Diana Linnekin

Normal and Oncogenic Forms of the Receptor Tyrosine Kinase Kit

Kit is a receptor tyrosine kinase (RTK) that binds stem cell factor. This receptor ligand combination is important for normal hematopoiesis, as well as pigmentation, gut function, and reproduction. Structurally, Kit has both an extracellular and intracellular region. Theintra‐cellular region is comprised of a juxtamembrane domain (JMD), a kinase domain, a kinase insert, and a carboxyl tail. Inappropriate expression or activation of Kit is associated with a variety of diseases in humans. Activating mutations in Kit have been identified primarily in the JMD and the second part of the kinase domain and have been associated with gastrointestinal stromal cell tumors and mastocytosis, respectively. There are also reports of activating mutations in some forms of germ cell tumors and core binding factor leukemias. Since the cloning of the Kit ligand in the early 1990s, there has been an explosion of information relating to the mechanism of action of normal forms of Kit as well as activated mutants. This is important because understanding this RTK at the biochemical level could assist in the development of therapeutics to treat primary and secondary defects in the tissues that require Kit. Furthermore, understanding the mechanisms mediating transformation of cells by activated Kit mutants will help in the design of interventions for human disease associated with these mutations. The objective of this review is to summarize what is known about normal and oncogenic forms of Kit. We will place particular emphasis on recent developments in understanding the mechanisms of action of normal and activated forms of this RTK and its association with human disease, particularly in hematopoietic cells.

Stem Cell Factor, the JAK-STAT Pathway and Signal Transduction

Recent work has demonstrated the importance of Janus family kinases (JAKs) and signal transducers and activators of transcription (STATs) in the stimulus-response coupling of receptors lacking intrinsic tyrosine kinase activity. In particular, the JAK-STAT pathway appears critical in signal transduction by interferon as well as numerous hematopoietic growth factors interacting with members of the hemapoietin receptor superfamily. Although ligands that interact with receptor tyrosine kinases (RTK), such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and colony stimulating factor-1 (CSF-1), have been shown to induce increases in phosphorylation of both JAKs and STATs, little is known about activation of this pathway by stem cell factor (SCF). This review will summarize what is known about the JAK/STAT pathway in relation to SCF signal transduction.

JAK2 is Constitutively Associated with C-Kit and is Phosphorylated in Response to Stem Cell Factor

Stem cell factor (SCF) interacts with the receptor tyrosine kinase c-Kit and has potent effects on hematopoiesis. We have examined the role of JAK2 in the SCF signal transduction pathway. JAK2 and c-Kit were constitutively associated, and treatment with SCF resulted in rapid and transient tyrosine phosphorylation of JAK2. Incubation of cells with JAK2 antisense oligonucleotides resulted in significant decreases in SCF-induced proliferation. These data suggest that JAK2 plays a role in SCF-induced proliferation.

The Csk-like proteins Lsk, Hyl, and Matk represent the same Csk homologous kinase (Chk) and are regulated by stem cell factor in the megakaryoblastic cell line MO7e

Recently, the cDNAs for Lsk, Matk and Hyl, three Csk-related protein tyrosine kinases, have been cloned. We have examined the relationship of Lsk, Matk and Hyl, and found that the gene for each of these proteins is localized to the same region of human chromosome 19. Further, the proteins encoded by Lsk and Matk cDNAs are immunologically similar. These data strongly suggest that Lsk, Hyl and Matk are the same gene product. Previous reports demonstrating expression of Hyl and Matk in hematopoietic lineages led us to investigate the regulation of Lsk expression in response to stem cell factor (SCF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in M07e, a human leukemic cell line. Induction of Lsk/Hyl/Matk protein and mRNA was observed after treatment with SCF but not with GM-CSF. GM-CSF and IL-3, potent mitogens, had no effect on Lsk/Hyl/Matk expression. In contrast, PMA induced Lsk/Hyl/Matk but did not stimulate proliferation. Therefore, induction of Lsk/ Hyl/Matk does not correlate with the capacity to stimulate proliferation. None of the stimuli examined increased Csk protein or mRNA expression. These data demonstrate differential regulation of Csk family members by cytokines and suggest a role for Lsk/ Hyl/Matk in responses mediated by SCF and PMA. Further, our data demonstrate that, as has been seen in blood monocytes, cytokine driven translational control of Lsk/Hyl/ Matk is likely a critical mode of regulation. Lastly, since our studies strongly suggest that the Lsk, Hyl and Matk kinases are related and regulated distinctly from Csk, we and several of the original authors have agreed to rename this kinase the Csk homologous kinase (Chk).

Stem Cell Factor Induces Phosphorylation of a 200 kDa Protein which Associates with c-kit

Stem cell factor (SCF) promotes limited proliferation and differentiation of hematopoietic progenitor cells and is potently synergistic in combination with growth factors such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) or erythropoietin (Epo). We have examined tyrosine phosphorylation induced by SCF in the megakaryoblastic cell line Mo7e and found phosphorylation of proteins of 200, 145, 120, 58 and 55 kDa. The dominant phosphotyrosylproteins in SCF treated cells were 200 and 145 kDa. Our studies indicated that the 145 kDa protein was c-kit, the receptor for SCF. Subsequent work was directed towards further characterizing the 200 kDa protein. Surface labeling of Mo7e cells suggested that p200 had an extracellular domain and could be induced to associate with c-kit after stimulation with SCF. The rapid phosphorylation of p200 and its immediate association with c-kit suggest that p200 is potentially a component of the SCF signal transduction pathway.

PI3 Kinase mediates transformation of hematopoietic cells by the V816 c-kit mutant

Abstract C-Kit is a receptor tyrosine kinase that binds stem cell factor. Substitution of valine for aspartic acid 816 (V816) constitutively activates human c-Kit and this mutation is found in patients with mastocytosis, leukemia and germ cells tumors. Transduction of immortalized murine progenitor cells with wild-type c-Kit (MIHC-Kit) results in stem cell factor-induced growth (SCF), while cells expressing V816 c-Kit (MIHC-V816) are factor-independent and tumorigenic. The mechanisms mediating transformation by V816 c-Kit are unknown. SCF activates Erk 1, Erk2 and P13 kinase in MIHC-Kit cells, and P13 kinase contributes to activation of Akt and Jnks. In MIHC-V816 cells, P13 kinase, Jnk 1 and Jnk 2 were activated, but Akt, Erk 1 and Erk 2 were not. Thus, V816 c-Kit constitutively activates P13 kinase, but not all signaling pathways activated by wild-type c-Kit. Further, pathways downstream of P13 kinase are altered in MIHC-V816 cells. Studies with a P13 kinase inhibitor and V816/F721 c-Kit, a mutant incapable of recruiting P13 kinase, indicate that constitutive activation of P13 kinase plays a role in factor-independent growth mediated by V816 c-Kit and is critical for tumorigenicity of MIHC. These data are the first to demonstrate the role of P13 kinase in transformation of hematopoietic cells by this oncogenic c-Kit mutant.

Chapter 17 Induction of Protein Phosphorylation during Leukocyte Activation

Publisher Summary This chapter discusses the roles of various members of the major protein kinase families that have been suggested to play a role in leukocyte hematopoiesis and in the activation of cells participating in the mature antigen-specific immune response. It also discusses protein kinases in leukocyte development andactivation and some of the general findings of the major enzyme families believed to participate in the regulation of leukocyte biology. The receptors that control leukocyte function can be generally separated into three structure–functional categories. Class I receptors may be directly coupled to “classical” second messenger systems, such as phosphoinositol hydrolysis or cyclic adenosine monophosphate (cAMP). Class II receptors have a protein kinase activity intrinsic to the ligand binding protein, almost exclusively a tyrosine kinase. Class III receptors, probably the most enigmatic, are those that couple to serine and tyrosine phosphotransferase systems without any apparent kinase domain intrinsic to the receptor structure.