of oncogenic KIT mutations reveal that the strength of homotypic contacts and the cooperativety in the action of D4D5 regions determines whether KIT is normally regulated or constitutively activated in cancers. We propose that cooperative interactions mediated by multiple weak homotypic contacts between receptor molecules are responsible for regulating normal ligand-dependent or oncogenic RTK activation via a `zipper-like' mechanism for receptor activation.

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition

“…Substitution of individual amino acids involved in the formation of salt bridge-mediated homotypic contacts strongly compromises activation and cell signaling via KIT, PDGF receptors, and VEGF receptor 2 (6,33,34). Moreover, we have recently determined the crystal structure of D4-D5 of KIT harboring the oncogenic T417IΔ418-419 mutation to demonstrate how strong homotypic contacts taking place between an oncogenic D5 mutant lead to constitutively activated (ligand-independent) KIT stimulation in cancer cells (11).…”

“…We have demonstrated that tyrosine kinase activities of class I oncogenic mutations in D5 rely on the formation of homotypic D4 contacts between Arg381 and Glu386, a salt bridge known to play an important role in SCF-induced KIT activation (6). However, the constitutive kinase activity of class II oncogenic mutants does not depend on formation of D4 homotypic contacts (11).…”

“…Class I KIT oncogenic mutants exhibit ligand-sensitized tyrosine kinase activity that is fully activated at low ligand concentration. Class II possess constitutive, ligand-independent tyrosine kinase activity (11). The experiment presented in Fig.…”

“…The most common cytoplasmic mutations in the JM domain and the TKD have been proposed to disrupt JM domain-mediated autoinhibition and favor an active kinase domain conformation (9). Oncogenic mutations in D5 of the ectodomain enhance the binding affinity of homotypic contacts, a step necessary for SCF-induced KIT activation (6,10,11).…”

MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB

“…It has been known for over 20 years that the transmembrane helices of receptor tyrosine kinases including MET bear a common sequence, the GXXXG motif, which might self-dimerize the transmembrane helices (46). The full-length structures of KIT and PDGF receptor bound to their ligands stem cell factor and PDGF-B, respectively, revealed that in the case of class III receptor tyrosine kinases, multiple regions of the extracellular receptor contribute to the actively dimerized kinase complex (47)(48)(49)(50). Several oncogenic mutations of the KIT receptor have been analyzed, implying that normal receptor activation is an interplay between ligand binding, formation of homotypic receptor contacts, and transphosphorylation (49).…”