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Structure and regulation of Kit protein-tyrosine kinase—The stem cell factor receptor

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Abstract

Signaling by stem cell factor and Kit, its receptor, play important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a glycoprotein receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, mastocytomas, and nasal T-cell lymphomas. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. Kit activates Akt, Src family kinases, phosphatidylinositol 3-kinase, phospholipase Cγ, and Ras/mitogen-activated protein kinases. Kit exists in active and inactive conformations as determined by X-ray crystallography. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane domain, and a protein kinase domain that contains an insert of about 80 amino acid residues. The juxtamembrane domain inhibits enzyme activity in cis by maintaining the control αC-helix and the activation loop in their inactive conformations. The juxtamembrane domain also inhibits receptor dimerization. STI-571, a clinically effective targeted protein-tyrosine kinase inhibitor, binds to an inactive conformation of Kit. The majority of human gastrointestinal stromal tumors have Kit gain-of-function mutations in the juxtamembrane domain, and most people with these tumors respond to STI-571. STI-571 binds to Kit and Bcr-Abl (the oncoprotein of chronic myelogenous leukemia) at their ATP-binding sites.

Section snippets

Kit—the stem cell factor receptor

Kit is a type III receptor protein-tyrosine kinase [5] (see [6] for a description of type I through IX receptor protein-tyrosine kinases). The type III class also includes the platelet-derived growth factor (PDGF) receptor (α- and β-chains), the macrophage colony-stimulating-factor receptor (CSF-1), and the Fl cytokine receptor (Flt3). Receptor protein-tyrosine kinases all share the same topology: an extracellular ligand-binding domain, a single transmembrane segment, and a cytoplasmic kinase

Stem cell factor and Kit signaling pathways

Binding of SCF to Kit leads to receptor dimerization and activation of protein kinase activity [10]. The receptor becomes autophosphorylated at tyrosine residues during activation; the resulting phosphotyrosine residues serve as docking sites for signal transduction molecules containing SH2 and phosphotyrosine-binding (PTB) domains. Activated Kit also catalyzes the phosphorylation of substrate proteins.

Kit has the potential to participate in multiple signal transduction pathways as a result of

Overview of Kit protein kinase structure

The Kit protein-tyrosine kinase domain has the characteristic bilobed architecture observed in all protein kinases (Fig. 2) [14], [15]. Residues 582–671 make up the small N-terminal lobe of the kinase, and residues 678-953 make up the large C-terminal lobe with a hinge segment between them. The small lobe has a predominantly antiparallel β-sheet structure and is involved in anchoring and orienting ATP. It contains a glycine-rich (GAGAFG) ATP-phosphate-binding loop composed of residues 596–601.

Kit activation by stem cell factor

In the absence of SCF, which is the stimulatory Kit ligand, Kit exists in a monomeric dormant state. The general mechanism for activation of dormant receptor protein-tyrosine kinases involves binding of the appropriate ligand to the extracellular domain of two receptor monomers, bringing them together, and producing a receptor dimer. SCF exists as a non-covalent dimer, and this dimer binds to two Kit monomers thereby promoting Kit dimer formation [7]. For most receptor protein-tyrosine kinases,

Structure of active Kit

Mol et al. [14], [15] determined the structure of an active and inactive conformation of the human Kit intracellular domain. The construct contained a truncated kinase insert. The active form of Kit was obtained by incubating the enzyme with MgATP to initiate the transphosphorylation reaction. Mass spectrographic analysis of the products revealed that Tyr568 and Tyr570 were the first residues to be phosphorylated. Thus, autophosphorylation of residues in the JM domain occurs before that of the

Structure of inactive Kit

The Kit juxtamembrane domain inhibits kinase activity in cis. The JM segment of inactive Kit forms a V-shaped loop that inserts directly into the interface between the small and large lobes of the kinase (Figs. 2B and 3) [15]. This snapshot of the enzyme suggests that the JM domain has the potential to inhibit Kit by displacing the αC-helix, preventing the activation loop from assuming its extended and active conformation, and preventing the movement of the small and large lobes necessary for

Kit mutations and human neoplasms

Gain-of-function mutations occur in a percentage of human neoplasms including mastocytomas (>90%), gastrointestinal stromal tumors (>70%), sinonasal T-cell lymphomas (17%), seminomas/dysgerminomas (9%), and acute myelogenous leukemia (1%) [25]. Furthermore, autocrine or paracrine activation of Kit has been postulated in numerous other human malignancies including ovarian neoplasms and small-cell lung cancer [25], [26]. Furthermore, a large number of human cancers express Kit. Activating Kit

STI-571 targeted therapy of selected human neoplasms

STI-571 (Gleevec, imatinib) is an important and clinically useful protein-tyrosine kinase inhibitor (see [37] for an overview). In contrast to agents such as cisplatin, doxorubicin, and 5-fluorouracil, which are general cytotoxic compounds, STI-571 is a targeted cancer chemotherapeutic drug. This compound inhibits each of the following protein-tyrosine kinases: Abl, Bcr-Abl, Kit, and the PDGF receptor (α and β). Its clinical efficacy was established first in the treatment of chronic myelogenous

STI-571 binding to Kit

Mol et al. [15] determined the structure of the Kit/STI-571 complex by X-ray crystallography. They report that the drug enters the adenine-binding portion of the active site in the cleft between the small and large lobes; a portion of the drug extends into an adjacent hydrophobic pocket. STI-571 interacts with an inactive conformation by binding to the Phe811 “out” [15] or “off” [22] conformation; it is unable to bind to the active “in” or “on” conformation. The drug forms hydrogen bonds with

Epilogue

Protein phosphorylation and dephosphorylation must be stringently regulated both in time and place. Most protein kinases occur physiologically in an inactive or less active basal state. Non-receptor protein kinases such as Abl and Src are maintained in a basal state by inhibitory intramolecular interactions involving SH2 and SH3 domains [4], [37]. Receptor protein kinases require a stimulatory ligand to convert them from a dormant to an active state. The ErbB/HER family of kinases are the

Acknowledgment

I thank Dr. Clifford D. Mol for providing Fig. 2.

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    Abbreviations: GIST, gastrointestinal stromal tumors; JM, juxtamembrane; PDGF, platelet-derived growth factor; pTyr, phosphotyrosine; PTB, phosphotyrosine binding; SCF, stem cell factor; SH2, Src homology 2; SH3, Src homology 3.

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