Leukocyte Tyrosine Kinase Research Articles

Multifaceted Roles of ALK Family Receptors and Augmentor Ligands in Health and Disease: A Comprehensive Review.

This review commemorates the 10-year anniversary of the discovery of physiological ligands Augα (Augmentor α; ALKAL2; Fam150b) and Augβ (Augmentor β; ALKAL1; Fam150a) for anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK), previously considered orphan receptors. This manuscript provides an in-depth review of the biophysical and cellular properties of ALK family receptors and their roles in cancer, metabolism, pain, ophthalmology, pigmentation, central nervous system (CNS) function, and reproduction. ALK and LTK receptors are implicated in the development of numerous cancers, and targeted inhibition of their signaling pathways can offer therapeutic benefits. Additionally, ALK family receptors are involved in regulating body weight and metabolism, modulating pain signaling, and contributing to eye development and pigmentation. In the CNS, these receptors play a role in synapse modulation, neurogenesis, and various psychiatric pathologies. Lastly, ALK expression is linked to reproductive functions, with potential implications for patients undergoing ALK inhibitor therapy. Further research is needed to better understand the complex interactions of ALK family receptors and Aug ligands and to repurpose targeted therapy for a wide range of human diseases.

Leukocyte Tyrosine Kinase (Ltk) Is the Mendelian Determinant of the Axolotl Melanoid Color Variant.

The great diversity of color patterns observed among amphibians is largely explained by the differentiation of relatively few pigment cell types during development. Mexican axolotls present a variety of color phenotypes that span the continuum from leucistic to highly melanistic. The melanoid axolotl is a Mendelian variant characterized by large numbers of melanophores, proportionally fewer xanthophores, and no iridophores. Early studies of melanoid were influential in developing the single-origin hypothesis of pigment cell development, wherein it has been proposed that all three pigment cell types derive from a common progenitor cell, with pigment metabolites playing potential roles in directing the development of organelles that define different pigment cell types. Specifically, these studies identified xanthine dehydrogenase (XDH) activity as a mechanism for the permissive differentiation of melanophores at the expense of xanthophores and iridophores. We used bulked segregant RNA-Seq to screen the axolotl genome for melanoid candidate genes and identify the associated locus. Dissimilar frequencies of single-nucleotide polymorphisms were identified between pooled RNA samples of wild-type and melanoid siblings for a region on chromosome 14q. This region contains gephyrin (Gphn), an enzyme that catalyzes the synthesis of the molybdenum cofactor that is required for XDH activity, and leukocyte tyrosine kinase (Ltk), a cell surface signaling receptor that is required for iridophore differentiation in zebrafish. Wild-type Ltk crispants present similar pigment phenotypes to melanoid, strongly implicating Ltk as the melanoid locus. In concert with recent findings in zebrafish, our results support the idea of direct fate specification of pigment cells and, more generally, the single-origin hypothesis of pigment cell development.

Zebrafish pigment cells develop directly from persistent highly multipotent progenitors

Neural crest cells are highly multipotent stem cells, but it remains unclear how their fate restriction to specific fates occurs. The direct fate restriction model hypothesises that migrating cells maintain full multipotency, whilst progressive fate restriction envisages fully multipotent cells transitioning to partially-restricted intermediates before committing to individual fates. Using zebrafish pigment cell development as a model, we show applying NanoString hybridization single cell transcriptional profiling and RNAscope in situ hybridization that neural crest cells retain broad multipotency throughout migration and even in post-migratory cells in vivo, with no evidence for partially-restricted intermediates. We find that leukocyte tyrosine kinase early expression marks a multipotent stage, with signalling driving iridophore differentiation through repression of fate-specific transcription factors for other fates. We reconcile the direct and progressive fate restriction models by proposing that pigment cell development occurs directly, but dynamically, from a highly multipotent state, consistent with our recently-proposed Cyclical Fate Restriction model.

Tyrosine Kinase Inhibitors Target B Lymphocytes.

Autoimmune disorders and some types of blood cancer originate when B lymphocytes malfunction. In particular, when B cells produce antibodies recognizing the body's proteins, it leads to various autoimmune disorders. Additionally, when B cells of various developmental stages transform into cancer cells, it results in blood cancers, including multiple myeloma, lymphoma, and leukemia. Thus, new methods of targeting B cells are required for various patient groups. Here, we used protein kinase inhibitors alectinib, brigatinib, ceritinib, crizotinib, entrectinib, and lorlatinib previously approved as drugs treating anaplastic lymphoma kinase (ALK)-positive lung cancer cells. We hypothesized that the same inhibitors will efficiently target leukocyte tyrosine kinase (LTK)-positive, actively protein-secreting mature B lymphocytes, including plasma cells. We isolated CD19-positive human B cells from the blood of healthy donors and used two alternative methods to stimulate cell maturation toward plasma cells. Using cell proliferation and flow cytometry assays, we found that ceritinib and entrectinib eliminate plasma cells from B cell populations. Alectinib, brigatinib, and crizotinib also inhibited B cell proliferation, while lorlatinib had no or limited effect on B cells. More generally, we concluded that several drugs previously developed to treat ALK-positive malignant cells can be also used to treat LTK-positive B cells.

Structural basis of cytokine-mediated activation of ALK family receptors.

Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGβ and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.

P-Glycoprotein Inhibitor Tariquidar Plays an Important Regulatory Role in Pigmentation in Larval Zebrafish.

Zebrafish has emerged as a powerful model in studies dealing with pigment development and pathobiology of pigment diseases. Due to its conserved pigment pattern with established genetic background, the zebrafish is used for screening of active compounds influencing melanophore, iridophore, and xanthophore development and differentiation. In our study, zebrafish embryos and larvae were used to investigate the influence of third-generation noncompetitive P-glycoprotein inhibitor, tariquidar (TQR), on pigmentation, including phenotype effects and changes in gene expression of chosen chromatophore differentiation markers. Five-day exposure to increasing TQR concentrations (1 µM, 10 µM, and 50 µM) resulted in a dose-dependent augmentation of the area covered with melanophores but a reduction in the area covered by iridophores. The observations were performed in three distinct regions—the eye, dorsal head, and tail. Moreover, TQR enhanced melanophore renewal after depigmentation caused by 0.2 mM 1-phenyl-2-thiourea (PTU) treatment. qPCR analysis performed in 56-h post-fertilization (hpf) embryos demonstrated differential expression patterns of genes related to pigment development and differentiation. The most substantial findings include those indicating that TQR had no significant influence on leukocyte tyrosine kinase, GTP cyclohydrolase 2, tyrosinase-related protein 1, and forkhead box D3, however, markedly upregulated tyrosinase, dopachrome tautomerase and melanocyte inducing transcription factor, and downregulated purine nucleoside phosphorylase 4a. The present study suggests that TQR is an agent with multidirectional properties toward pigment cell formation and distribution in the zebrafish larvae and therefore points to the involvement of P-glycoprotein in this process.

Comparative Genomics within and across Bilaterians Illuminates the Evolutionary History of ALK and LTK Proto-Oncogene Origination and Diversification.

Comparative genomic analyses have enormous potential for identifying key genes central to human health phenotypes, including those that promote cancers. In particular, the successful development of novel therapeutics using model species requires phylogenetic analyses to determine molecular homology. Accordingly, we investigate the evolutionary histories of anaplastic lymphoma kinase (ALK)—which can underlie tumorigenesis in neuroblastoma, nonsmall cell lung cancer, and anaplastic large-cell lymphoma—its close relative leukocyte tyrosine kinase (LTK) and their candidate ligands. Homology of ligands identified in model organisms to those functioning in humans remains unclear. Therefore, we searched for homologs of the human genes across metazoan genomes, finding that the candidate ligands Jeb and Hen-1 were restricted to nonvertebrate species. In contrast, the ligand augmentor (AUG) was only identified in vertebrates. We found two ALK-like and four AUG-like protein-coding genes in lamprey. Of these six genes, only one ALK-like and two AUG-like genes exhibited early embryonic expression that parallels model mammal systems. Two copies of AUG are present in nearly all jawed vertebrates. Our phylogenetic analysis strongly supports the presence of previously unrecognized functional convergences of ALK and LTK between actinopterygians and sarcopterygians—despite contemporaneous, highly conserved synteny of ALK and LTK. These findings provide critical guidance regarding the propriety of fish and mammal models with regard to model organism-based investigation of these medically important genes. In sum, our results provide the phylogenetic context necessary for effective investigations of the functional roles and biology of these critically important receptors.

LTK is an ER-resident receptor tyrosine kinase that regulates secretion.

The endoplasmic reticulum (ER) is a key regulator of cellular proteostasis because it controls folding, sorting, and degradation of secretory proteins. Much has been learned about how environmentally triggered signaling pathways regulate ER function, but only little is known about local signaling at the ER. The identification of ER-resident signaling molecules will help gain a deeper understanding of the regulation of ER function and thus of proteostasis. Here, we show that leukocyte tyrosine kinase (LTK) is an ER-resident receptor tyrosine kinase. Depletion of LTK as well as its pharmacologic inhibition reduces the number of ER exit sites and slows ER-to-Golgi transport. Furthermore, we show that LTK interacts with and phosphorylates Sec12. Expression of a phosphoablating mutant of Sec12 reduces the efficiency of ER export. Thus, LTK-to-Sec12 signaling represents the first example of an ER-resident signaling module with the potential to regulate proteostasis.

Identification of a biologically active fragment of ALK and LTK-Ligand 2 (augmentor-α)

Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,β) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor-receptor interactions.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

ALKALs are in vivo ligands for ALK family receptor tyrosine kinases in the neural crest and derived cells

Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.

Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand–receptor interactions

Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-β) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states.

Deorphanization of the human leukocyte tyrosine kinase (LTK) receptor by a signaling screen of the extracellular proteome.

There are many transmembrane receptor-like proteins whose ligands have not been identified. A strategy for finding ligands when little is known about their tissue source is to screen each extracellular protein individually expressed in an array format by using a sensitive functional readout. Taking this approach, we have screened a large collection (3,191 proteins) of extracellular proteins for their ability to activate signaling of an orphan receptor, leukocyte tyrosine kinase (LTK). Only two related secreted factors, FAM150A and FAM150B (family with sequence similarity 150 member A and member B), stimulated LTK phosphorylation. FAM150A binds LTK extracellular domain with high affinity (K(D) = 28 pM). FAM150A stimulates LTK phosphorylation in a ligand-dependent manner. This strategy provides an efficient approach for identifying functional ligands for other orphan receptors.

A Simple, Highly Visual in Vivo Screen for Anaplastic Lymphoma Kinase Inhibitors

Anaplastic lymphoma kinase (ALK) is an important drug target in many cancers, including lymphoma, neuroblastoma, and lung cancer. Here, we demonstrate proof-of-principle for a novel and inexpensive assay for ALK inhibitor activity and identification in zebrafish. We demonstrate that the human oncogenic ALK fusion, NPM-ALK, drives overproduction of iridophores, a highly visible, shiny pigment cell-type in zebrafish. Treatment with the potent ALK inhibitor, TAE684, fully inhibits production of ALK-dependent iridophores. Using our assay, we test multiple properties of TAE684 in vivo, including efficacy, specificity, and toxicity. We note that TAE684 also inhibits the closely related leukocyte tyrosine kinase (Ltk) that is required for endogenous iridophore development. Similar effects are observed with an independent inhibitor, Crizotinib. Our assay can thus be utilized to identify ALK or LTK inhibitors. Importantly, the natural reflectivity of iridophores lends itself to automation for high throughput assessment of ALK and LTK inhibitor compounds in vivo.

ALK-Activating Homologous Mutations in LTK Induce Cellular Transformation

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase reported to be overexpressed in human leukemia. Though much regarding the function of LTK remains unknown, it shares a high degree of similarity with anaplastic lymphoma kinase (ALK), which is found mutated in human cancer. In order to determine if LTK has transforming potential, we created two LTK mutants, F568L and R669Q, that correspond to two well-characterized activating mutations of ALK (F1174L and R1275Q). LTK-F568L, but not wildtype LTK or LTK-R669Q, transformed hematopoietic cells to cytokine independence. LTK-F568L exhibited a stronger ability to induce loss of contact inhibition and anchorage-independent growth of epithelial cells compared to LTK-R669Q, while wildtype LTK was non-transforming in the same cells. Likewise, LTK-F568L induced greater neurite outgrowth of PC12 cells than R669Q, while wildtype LTK could not. Correlating with transforming activity, LTK-F568L displayed significantly enhanced tyrosine phosphorylation compared to wildtype LTK and LTK-R668Q and induced activation of various signaling proteins including Shc, ERK and the JAK/STAT pathway. Expression of wildtype LTK or LTK-R669Q generally led to weaker activation of signaling proteins than expression of LTK-F568L, or no activation at all. Thus, mutating LTK at residue F568, and to a lesser extent at R669, activates the receptor tyrosine kinase, inducing cell signaling that results in transforming properties. These studies suggest that aberrant activation of LTK may contribute to neoplastic cell growth.

ALK receptor activation, ligands and therapeutic targeting in glioblastoma and in other cancers.

The intracellular anaplastic lymphoma kinase (ALK) fragment shows striking homology with members of the insulin receptor family and was initially identified as an oncogenic fusion protein resulting from a translocation in lymphoma and more recently in a range of cancers. The full-length ALK transmembrane receptor of ~220 kDa was identified based on this initial work. This tyrosine kinase receptor and its ligands, the growth factors pleiotrophin (PTN) and midkine (MK) are highly expressed during development of the nervous system and other organs. Each of these genes has been implicated in malignant progression of different tumor types and shown to alter phenotypes as well as signal transduction in cultured normal and tumor cells. Beyond its role in cancer, the ALK receptor pathway is thought to contribute to nervous system development, function, and repair, as well as metabolic homeostasis and the maintenance of tissue regeneration. ALK receptor activity in cancer can be up-regulated by amplification, overexpression, ligand binding, mutations in the intracellular domain of the receptor and by activity of the receptor tyrosine phosphatase PTPRz. Here we discuss the evidence for ligand control of ALK activity as well as the potential prognostic and therapeutic implications from gene expression and functional studies. An analysis of 18 published gene expression data sets from different cancers shows that overexpression of ALK, its smaller homolog LTK (leukocyte tyrosine kinase) and the ligands PTN and MK in cancer tissues from patients correlate significantly with worse course and outcome of the disease. This observation together with preclinical functional studies suggests that this pathway could be a valid therapeutic target for which complementary targeting strategies with small molecule kinase inhibitors as well as antibodies to ligands or the receptors may be used.

Anaplastic Lymphoma Kinase and Leukocyte Tyrosine Kinase: Functions and genetic interactions in learning, memory and adult neurogenesis

Anaplastic Lymphoma Kinase (Alk) is a receptor tyrosine kinase expressed throughout the adult mammalian hippocampus. Recent studies in Drosophila and prior studies in Caenorhabditis elegans have implicated Alk signaling in learning and neurogenesis. We have studied the roles of Alk and the closely related receptor Leukocyte Tyrosine Kinase (Ltk) in learning, behavior and neurogenesis. In the hippocampus, both receptors are expressed throughout the dentate gyrus, CA1 and CA3. To assess the functional roles of Alk and Ltk in the mammalian brain, we analyzed phenotypes in Alk mutant, Ltk mutant and Alk/Ltk double-mutant mice compared to wild-type littermates. Similar to Drosophila, we found enhanced performance in spatial memory in Alk mutant mice. Also similar to Drosophila, we observed reduced neurogenesis associated with loss of Alk function. We also report genetic interactions between Alk and Ltk with respect to neurogenesis and behavioral measures such as activity, anxiety levels, and retention of spatial memory.

Cytoplasmic ACK1 Interaction with Multiple Receptor Tyrosine Kinases Is Mediated by Grb2

ACK1 (activated Cdc42-associated kinase 1), a cytoplsmic tyrosine kinase, is implicated in metastatic behavior, cell spreading and migration, and epidermal growth factor receptor (EGFR) signaling. The function of ACK1 in the regulation of receptor tyrosine kinases requires a C-terminal region that demonstrates a significant homology to the EGFR binding domain of MIG6. In this study, we have identified additional receptor tyrosine kinases, including Axl, leukocyte tyrosine kinase, and anaplastic lymphoma kinase, that can bind to the ACK1/MIG6 homology region. Unlike the interaction between MIG6 and EGFR, our data suggest that these receptor tyrosine kinases require the adaptor protein Grb2 for efficient binding, which interacts with highly conserved proline-rich regions that are conserved between ACK1 and MIG6. We have focused on Axl and compared how ACK1/Axl differs from the ACK1/EGFR axis by investigating effects of knockdown of endogenous ACK1. Although EGFR activation promotes ACK1 turnover, Axl activation by GAS6 does not; interestingly, the reciprocal down-regulation of GAS6-stimulated Axl is blocked by removing ACK1. Thus, ACK1 functions in part to control Axl receptor levels. Silencing of ACK1 also leads to diminished ruffling and migration in DU145 and COS7 cells upon GAS6-Axl signaling. The ability of ACK1 to modulate Axl and perhaps anaplastic lymphoma kinase (altered in anaplastic large cell lymphomas) might explain why ACK1 can promote metastatic and transformed behavior in a number of cancers.

Expression of a chimeric CSF1R-LTK mediates ligand-dependent neurite outgrowth

In an earlier screening, we identified several genes for kinases that might control the extension of neurites. One of these genes encoded a leukocyte tyrosine kinase (LTK), which is a receptor tyrosine kinase whose ligands remain to be identified. To examine the possible role of this LTK in neurite outgrowth, we constructed a chimeric receptor, in which the extracellular domain of the receptor for colony-stimulating factor-1 was fused to the cytoplasmic domain of LTK, which allowed the selective activation of LTK by colony-stimulating factor-1. Our studies using this chimeric receptor suggest that activation of the tyrosine kinase activity of LTK is sufficient to promote neurite outgrowth through pathways that include reactions catalyzed by phosphatidylinositol 3-kinase and MAPK.

Leukocyte tyrosine kinase functions in pigment cell development.

A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.