Thesis
Reference
Mutational and pharmacological analysis of C-Kit-mediated, integrin-dependent cell spreading
CALDERIN SOLLET, Zuleika
Abstract
KitL and its receptor c-Kit form a paracrine system that permits cells to respond to their microenvironment. The c-Kit kinase activating mutations can cause mastocytosis as well as Gastrointestinal Stromal Tumors and Leukemias. Although successful clinical treatments with kinase inhibitors, notably imatinib, a tumor cell population persist within the tumoral microenvironment. Does KitL/c-Kit contribute to the niche-based interactions even in the presence of kinase inhibition? By using a “niche model” we showed that bound-KitL might bind to and stabilize c-Kit in an active conformation, which is insensitive to imatinib but not to dasatinib. In this “mechanically active” state, c-Kit might recruit the PI3K which in turns induces a kinase-independent spreading response. Moreover, we showed that the constitutively active kinases W556A and D814V caused a non-spread phenotype which seems to be reverted with imatinib. A better understanding of tumor cell interactions is essential to design drugs that specifically target oncogenic proteins and the tumor microenvironment.
CALDERIN SOLLET, Zuleika. Mutational and pharmacological analysis of
C-Kit-mediated, integrin-dependent cell spreading. Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5187
DOI : 10.13097/archive-ouverte/unige:103204 URN : urn:nbn:ch:unige-1032040
Available at:
http://archive-ouverte.unige.ch/unige:103204
Disclaimer: layout of this document may differ from the published version.
UNIVERSITE DE GENEVE
Département de génétique et évolution FACULTE DES SCIENCES Professeur Brigitte Galliot
Département de physiologie cellulaire et métabolisme FACULTE DE MEDECINE
Professeur Bernhard Wehrle-Haller ___________________________________________________________________________
Mutational and Pharmacological Analysis of C-Kit-mediated, Integrin-dependent Cell Spreading
THESE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Zuleika PYTHOUD de
Cheiry (Fribourg)
Thèse N°5187
GENEVE No Print Limit SA
2018
Table of Contents
Acknowledgements...5
Abbreviations ...7
Abstract...8
Résumé ...10
Introduction ...12
I. Physiological functions of KitL and c-Kit ... 12
I.1. The KitL/c-Kit function during embryogenesis ... 13
I.2. The KitL/c-Kit function in adulthood ... 15
II. The c-Kit ligand: Stem Cell Factor (SCF) or Kit Ligand (KitL) ... 18
III. The tyrosine kinase receptor c-Kit ... 21
III.1. Overall structure of c-Kit... 21
III.2. The c-Kit protein: isoforms and Post Translational Modifications ... 25
III.3. Mechanism of c-Kit activation ... 26
IV. KitL/c-Kit signaling ... 28
IV.1. Recruitment of signaling proteins: adapters and enzymes ... 28
IV.2. Signaling pathways activated downstream of c-Kit ... 30
IV.3. Regulation of c-Kit signaling ... 37
V. KitL/c-Kit in malignant transformation ... 42
V.1. Regulatory and catalytic mutations affecting c-Kit ... 44
V.2. Oncogenic signaling by activated c-Kit ... 46
V.3. c-Kit related-diseases and malignancies ... 50
VI. Pharmacological inhibition of c-Kit ... 58
VII.The Cellular environment ... 63
VII.1. The ExtraCellular Matrix (ECM) ... 63
VII.2. The integrins ... 65
Objectives ...69
Materials and methods ...70
Results ...74
I. Effects of KitL presentation to c-Kit in cell-matrix adhesion and spreading. Differential roles of soluble- and immobilized-KitL in cell-matrix adhesion and spreading (S. Tabone-Eglinger et al.,
2014)... 75
II. Role of the c-Kit kinase activity in KitL-induced cell spreading on fibronectin ... 77
III. Screening of tyrosine kinase inhibitors to block immobilized-KitL-mediated spreading on low fibronectin concentrations ... 78
III.1. Effect of c-Kit inhibition in cell adhesion and spreading ... 79
III.2. Effect of SFKs inhibition in cell adhesion and spreading ... 79
III.3. Effect of FAK inhibition on cell adhesion and spreading ... 81
IV. Mutagenesis of c-Kit to identify domains or residues involved in mechanical activation ... 82
IV.1. PI3K recruitment is required for immobilized-KitL-induced spreading on low fibronectin concentrations ... 83
IV.2. Function of the c-Kit c-terminal tail in cell spreading ... 83
IV.3. Function of the di-tyrosine motif Y567/Y569 in cell spreading ... 85
V. Effect of oncogenic c-Kit mutations in the JMD and the TKD in cell spreading on fibronectin 87 V.1. Function of the di-tyrosine motif in the context of the activated receptor c-KitW556A and c-KitD814V ... 88
V.2. Spreading sensitivity of c-KitW556A and c-KitD814V to imatinib and dasatinib ... 90
Discussion ...92
I. Adhesive interactions within the hematopoietic niche ... 92
II. Crosstalk between c-Kit and integrins in mediating cell-matrix adhesions ... 95
III. Profiles of different tyrosine kinase inhibitors in cell spreading ... 98
IV. Mutagenesis identifies domains involved in the mechanical activation of c-Kit ... 101
V. Oncogenic c-KitW556A and c-KitD814V receptors inhibit KitL-induced cell spreading on fibronectin ... 102
References ... 106
Acknowledgements
The acknowledgments are the subtlest part of this manuscript. I will do my utmost not to forget anyone.
I would like to first express my gratitude to Prof. Wehrle-Haller for his continuous support, insights and encouragement. His in-depth knowledge, motivation and guidance were of incomparable helps in all aspects of my research and in the writing of this thesis. I could not have had a better advisor for my Ph.D study.
I thank the members of the jury, namely Prof. Brigitte Galliot and Prof. Martin Bastmeyer for their recommendations and insightful comments which were truly appreciated.
My sincere thanks go to the Geneva cancer league and the Dr. Henri Dubois-Ferrière Dinu Lipatti Foundation for financing this project. It wouldn’t have been possible to complete this research without their precious support.
I would also like to thank Prof. Collart and Prof. Imhof, who acted as my Godfather thesis, for their professional guidance and useful recommendations on this project and my career.
My special thanks to Prof. Matthes for his collaboration and advise on this project and for providing the necessary supporting material (i.e. leukemia cells). My thanks also to Prof.
Scapozza for his suggestions in the pharmacological field.
Thanks to my fellow colleagues and labmates for their cooperation, stimulating discussions, and for the great work we have done together and for the funny moments we shared over the last five years. My sincere thanks to Séverine for allowing me to take over this very exciting research project and for providing me with all the assistance for a success completion. My special thanks to Nicole for her unconditional helps, her kindness, friendship, guidance and for the funny coffee-break tête-à-têtes. My sincere thanks to Marie-Claude for teaching me the tips and tricks of laboratory technics, for her professionalism and commitment as well for the enjoyable lunch and coffee-break chats and her assistance in helping me improving my French.
Thanks to Patricia for her friendship and for the great French-Spanish-English-based after work hours lab and non-lab related discussions. Thanks for sharing with the team the delicious and tastefully plates her mother prepared. Thanks also to Caro for her scientific advices and friendship as well as for her availability for countless scientific idea sharing and late discussions. It was also a pleasure working with the recently hired next generation of new scientists, namely Kenza, Monica, Marta and Seimia. I wish them good luck in their respective projects. It was also a pleasure to meet Adama and Kathy. Thanks for their being always in good mood and for the funny moments.
I would like also to thank Isa for the help provided with the PamGene machine. Thanks to my friends from the biostock, namely Dominique and Gamis for their professionalism. Thanks to Cyril for sharing the culture room and waking me up in the morning with his perspicacious jokes and smile. Thanks to the bioimaging team for the precious help they provide at the facility in the images acquisition. Thanks also to Roberto and the other technical team for being always there when needed. Thanks also to the administrative staff Tamara and Corinne for helping me with the external orders.
I would like also to thank all CMU’s collaborators, employees and friends I worked with over these years for their cooperation, friendship and professionalism.
Last but not least, a special thanks to my family who encouraged me a lot and for the significant efforts they made for my future. Sincere thanks go to all my friends in and out of Switzerland for their support as well as my family in law for their strong attention and endorsement.
I dedicated this achievement to my husband, Olivier, for his unconditional help and support.
He transmitted me the sense of rigor and perseverance. My sincere thanks to him for being always present during these years of intense work and in my live in general.
Abbreviations
A-loop: Activation loop KitL: Kit Ligand Akt: RAC-alpha serine/threonine protein
kinase
LOF: Loss-Of-Function MLC: Myosin Light Chain
AML: Acute Myeloid Leukemia m-KitL: membrane-bound Kit ligand APS: Adaptor molecule containing PH and
SH2 domains
PDGFR: Platelet Derived-Growth Factor Receptor
Bcr-Abl: Breakpoint Cluster Region protein- Abelson protein
PDK1: 3-Phosphoinositide-dependent protein kinase 1
BMMC: Bone Marrow Derived Mast Cells CBF: Core Binding Factor
PI3K: Phosphatidylinositol-3-Kinase
PIP2: PhosphatidylInositol 4,5-biPhosphate CBL: E3 ubiquitin-protein ligase CBL
CD34: Cluster of Differentiation 34
PIP3: PhosphatidylInositol 3,4,5- triPhosphate
CML: Chronic Myeloid Leukemia PKC: Protein Kinase C CXCR-4: C-X-C chemokine receptor type 4 PLC: Phospholipase C
DAG: DiAcylGlycerol PTB: Phospho Tyrosine-Binding domain DFG: Asp-Phe-Gly motif PTP: Protein-Tyrosine Phosphatase DMSO: DiMethyl SulfOxide SCF: Stem Cell Factor
ECD: ExtraCellular Domain SDF-1: Stromal-derived factor-1 ECM: ExtraCellular Matrix s-KitL: soluble-Kit ligand
EGFR: Epidermal Growth Factor Receptor Sl: murine steel locus EPO: Erythropoietin SFKs: Src Family of Kinases ERK: Extracellular Signal-Regulated Kinase SH2: Src homology domain 2 FGFR: Fibroblast Growth Factor Receptor Sld: Steel dickie
FAK: Focal Adhesion Kinase FLT3: Fms-Like Tyrosine kinase 3
STAT: Signal Transducers and Activators of Transcription
GAB2: GRB2-associated-binding protein 2 SOCS: Suppressor Of Cytokine Signlaing GIST: Gastrointestinal Stromal Tumors SOS: Son Of Sevenless
GNNK: Gly-Asn-Asn-Lys motif TKD: Tyrosine Kinase Domain GOF: Gain-Of-Function
Grb2: Growth factor receptor-bound protein 2
TKI: Tyrosine Kinase Inhibitor TKR: Tyrosine Kinase Receptor Tr-c-Kit: Truncated c-Kit ICC: Interstitial cells of Cajal
ITD: Internal Tandem Duplication JAK: Janus Kinase
ROCK: Rho-Associated Protein Kinase VEGFR: Vascular endothelial Growth Factor Receptor
JMD: JuxtaMembrane Domain v-Kit: viral c-Kit homolog JNK: c-Jun N-terminal Kinase (MAPK8) W: murine white spotting locus KID: Kinase Insert Domain
Abstract
In the hematopoietic stem cell niche, KitL and its receptor c-Kit form a paracrine system that permits cells to have a biological response adapted to their microenvironment. KitL exists in two isoforms (soluble and membrane-bound) binding to and activating c-Kit, which causes reciprocal phosphorylation of the receptor, recruitment of signaling proteins and the initiation of multiple signal transduction pathways that lead to diverse cellular responses such as cell proliferation, survival, and migration. The KitL/c-Kit system is involved in gametogenesis, melanogenesis, and hematopoiesis during development and adulthood.
However, c-Kit overexpression or c-Kit kinase activating mutations can cause various forms of cancer, such as Gastrointestinal Stromal Tumors, Acute Myeloid Leukemia, and mastocytosis.
Although successful treatments in the clinic with kinase inhibitors, notably imatinib, a tumor stem cell population persist in the microenvironment leading to the development of kinase resistant mutants and tumor relapse. Thus, cancer cells can exist in a niche-anchored state, which appears to be drug resistant. Unfortunately, it is still unknown how such a dormant and niche-anchored phenotype can persist in the presence of kinase inhibition. The c-Kit receptor and integrins can mediate the communication of cells with their respective niche by interacting with KitL and proteins of the extracellular matrix, respectively. Integrins are transmembrane proteins that bind extracellular ligands including fibronectin, laminin or collagen. They regulate various cellular functions such as cell survival, adhesion, and migration. It is, therefore, possible that both systems also function in the presence of kinase inhibition and contribute to the adhesion of malignant cells to the niche. Insight into this problem will help to find new therapeutic targets to overcome resistance. For this end, the primary goal of this thesis is to investigate the adhesive properties of the KitL/c-Kit pair and how it is functioning in the presence of small molecule inhibitors or c-Kit mutations.
By using a “niche model” we showed that activation of c-Kit by KitL caused adhesion and spreading of c-Kit expressing cells on fibronectin. However, depending on which KitL isoform activated c-Kit, different responses were provoked. While a soluble form stimulated spreading on fibronectin-rich surfaces, an immobilized-KitL induced a stronger response on a fibronectin-poor matrix. Moreover, treatment of c-Kit expressing cells with tyrosine kinase inhibitors, notably imatinib efficiently inhibited soluble- but not immobilized-KitL-mediated spreading. Interestingly, only dasatinib (another drug that targets tyrosine kinase proteins)
was capable of blocking spreading towards immobilized-KitL. Further characterization of cell spreading on immobilized-KitL showed that it depended on the PI3K recruitment on c-Kit pY719 and on the kinase activity as assessed by the overexpression of a c-Kit kinase-dead mutant (D790N). On the other hand, the use of c-Kit oncogenic mutants W556A and D814V demonstrated that an active kinase caused a non-spreading phenotype. Finally, treatment with imatinib seemed to revert the response of the W556A and to a lesser extent D814V mutants. Consequently, bound-KitL might bind to and stabilize c-Kit in an active conformation, which is insensitive to imatinib but can be blocked by dasatinib. In this
“mechanically active” state, c-Kit might recruit intracellular adaptors which in turns activate a c-Kit kinase-independent spreading response. The mechanisms related to drug sensitivity of certain malignancies is still not well-understood. They often involve activating mutations of proto-oncogenes, as well as components (cells or molecules) of the tumor microenvironment.
Current therapies have limitations as they have multiple targets. A better understanding of the molecular biology of tumors is essential to design new drugs to specifically target oncogenic proteins involved in tumorigenesis and thus overcome resistance and improve the management of affected patients.
Résumé
Au sein de la niche hématopoïétiques, KitL et son récepteur tyrosine kinase c-Kit forment un système paracrine qui permet aux cellules d'avoir une réponse biologique adaptée à leur microenvironnement. KitL existe en deux isoformes (une soluble et une immobilisée) capables toutes les deux d’interagir et d'activer c-Kit. Cette interaction provoque l’auto phosphorylation du récepteur c-Kit et le recrutement de protéines de signalisation. Cela active de multiples voies de transduction du signal qui conduisent à diverses réponses cellulaires telles que la prolifération, la survie et la migration. Au cours du développement embryonnaire et l’âge adulte, le système KitL/c-Kit est impliqué dans la gamétogenèse, la mélanogenèse et l'hématopoïèse. La surexpression de c-Kit ou les mutations activatrices du récepteur peuvent provoquer diverses formes de cancer, telles que les tumeurs stromales gastro-intestinales, la leucémie aiguë myéloïde, ou encore la mastocytose. Des traitements avec des inhibiteurs de kinases, notamment l'imatinib, ont montré des résultats cliniques satisfaisants, cependant une population de cellules souches tumorales persiste dans le microenvironnement entraînant le développement de mutants résistants aux inhibiteurs et une récidive du cancer. Cette persistance serait liée aux propriétés adhésives des cellules cancéreuses à la niche. Malheureusement, on ignore encore comment une cellule ancrée et
« dormante » peut persister dans la niche et ce, malgré l’inhibition de l’activité kinase du récepteur c-Kit. L'interaction entre les cellules et le tissu environnant est en partie médiée par c-Kit lié au KitL, et des protéines d’encrage à la MEC, les intégrines. Les intégrines sont des protéines transmembranaires directement liées aux composants de la MEC (comme la fibronectine, laminine ou collagène) au niveau extracellulaire et connues pour réguler diverses fonctions cellulaires comme l’adhésion, la migration ou la survie. Nous pensons que les deux systèmes, KitL/c-Kit et intégrines, contribuent à l'adhésion des cellules tumorales à la niche et à leur résistance aux traitements. Pour ces raisons, l'objectif principal de cette thèse est d'étudier les propriétés adhésives des cellules médiées par KitL/c-Kit et de comprendre comment ce système fonctionne en présence de petites molécules inhibitrices de l’activité kinase ou de mutations c-Kit.
En utilisant un « modèle expérimental de niche hématopoïétique », nous avons montré que l'activation de c-Kit par KitL déclenche l'adhésion et l’étalement des cellules exprimant c-Kit sur la fibronectine. Cependant, en fonction de l’isoforme du KitL utilisée pour stimuler et
activer c-Kit nous avons observés différentes réponses. Alors qu'une forme soluble du KitL a induit l’étalement sur des surfaces riches en fibronectine, le ligand immobilisé sur une matrice pauvre en fibronectine a entrainé une réponse plus forte. De plus, le traitement de ces cellules avec l'imatinib a montré une inhibition significative de l'étalement induit par le KitL soluble mais pas lorsque celui est immobilisé. De façon intéressante, seul le dasatinib (un autre inhibiteur de protéines tyrosine kinase) a été capable de bloquer l’étalement cellulaire lorsque KitL immobilisé. Nous avons observé que l'étalement cellulaire sur KitL immobilisé dépend du recrutement de PI3K sur la Y719 phosphorylée de c-Kit. Par ailleurs, l’activité kinase du c-Kit semble également nécessaire pour cet étalement. En effet, la surexpression d'un mutant de c-Kit sans activité kinase (D790N) inhibe l’étalement des cellules. D'autre part, des mutants oncogéniques c-Kit W556A et D814V possèdent une activité kinase trop importante provoquant un « non étalement » des cellules. Enfin, le traitement par l'imatinib semble inverser la réponse du mutant W556A et, dans une moindre mesure, celle du mutant D814V. Par conséquent, nous suggérons que la stimulation par un ligand immobilisé pourrait stabiliser le c-Kit dans une conformation active, insensible à l'imatinib mais sensible à la dasatinib. Dans cet état « mécaniquement actif », c-Kit pourrait recruter des adaptateurs intracellulaires qui, à leur tour, activeraient une réponse d'étalement indépendante de l’activité kinase.
Les mécanismes liés à la résistance aux médicaments de certaines tumeurs malignes sont encore mal compris. Ils impliquent souvent des mutations activatrices des proto-oncogènes ainsi que des éléments (cellulaire ou moléculaires) du microenvironnement tumoral tels que les cellules stromals ou les protéines de la matrice extracellulaire. Les thérapies actuelles ont des limites car elles sont souvent peu spécifiques. Une meilleure compréhension de la biologie moléculaire des tumeurs est essentielle pour la conception de nouveaux médicaments ciblant plus spécifiquement des protéines oncogéniques, comme c-kit, impliquées dans la tumorigenèse pour ainsi surmonter la résistance et améliorer la prise en charge des patients atteints.
Introduction
Tyrosine kinase signaling pathways involve growth factors which can be found soluble, membrane- or matrix-bound. Tyrosine Kinase Receptors (TKR) are proteins that have a high affinity for growth factors and can analyze information and convert these stimuli into a suitable biological response.
The Kit Ligand (KitL) and its cognate receptor c-Kit are one of them. It is an intercellular communication system that is essential to the development and function of several cell lineages during embryonic development and adulthood. The information transmitted by KitL is analyzed and processed by the cells which express c-Kit activating diverse intracellular signaling pathways. These signaling cascades allow the cell to have a biological response adapted to its environment. Anomalies in the expression or function of KitL/c-Kit cause various human pathologies. Some rare diseases are linked to a decrease in the overall function of the pathway, while other ones are due to a hyperactivation of the pathway. These latter conditions include benign or malignant neoplasia. The pharmaceutical industry has developed different targeted therapies including small inhibitors which are being used in the clinic to treat c-Kit associated malignancies.
In this work, we wanted to address the molecular mechanisms of oncogenic signaling of constitutively activated forms of the c-Kit receptor and their involvement in cell adhesion and spreading on extracellular matrix components. The specificity of oncogenic signaling can be understood by studying the mechanisms that ensure the intracellular signaling under physiological conditions. In the introduction to this thesis, we first discuss the physiological role of the KitL/c-Kit pathway as well as the molecular mechanisms of signaling in normal conditions. Second, we present the involvement of the KitL/c-Kit axis in oncogenic signaling, the associated pathologies, mainly cancerous conditions, and the specific targeted therapies.
I. Physiological functions of KitL and c-Kit
The c-Kit gene, which encodes a receptor with tyrosine kinase activity, was discovered in 1987 (Yarden et al., 1987). It is the cellular homolog of the viral oncogene v-Kit of the Hardy- Zuckerman 4 feline sarcoma virus (Besmer et al., 1986). One year later c-Kit was established as the gene product of the White murine spotting (W) locus (Chabot et al., 1988; Geissler et al., 1988). In the mouse, the c-Kit ligand is encoded by a gene which maps near the Steel (Sl) locus (Copeland et al., 1990; Williams et al., 1990). In mice, many natural mutations affect the
Sl and the W loci (Russell, 1979). The molecular characterization of these mutations (reviewed in (Besmer et al., 1993)) revealed that the severity of the W phenotype correlates with the magnitude of the c-Kit kinase activity (Reith et al., 1993).
The physiological relevance of the KitL/c-Kit pathway can be apprehended with the phenotype of the Sl and W mutant mice. The natural diversity of these mutants with their spectrum of associated alterations is a valuable tool to study the structure-function relationship of KitL/c-Kit and their role in development. Thus, Sl and W mice revealed the fundamental role of the KitL/c-Kit pair in the physiology of primordial germ cells, hematopoietic cells, mast cells, intestinal Cajal cells (ICCs), and melanocytes.
The loss of total expression of c-Kit or KitL proteins leads to death in utero or the perinatal period due to severe macrocytic anemia. In the case of hypomorphic mutations, -poor KitL production or little c-Kit activity- several manifestations appear such as anemia, infertility, lack of pigmentation, low number of tissue mast cells and lack of intestinal pacemaker activity. The different mutants and phenotypes of the corresponding mice were reviewed in (Galli et al., 1994) and will be discussed later.
I.1. The KitL/c-Kit function during embryogenesis
During embryogenesis, the messenger RNAs of c-Kit and KitL are expressed on the migratory paths and final targets of primordial germ cells and melanocytes. The KitL and c-Kit mRNAs are also in the sites of embryonic hematopoiesis, the intestine and the central nervous system (Keshet et al., 1991; Matsui et al., 1990; Orr-Urtreger et al., 1990). Therefore, the KitL/c-Kit pair are not only involved in development but also influences the migration of primordial cells to their destination, called "niche."
Crossed transplantation experiments of spleen or splenic tissue cells in W or Sl animals demonstrated that the W phenotype was an intrinsic defect in the hematopoietic compartment, whereas the Sl ones affected the microenvironment (Wolf, 1978). Bone marrow of the W/Wv and Sl/Sld mice contains few early progenitor Colonies Forming Units (CFU-Spleen), CFU of erythroid lineage (CFU-E) and granulocyte/macrophage (CFU-GM) (Broudy, 1997).
The KitL/c-Kit pathway acts in coordination with the erythropoietin (Epo) and its receptor (Epo/EpoR) pathway during embryonic and adult erythropoiesis (Munugalavadla & Kapur, 2005; von Lindern et al., 2004). The role of the KitL/c-Kit couple in hematopoiesis is a broad
subject. We briefly present some aspects of their role in the maintenance of embryonic Hematopoietic Stem Cells (HSCs). In vivo, fetal liver progenitors express c-Kit although they are dependent on its activation only at certain stages of development (Ogawa et al., 1993).
This activation appears to function in the ability of HSCs to reconstitute long-term hematopoiesis (Miller et al., 1997). In vitro, symmetrical divisions of fetal HSCs depend on the stimulation of c-Kit (Bowie et al., 2007). Thus, it appears that the KitL/c-Kit pathway is involved in the growth and maintenance of the HSC compartment in the fetal liver, explaining the reduced number of hematopoietic progenitors in deficient mice.
Other than the hematopoietic function, KitL and c-Kit have additional embryonic roles. Non- hematopoietic cells that depend on c-Kit are the primordial germ cells (PGCs), melanoblasts (melanocyte precursors), and mesenchymal precursors which give rise to the interstitial cells of Cajal (ICCs). For each cell population, the activation of the KitL/c-Kit pathway and its functional consequences may vary during differentiation.
The KitL/c-Kit signaling pathway is crucial for the proliferation, survival, and migration of melanocytes both during embryonic and adult life (Mackenzie et al., 1997; Wehrle-Haller, 2003). Melanoblasts depend on KitL/c-Kit for their survival and later for their proliferation, but it does not cause their differentiation (Cable et al., 1995; Mackenzie et al., 1997). During the embryonic development, the pathway also induces the migration of melanoblasts from the neural crest to the nascent dermis. Moreover, the translocation of melanoblast from the dermis to the epidermis is also under the control of KitL/c-Kit during the neonatal period (Jordan & Jackson, 2000; Nishikawa et al., 1991).
Interstitial cells of Cajal (ICCs) are pacemaker cells of mesenchymal origin located in the intestinal wall and provide the electrical rhythm for peristalsis (Huizinga & Lammers, 2008).
In ICCs, the role of KitL/c-Kit depends on the stage of development. It is initially involved in the migration of precursors, and then it is required for the neo-natal proliferation phase. The loss of c-Kit expression correlates with the differentiation of common precursors in smooth muscle cells while future ICCs maintain c-Kit expression, highlighting the c-Kit function in the differentiation of mature ICCs (Ward & Sanders, 2001).
Primary germ cells are embryonic cells, precursors of male and female gametes. The KitL/c- Kit axis allows these cells to migrate to the genital crests and ensures their survival and proliferation (Farini et al., 2007; Y. Gu et al., 2009; Kissel et al., 2000).
I.2. The KitL/c-Kit function in adulthood
During adult life, KitL and c-Kit still expressed in the hematopoietic compartment.
Differentiated cells such as ICCs, melanocytes, mast cells and endothelial cells continue to express c-Kit. The receptor is also present in neural (Das et al., 2004; L. Sun et al., 2004) and cardiac stem cells (Miyamoto et al., 2010; Tallini et al., 2009).
KitL-stimulated signaling is necessary for the maintenance and functionality of ICCs (Reviewed in (K. M. Sanders & Ward, 2007)). It should be noted that the intraperitoneal injection of blocking antibodies directed against c-Kit (ACK2 antibody) altered the contractile capacity of the intestine in wild-type BALB/c mice (Maeda et al., 1992).
KitL/c-Kit are also involved in the proliferation of melanocytes in the skin during the postnatal period (Okura et al., 1995) and adulthood (Grichnik et al., 1998). They also mediate the UV- induced pigmentation (Hachiya et al., 2004) and the cyclic regeneration of the hair pigment units (Hachiya et al., 2009). The expression of KitL/c-Kit also controls the migration and survival of melanocytes (Yoshida et al., 2001).
In adults, Leydig’s testicular interstitial cells (Manova et al., 1990), as well as spermatogonia at stage A1-A4 (Dym et al., 1995) and Sertoli cells (Sandlow et al., 1996) express c-Kit.
Significant modulations of KitL expression occur during maturation of spermatogonia (Hakovirta et al., 1999). After birth, the KitL/c-Kit pathway is involved in the proliferation, survival, and differentiation of spermatogonia (reviewed in (Mithraprabhu & Loveland, 2009)). It is also required in oogenesis via expression KitL and c-Kit by oocytes and granulosa cells, respectively (reviewed in (Merkwitz et al., 2011)).
Hematopoiesis
The c-Kit receptor is expressed at all developmental periods by HSCs (Ogawa et al., 1991), mast cells (Mayrhofer et al., 1987; Nocka, Buck, et al., 1990), and by lymphoid cells and granulocytes (Da Silva et al., 2006). The c-Kit activity is required for adult hematopoiesis (Ogawa et al., 1991). It is also essential for symmetrical divisions of HSCs in vitro (Bowie et al., 2007; Keller et al., 1995) and the survival of HSCs in vivo (Thoren et al., 2008).
KitL is produced by fibroblast (Nocka, Buck, et al., 1990) stromal cells and cells in the airways (Da Silva et al., 2006). Other than the survival of HSCs, KitL is involved in the retention and recirculation of HSCs. In the hematopoietic niche, HSCs adhere to the extracellular matrix and stromal cells. KitL stimulates the adhesion to fibronectin (Levesque et al., 1995) and may,
therefore, play a role in niche retention. As a small percentage of HSCs circulates in the blood (Mendez-Ferrer et al., 2008), a KitL gradient (as well as that of other cytokines) may take part in the return of HSCs to the niche (Nervi et al., 2006). Thus, the KitL/c-Kit couple has a major role in the HSCs homeostasis within the bone marrow niche (reviewed in (Kent et al., 2008)).
The KitL/c-Kit pathway also plays a fundamental role in adult erythropoiesis. In combination with the Epo receptor, KitL/c-Kit is required in normal erythropoiesis as well as reconstitution of the erythroid compartment after anemia (reviewed in (Munugalavadla & Kapur, 2005)).
Recently, it has been suggested that the KitL/c-Kit may influence the adaptive immune response by its action on dendritic cells (Ray et al., 2010).
Mast cells physiology
Mast cells are effectors of the innate immunity which are primarily located in the connective tissue. When activated, mast cells release the content of their granules by exocytosis. These events initiate the inflammatory response observed in diseases such as asthma (reviewed in (Malbec & Daeron, 2007). Mast cells are also related to mastocytosis, a benign condition characterized by an increase in their proliferation (Da Silva et al., 2006; Reber et al., 2006).
The role of the KitL/c-Kit pathway in mast cell biology was exposed thanks to the study of W and Sl mice. Indeed, these mice have little or no mast cells (Kitamura & Go, 1979; Kitamura et al., 1978).
In mice, KitL is a survival factor for mast cells, with a demonstrated anti-apoptotic effect in vitro (Yee, Paek, et al., 1994) and in vivo (Finotto et al., 1997). In humans, KitL acts in synergy with other cytokines such as NGF (Kanbe et al., 2000) or IL-3 (Gebhardt et al., 2002) to promote the survival of mature mast cells. The presence of KitL alone is sufficient to induce the differentiation of hematopoietic progenitors into mast cells (Valent et al., 1992). In the presence of KitL, human CD34+ cells of the bone marrow (Kirshenbaum et al., 1992), cord blood (Durand et al., 1994; Moriyama et al., 1996) or fetal liver cells (Irani et al., 1992) differentiate into mature mast cells. The cytokine IL3 is required for the development and maturation of mouse mast cells (Rennick et al., 1995).
KitL also acts as a chemoattractant for murine and human mast cells in vitro (Kiener et al., 2000; Meininger et al., 1992). It was shown that the broncho-alveolar fluid of asthmatic patients exerts a chemo-attracting activity on mast cells which can be blocked by an anti-KitL antibody (Olsson et al., 2000). KitL increases the adhesion of murine mast cells to fibronectin in an integrin-dependent manner (Dastych & Metcalfe, 1994; S. Tabone-Eglinger et al., 2014).
Adhesion to fibronectin seems to require the c-Kit kinase activity because a c-Kit inhibitor can block this response (Takeuchi et al., 2003) induced by s-KitL (S. Tabone-Eglinger et al., 2014).
KitL also stimulates the secretory functions of human mast cells. Indeed, it can induce degranulation (Bischoff & Dahinden, 1992; Columbo et al., 1992; Takaishi et al., 1994) and the production of pro-inflammatory and chemo-attractive cytokines (Baghestanian et al., 1997;
Gibbs et al., 1997; Oliveira et al., 2002). Thus, KitL/c-Kit pair plays a major role in homeostasis and function of mast cells. For a review see reference (Okayama & Kawakami, 2006).
As we saw, the KitL/c-Kit pathway is involved in the development and homeostasis of several cell lineages during embryogenesis and in adulthood as well as in hematopoiesis. As a result, deregulation of the pathway causes pathological disorders. We can distinguish pathologies related to a loss of function of the pathway or a decrease in its activity; or to a gain in function due to hyperactivation or uncontrolled activation. The involvement of KitL/c-Kit in disease is addressed in Section V.3.
II. The c-Kit ligand: Stem Cell Factor (SCF) or Kit Ligand (KitL)
The KitL/c-Kit pathway constitutes a cellular communication system where the KitL signal acts on its receptor cells mostly in a paracrine fashion. When bound to the membrane, KitL can also signal in a “juxtacrine” manner; this is in the contacting cell. What are the mechanisms that transform the KitL signal into a specific biological response? How can the same signal lead to different biological responses in different cells or even within the same cell? This section describes various aspects of KitL.
The c-Kit ligand is the Stem Cell Factor (SCF) (Williams et al., 1990), also called "mast cell growth factor," "steel factor," or "kit ligand" hereafter KitL. Various cell types produce KitL including fibroblasts, endothelial cells, keratinocytes, hematopoietic stromal cells, Sertoli cells and some tumor cells (Ashman, 1999). It is encoded by the Sl locus in the mouse chromosome 10 (human chromosome 12) (reviewed in (Broudy, 1997). It has been included in the family of helical cytokine structural superfamily (Jiang et al., 2000). KitL primary structure consists of an extracellular domain, followed by a transmembrane region and an intracellular c-tail (Zsebo et al., 1990).
KitL is a 30 kDa glycoprotein that exists in soluble or membrane-bound form. Both isoforms result from alternative splicing and proteolytic cleavage (Anderson et al., 1991).
The soluble KitL (s-KitL) derives from the proteolytic cleavage in exon 6 of a 248-amino acid membrane protein (KitL248) (Figure 1). This proteolytic cleavage occurs on the cell surface (Huang et al., 1992) and may depend on the action of metalloproteinases including MMP-9 (Heissig et al., 2002). Soluble-KitL circulates as glycosylated noncovalent homodimers at a plasma concentration of about 3.3 ng/ml (Langley et al., 1993; Langley et al., 1992).
The membrane-bound KitL (m-KitL) originates from the alternative splicing of exon six which leads to a glycoprotein lacking the proteolytic site and thus remains anchored to the membrane (KitL220) (Figure 1). Nevertheless, this form can be cleaved at exon 7 to generate a soluble form (Majumdar et al., 1994).
Figure 1. Alternative splicing of KitL. Two transmembrane precursors arise from alternative splicing of exon 6 of the mRNA encoding KitL: KitL248 and KitL220. KitL248 is cleaved at a proteolytic site (*) contained in exon 6 and releases the soluble-KitL (s-KitL). The KitL220 lack the cleavage site and remains mostly anchored to the membrane (m-KitL). A minor cleavage can occur in exon 7 to release a soluble form which is insignificant.
Both KitL isoforms are biologically active, but their level of expression is tissue-dependent (Huang et al., 1992). In adults, s-KitL is the predominant form in many organs. Under physiological conditions, s-KitL is produced by pulmonary fibroblasts (Kiener et al., 2000) and bronchial smooth muscle cells (Kassel et al., 1999). In contrast, mast cells and mouse testis and Sertoli cells preferentially produce m-KitL (Mauduit, Chatelain, et al., 1999; Welker et al., 1999). Within the same tissue, the level of expression of the two KitL isoforms may vary according to the stage of development. These differences are exemplified in Sl/Sld mice which revealed that germinal cells do not proliferate at puberty. During the development of male gonads, Sertoli cells express both KitL isoforms at different stages. While the soluble form is expressed just before and after birth, the membrane form persists. Interestingly, the division of PGCs correlates well with m-KitL expression (Mauduit, Hamamah, et al., 1999). In Sertoli cells, the shift between the two isoforms depends on the pH, with the m-KitL being produced at acidic pH (Mauduit, Chatelain, et al., 1999). Therefore, in adulthood, the developmental cycle of spermatogonia could be directed by the expression of different forms of KitL at various stages.
Although both KitL isoforms bind to and activate the receptor c-Kit, their functions are not equivalent and cannot be mutually compensated in vivo. Steel dickie (Sld) mice carry an intragenic deletion that restricts the expression of KitL to the soluble form (Brannan et al., 1991; Flanagan et al., 1991). The Sld/Sld mice are viable but have a severe phenotype, with pigmentation defects, macrocytic anemia, reduced the number of tissue mast cells and sterility (Lev et al., 1994; Nakayama et al., 1988). Conversely, the expression of only a membrane-restricted from of KitL in mice reduces the number of mast cells and increased sensitivity to irradiation (Tajima et al., 1998). The complete absence of KitL expression is embryonic lethal because of anemia.
The differential effects of KitL isoforms were also studied in vitro by using fetal liver stromal cells from Sl mice expressing s-KitL or m-KitL. The study demonstrated that growth of HSCs and progenitor cells is maintained longer with the membrane form than with the soluble one (Toksoz et al., 1992). Moreover, expression of KitL by endothelial and perivascular cells promotes the maintenance of HSCs in the bone marrow (Ding et al., 2012). In contrast, the release of stem and progenitor cells from the bone marrow requires the soluble form (Heissig et al., 2002). Biochemically, c-Kit stimulation by m-KitL drives to a long-lasting activation and half-life of the receptor. In contrast, s-KitL leads to faster receptor degradation (Miyazawa et al., 1995).
Therefore, the activation levels of c-Kit may have different consequences regarding endocytosis and intracellular signaling that in turns may affect the biological response.
Indeed, it is possible that if s-KitL stimulation of c-Kit can occur on the whole cell surface, m- KitL stimulation must be focal. Indeed, m-KitL stimulates adhesion of c-Kit expressing mast cells (Flanagan et al., 1991) a phenomenon that is accompanied by the accumulation of KitL at the point of contact between two cell types comparable to an immunological synapse (S.
Tabone-Eglinger et al., 2014).
III. The tyrosine kinase receptor c-Kit
The protein c-Kit is a tyrosine kinase receptor (TKR) (Yarden et al., 1987). These are membrane receptors of high affinity for their ligands, which may be growth factors or hormones (e.g., insulin) Depending on the ligand; they can induce diverse cellular responses such as cell proliferation, survival, differentiation, metabolism, adhesion or migration. Ligand binding initiates activation of the TKRs that in turns begins a signal transduction cascade that ultimately leads to a specific biological response.
The c-Kit gene was first identified as the normal homolog the viral oncogene v-Kit (Besmer et al., 1986). It is encoded by the white spotting (W) locus on chromosome 5 in mice (chromosome 4 in human) (Chabot et al., 1988; Geissler et al., 1988; Spritz et al., 1994). Like in the case of the Sl locus, naturally occurring mutations in the W cause deficiencies in gametogenesis, melanogenesis, and hematopoiesis in the mouse (see Table 2). Complete loss leads to death in utero and perinatal (Rossi et al., 1992). The c-Kit receptor is expressed in mast cells, hematopoietic stem and progenitor cells, ICCs, melanocytes, PGCs, natural killer cells, and a subset of neurons (Keshet et al., 1991; Matsui et al., 1990; Miettinen & Lasota, 2005; Nocka et al., 1989; Orr-Urtreger et al., 1990). It has also been detected in breast epithelial cells (Yared et al., 2004), and Non-Small Cell Lung Cancer (Levina et al., 2010). The physiological importance of c-Kit in these cells was demonstrated by the study of W and Sl mutations and were detailed in the previous section.
III.1. Overall structure of c-Kit
Kinases are enzymes capable of transferring the α-phosphate group of ATPs to a target substrate. Depending on the residue on which the transfer takes place, these proteins are classified as serine/threonine kinases and tyrosine kinases. Among the proteins with tyrosine kinase activity, 58 are membrane receptors (Manning et al., 2002). In humans, the Tyrosine Kinase Receptors, hereafter TKRs, are grouped into twenty classes or sub-families (I to XX) (Lemmon & Schlessinger, 2010) represented in Figure 2.
Figure 2. The family of TKR in humans. The TKRs family comprise 20 sub-families. Schematic representation of typical members of each family is provided, and the name of each member are listed below. The extracellular domains are represented according to a key given in the white box.
The intracellular domains are shown in red. It should be noted that c-Kit is a member of the PDGF receptor (Class III) family. Adapted from (Lemmon & Schlessinger, 2010).
The c-Kit receptor belongs to the class III subfamily of TKRs along with Platelet Derived Growth Factor Receptor (PDGFR) a and b, CSF-1R, and FLT3 (Chitu et al., 2015). This group of TKRs shares a common structural organization composed of an extracellular domain (ECD), one transmembrane domain (TMD), and an intracellular tyrosine kinase domain interrupted by an insert region. The schematic structure of c-Kit is shown in Figure 3.
Figure 3. Schematic representation of the structural domains of c-Kit. The extracellular domain (ECD) is composed of five immunoglobulin-like domains (Ig) which provide binding to KitL and dimerization of the receptor. The membrane-spanning region (TMD) is followed by the juxtamembrane domain (JMD) in the cytoplasmic side. The cytoplasmic region contains the tyrosine kinase domain (TKD), separated in two kinase domains (KD1 and KD2) by a kinase insert sequence, and the c-tail.
The Extracellular Domain (ECD) of c-Kit comprises five Ig-like domains (D) designated as D1 to D5. The crystal structures of D1-D3 revealed a role of these domains for ligand binding. In this model KitL and c-Kit form a 2:2 complex with each of the three c-Kit domains (D1-D3)
“wrapping” around KitL. Charge complementarity is responsible for ligand/receptor attraction and interaction. Moreover, no interactions between two monomers of c-Kit were observed (H. Liu et al., 2007). In another study, Yuzawa et al. obtained the crystal structure of the whole extracellular domain of c-Kit in complex or not with KitL. This model showed that after ligand fixation, there is a substantial conformational change that allows the establishment of two saline bridges between the D4 and D5 domain and thus the interactions between two monomers of c-Kit (Yuzawa et al., 2007).
The Juxtamembrane Domain (JMD) of c-Kit is a structural and functional region typical of the class III TKRs. Chan et al. described the auto-inhibitory function of this domain on the c-Kit kinase activity (Chan et al., 2003). The crystal structure of the JMD revealed a stable, auto- folded structure. Biochemically, the authors showed that the JMD interacts with the N-lobe of the kinase, increasing the time required for the kinase to reach its maximum activity. The JMD contains a di-tyrosine motif Y568/Y570. When these tyrosine residues are phosphorylated, the structure is lost, and thus the JMD is no longer able to interact with the kinase, resulting in inhibition of the kinase activity (Chan et al., 2003). Consequently, the JMD might be the first step allowing the transition to an active kinase. This hypothesis was demonstrated with the crystal structure of the inactive kinase (Mol et al., 2004). In the
inactive state (Figure 4), the JMD forms a pin that inserts into the catalytic cleft of the kinase.
Additional interactions not only block the two kinase lobes in a closed configuration but also inhibit the catalytic site. The JMD thus acts as a conformational autoinhibitory domain by a steric blockade in cis (Mol et al., 2004). The authors also demonstrated that Y568/Y570 are phosphorylated in trans following receptor activation. Then the JMD is released opening the access to the catalytic site (Mol et al., 2004).
Figure 4. Overall structures of activated and auto-inhibited c-Kit kinase. Ribbon structure of active c-Kit kinase showing the N and C termini, the JMD (fuchsia), the kinase insert sequence (KID). The C- helix (gold) and A-loop (green) of the kinase domain and are also shown. A) Active kinase: the active center is represented by the presence of ATP (blue) and Mg2+ ion (pink). In the active state, the C- helix is in a productive conformation, the A-loop is extended, and the JMD is released. B) Inactive (auto-inhibited) kinase: The entire JMD is visible and inserts into the catalytic cleft between the kinase lobes. The C-helix shifts and impedes the extended active conformation of the A-loop. The auto-inhibited A-loop folds back the C-kinase lobe and binds as a substrate. Adapted from (Mol et al., 2004).
The Tyrosine Kinase Domain (TKD) of c-Kit is divided into two lobes by an insert region. The kinase domain presents a bi-lobed architecture which is representative of the kinases (Mol et al., 2003). The N-terminal lobe, called "ATP-binding lobe" is mainly composed of beta sheets, a single helix (C-helix), and the P-loop which ensures the fixation of the ATP. The C-terminal lobe is composed of alpha helices and is involved in substrate binding. It contains the characteristic kinase activation loop (A-loop). It possesses a tyrosine residue (human Y823) whose phosphorylation stabilizes the loop in the open conformation and facilitate the fixation
of the substrates (Huse & Kuriyan, 2002). The Y823 residue appears to be the latter to be phosphorylated, and this modification stabilizes the active conformation of c-Kit (Mol et al., 2004). However, unlike the other kinases, it appears that Y823 phosphorylation is not required for the c-Kit kinase activity (DiNitto et al., 2010). The phosphotransferase activity is carried out by the catalytic loop, located in between the two lobes.
III.2. The c-Kit protein: isoforms and Post Translational Modifications
The c-Kit receptor is a transmembrane glycoprotein of 120-145 kDa depending on the PTM (d'Auriol et al., 1988; Yarden et al., 1987). It exists in several isoforms, the best characterized of which are the GNNK (+) and GNNK (-). Such isoforms present or not a GNNK sequence in the extracellular domain as a result of alternative splicing between exons 9 and 10. Both isoforms are often co-expressed in different tissues, but the shortest one seems to be predominant (Crosier et al., 1993). Although c-Kit isoforms have similar affinities for KitL, they differ regarding signaling (Caruana et al., 1999; Reith et al., 1991; J. Sun et al., 2008; Voytyuk et al., 2003). In general, after ligand stimulation, the GNNK(-) variant displayed stronger and faster receptor phosphorylation and activation (Montero et al., 2008) and enhanced chemotactic response to KitL (Young et al., 2007). Moreover, it strongly stimulates focus and tumor formation in nude mice compared to the GNNK(+) form (Caruana et al., 1999).
Differential expression of the two forms have been observed in malignant cells lines (Crosier et al., 1993), hematologic malignancies (Guerrini et al., 2007; Piao et al., 1994), systemic mastocytosis (E. C. Chan et al., 2013) and solid tumors (Sakuma et al., 2003; Theou et al., 2004). Therefore, the expression pattern of both variants can be a useful prognostic tool. It should be noted that in our experimental work we use the shortest c-Kit isoform GNNK(-).
A shorter transcript of c-Kit (truncated, tr-c-Kit) was reported in murine HSCs and progenitors (Zayas et al., 2008), haploid germ cells (Rossi et al., 1992; Sette et al., 2000), and human spermatozoa (Muciaccia et al., 2010). This form was also identified in colon carcinoma (Toyota et al., 1994) and human prostate cancer (Paronetto et al., 2004). The tr-c-Kit receptor lacks the ECD and the first part of the TKD. Consequently, it cannot bind the ligand and thus be activated and is not kinase active. Tr-c-Kit can be phosphorylated and can form a complex composed of various molecules such as the Fyn kinase and the PLCg (Paronetto et al., 2003).
Like KitL, the membrane-bound c-Kit can be cleaved and thus form a soluble form. In mast cells, the protease (ADAM17) could be involved in this cleavage (Cruz et al., 2004). The soluble
c-Kit can bind the soluble-KitL (Turner et al., 1995) and could compete with the membrane- bound form of c-Kit for ligand binding (Brizzi, Blechman, et al., 1994). In GIST patients, the plasma concentration of soluble c-Kit is one-hundred times higher than that of KitL. The ratio KitL/c-Kit increase after imatinib treatment (Bono et al., 2004). Whether KitL and c-Kit can interact when both are in soluble states is an open question of physiological interest. Indeed, either soluble c-Kit (i) reduces the KitL availability or (ii) prolongs the lifetime of KitL signal by protecting it from degradation. There is no data concerning the capacity of soluble c-Kit to fix the membrane KitL. Therefore, we do not know whether soluble c-Kit can activate cells expressing membrane KitL.
In the mature state, c-Kit is a highly glycosylated in the extracellular domain (Majumder et al., 1988). In general, glycosylation is initiated in the endoplasmic reticulum and then continues in the Golgi apparatus until the production of mature protein (poly-glycosylated) which is then transported to the cell membrane (Reviewed in (Aebi, 2013)). Besides glycosylation, c-Kit is phosphorylated in vivo in eight tyrosine residues: Y568, Y570, Y703, Y721, Y730, Y823, Y900, and Y936. Phosphorylation of serine residues (S741, S746) by PKC is also possible as a negative feedback mechanism (Blume-Jensen et al., 1995). Ubiquitination of c-Kit by Cbl can also occur as a downregulation mechanism (Masson et al., 2006; J. Sun et al., 2007).
III.3. Mechanism of c-Kit activation
The classic mechanism of TKRs activation consists of ligand-induced receptor dimerization (Schlessinger, 2002). Except for the insulin receptor, TKRs are present on the cell surface as monomers and their dimerization are induced by binding of their respective ligands and stabilized by interactions between the two monomers. The dimerization event depends on the nature of the ligand. In some cases, it results from binding of bivalent ligands (e.g., VEGF and KitL). Other receptors such as the EGFR exist in a pre-dimer state which is further stabilized by ligand fixation. Whatever the TKR concerned, there is a balance between the monomeric and the pre-dimerized forms. The equilibrium is in favor of the monomeric forms and is displaced to the towards the dimeric forms after fixation of the ligand (Jallal et al., 1992).
C-Kit dimerization is initiated by the dimeric nature of the ligand which induces the formation of a 1:2 complex (1 KitL dimer : 2 c-Kit monomers) (Lemmon et al., 1997). As for another member of the class III TKRs, the JMD acts as a conformational inhibitor. Therefore, complete
activation of the receptor requires the relieve of the JMD inhibition. Activation of c-Kit occurs in multiple steps (Figure 5). First, binding of KitL leads to a conformational change in the receptor that in turn initiates the kinase activity (Yuzawa et al., 2007). Then, a first series of slow tyrosine phosphorylation occurs, including the JMD Y568 and Y570. The JMD is then destabilized and releases the inhibition. Afterwards, the kinase becomes fully active, leading to the second phase of rapid tyrosine phosphorylation. Finally, the Y823 in the A-loop is phosphorylated stabilizing the active kinase conformation and opening the access to substrates (Chan et al., 2003).
Figure 5. Model of c-Kit activation. In the absence of ligand, c-Kit is in a monomeric state in which the kinase (blue) is inhibited (INACTIVE) by the JMD (orange). Binding of soluble- (s-KitL) or membrane-bound-KitL (m-KitL) (green) induces receptor dimerization and a partial activation of the c-Kit kinase (PARTIALLY-ACTIVE). The JMD is then phosphorylated ( ), and the auto-inhibition is relieved. The c-Kit kinase rearranges and becomes FULLY-ACTIVE resulting in the phosphorylation of several tyrosine residues including that of the A-loop (red).
It seems that the di-tyrosine motif of the JMD (Y568/Y570) are the first to be phosphorylated and that phosphorylation of Y823 in the A-loop is not required for the kinase activity (DiNitto et al., 2010). Upon phosphorylation, several intracellular proteins initiating different signal transduction pathways and cellular responses (Section IV).
IV. KitL/c-Kit signaling
IV.1. Recruitment of signaling proteins: adapters and enzymes
Binding of KitL to c-Kit induce conformational changes in the receptor that leads to kinase activation and the phosphorylation of several intracytoplasmic tyrosine residues. Once phosphorylated these tyrosine residues constitute the signal for the recruitment of proteins with Src-Homology 2 (SH2) and Phosphotyrosine-Binding (PTB) domains (Figure 6).
Figure 6. Tyrosine phosphorylation sites in c- Kit. The figure depicts the juxtamembrane domain (JMD), the tyrosine kinase domain (TKD) and the c-tail of c-Kit and the related phosphorylation sites. Upon receptor activation, the tyrosine residues Y567, Y569, Y703, Y719, Y728, Y821, Y900, Y934 act as docking sites for multiple adapter proteins and enzymes. Adapted from (Lennartsson &
Ronnstrand, 2012).
For abbreviations see Table 1.
The recruited proteins can be grouped into two categories: those having enzymatic activity, and those lacking enzymatic activity (adaptive proteins). Under certain conditions, enzymes can also function as adapters.
Adapter proteins contain protein-protein interaction domains that allow them to interact with several proteins at the same time, as well as with membrane phospholipids. These interactions create a signaling complex with the activated receptor.
Enzymatic effectors carry different kind of enzymatic activities: tyrosine kinase, tyrosine phosphatase, phospholipases, or ubiquitin ligase. The action of each of these enzymes generates and amplify the signal.
The proteins known to interact with c-Kit (human and mouse) are listed in Table 1. The adapters proteins include GRB2, GRB7, APS, LNK, CHK, and CRK. The enzymes engaged comprise members of the Src Family of Kinases, gamma phospholipases, tyrosine phosphatases, the PI3K and the E3-ubiquitin ligase Cbl. Reviewed in (Roskoski, 2005).
Table 1. Phospho-tyrosine residues of c-Kit and its known signaling partners.
Tyrosine residue
Protein References
Y568/Y570
APS: Adapter protein with pleckstrin homology and SH2 domains
LNK: Lymphocyte adapter protein
FYN, LYN: Tyrosine-protein kinase Fyn and Lyn SHC: SH2 domain-transforming protein C SHP1/2: SH2 domain Phosphatase 1/2
Cbl: Casitas B-Lineage lymphoma proto-oncogene CHK: Checkpoint homolog
SOCS: Suppressor Of Cytokine Signaling
(Bayle et al., 2004;
Kozlowski et al., 1998;
Lennartsson et al., 1999;
Linnekin et al., 1997;
Masson et al., 2006; D. J.
Price et al., 1997; Simon et al., 2008; Wollberg et al., 2003; Yi & Ihle, 1993)
Y703 GRB2: Growth Factor Receptor-Bound 2 (Thommes et al., 1999)
Y721 P85 (PI3K), CHK?
(Blume-Jensen et al., 1998; Kissel et al., 2000;
Serve et al., 1994; Serve et al., 1995)
Y730 PLCγ (Gommerman et al., 2000)
Y900 CRK (Lennartsson et al., 2003)
Y936 GRB2, GRB7, APS, Cbl (Thommes et al., 1999;
Wollberg et al., 2003)
The position of tyrosine residues is based on the human c-Kit sequence. Afterwards, the notation of c-Kit amino acid residues relies on the human sequence.
The signal emitted at the receptor level is amplified by the anchoring proteins which anchored in the plasma membrane by their N-terminus. Following receptor activation, these proteins can be directly or indirectly phosphorylated at tyrosine residues. In turn, these phospho-sites serve as a recruitment platform that increases the pool of signaling molecules (enzymes or adapters) near the activated RTK. In mast cells, for example, c-Kit directly phosphorylates the anchoring proteins GAB2 (Yu et al., 2006) and LAT2 (Iwaki et al., 2008). Upon phosphorylation, both LAT2 and GAB2 can recruit many signaling effectors that amplify the signal leading to different biological effects. For review articles on the role of GAB and LAT in signaling refer to (H. Gu & Neel, 2003; Horejsi et al., 2004; Sarmay et al., 2006).
In summary, receptor activation results in the recruitment of signaling effectors and their activation that finally stimulates specific intracellular signaling pathways. Signal transmission is controlled in space and time by the activity of kinases and phosphatases. Key signaling pathways downstream to c-Kit and the mechanisms of regulation will be discussed in sections IV.2 and IV.3, respectively.
IV.2. Signaling pathways activated downstream of c-Kit
The intracellular signaling pathways activated by c-Kit is one of the main subjects of this work.
Here we discuss some of these pathways and their functional implication in KitL/c-Kit axis in this section. It should be noted that much of the data obtained in vitro on the signaling pathways were done under soluble KitL stimulation. Functional results from transgenic animals are in a physiological context in which KitL can be soluble or membranous-bound depending on the tissue.
IV.2.i. The PI3K/AKT pathway
The phosphatidylinositol-3-kinases (PI3Ks) constitute a family of lipid kinases which phosphorylate phosphoinositide on the 3' hydroxyl group of the inositol ring (Morgan et al., 1990). Their lipid substrates are phosphatidylinositol (PtdIns), PtdIns-4-biphosphate (PtdIns4P) and PtdIns(4,5)P2 (Morgan et al., 1990; Okkenhaug, 2013). There are different classes of PI3Ks, but the Class I are preferentially activated by the TKRs, G protein-coupled receptors and Ras (Leevers et al., 1999). Class I PI3Ks catalyzes the conversion of PtdIns(4,5)P2
to PtdIns(3,4,5)P3. They are heterodimers composed of a p85 regulatory subunit and a p110 catalytic subunit (Carpenter et al., 1990). There are four p110 (a, b, d, g) and five p85 or p85- like (p85a, p55a, p50a, p85b, p55g) isoforms. In mammals, p110a and b are ubiquitously expressed while p110d and g are highly enriched in leukocytes (Vanhaesebroeck et al., 2010).
Below we refer to the activation of the class I PI3Ks, and more precisely to the p85a/p110 heterodimer.
The p85a subunit can interact via its SH2 domain with a phosphorylated tyrosine (pY) of an activated receptor. This tyrosine residue is part of the consensus binding motif (Y-X-X-M) for the SH2 domain of p85a (Songyang et al., 1993; Yoakim et al., 1994). Once engaged, the p85 subunit brings the p110 catalytic subunit near the membrane where it encounters its lipid substrates and produces PtdIns(3,4,5)P3. The generation of PtdInsP3 allows the recruitment
of effector proteins containing pleckstrin homology (PH) domains such as Akt, GAB2, PDK1, and modulators of the small GTPase activity. Following recruitment to PtdInsP3, PDK1 phosphorylates and activates Akt (Vanhaesebroeck & Alessi, 2000; Vanhaesebroeck et al., 2010).
The role of the PI3K/Akt pathway downstream of c-Kit activation has been studied in both hematopoietic and non-hematopoietic cells. It should be noted that the p85a subunit is the major isoform in hematopoietic cells. In the c-Kit receptor, the p85a subunit can bind directly to the murine pY719 (Serve et al., 1994; Serve et al., 1995) or human pY721 residue (Blume- Jensen et al., 1998) (Figure 7). Until now, p85a is the only known interactor for this c-Kit tyrosine residue. After KitL stimulation, PI3K can also be indirectly recruited to the tyrosine- phosphorylated GAB2 (Nishida et al., 2002). The c-Kit downstream activation of the PI3K is critical for mast cell proliferation and survival as well as mast cell secretion, adhesion, and actin polymerization (Kissel et al., 2000).
The cellular functions of class I PI3K were studied in mast cells derived from PI3K-/- mice lacking the p85a subunit. Mice defective for p85a have reduced number and function of mastocytes in the intestinal cavity and peritoneum (Fukao et al., 2002). Similarly, deficiency of p85a reduced fetal liver-derived mast cell proliferation and function in response to KitL.
Moreover, (Blume-Jensen et al., 1998)showed that PI3K is required for KitL-induced cell proliferation and survival in vitro. In agreement with this, KitL-dependent PI3K activity and Akt activation were blocked in p85a-deficient mast cells (Lu-Kuo et al., 2000). The function of PI3K/Akt pathway was also explored by disrupting the p85a binding site on c-Kit (mouse Y719). Indeed, KitL-induced PI3K signaling and Akt activation were almost completely abolished in c-KitY719F/Y719F mast cells (Blume-Jensen et al., 2000; Kissel et al., 2000). It was further shown that c-Kit could also activate the PI3K/Akt pathway through the indirect association of the scaffolding protein Gab2 (J. Sun et al., 2008).
The reconstitution of BMMC deficient for c-Kit with the Y719F mutant receptor demonstrated the role of the pathway in SCF-dependent mast cell-effector functions such as adhesion and degranulation. BMMCs from animals deficient for p85a have a chemotaxis defect to KitL (Tan, Yazicioglu, et al., 2003) whereas pure IgE-dependent degranulation is unaffected. The PI3K activity, therefore, appears to be involved in the KitL-mast cell-dependent effector functions in vitro and in vivo. Y719F knock-in mice revealed discrete phenotypes in hematopoietic
