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.cell signaling | surface receptors | phosphorylation | cancer | protein kinases R eceptor tyrosine kinases (RTKs) are cell surface receptors that serve as a signaling relay across the membrane for growth factors, cytokines, and hormones. They function to coordinate proliferation, differentiation, cell survival, and metabolism in multicellular organisms. The era of RTK study began more than half of a century ago (reviewed in ref. 1) and has significantly advanced over the last 3 decades, with numerous studies shedding light on the function, structure, and regulation of RTKs and their ligands (2-4).The RTK anaplastic lymphoma kinase (ALK) was originally identified in anaplastic large-cell non-Hodgkin's lymphoma as an oncogenic fusion protein with nucleophosmin resulting from a 2;5 chromosomal translocation (5, 6). The ALK gene is a hotspot for a variety of chromosomal translocations that result in the formation of fusion proteins that undergo spontaneous dimerization, leading to constitutive activation of the ALK kinase domain (reviewed in refs. 7 and 8). These chimeric ALK proteins were shown to drive numerous human cancers, both in hematopoietic malignancies and in solid tumors (7). Full-length, nonchimeric ALK is a driving force in neuroblastoma (NBL), where genetic studies have identified it as a major target of genetic alterations (i.e., gene amplification and somatic and germline mutations) (7,(9)(10)(11)(12). The majority of missense mutations in ALK found in NBL are located in the kinase domain and lead to constitutive receptor activation. Amplification of ALK and coamplification with the N-myc proto-oncogene (MYCN) (both genes are located on chromosome 2p) drive and cooperate in NBL progression (13). Collectively, these studies underscore the role of ALK in tumorigenesis, along with approval by the US Food and Drug Administration of an ALK inhibit...

Anaplastic lymphoma kinase (Alk) and leucocyte tyrosine kinase (Ltk) were identified as "orphan" receptor tyrosine kinases (RTKs) with oncogenic potential. Recently ALKAL1 and ALKAL2 (also named "augmentor-β" and "augmentor-α" or "FAM150A" and "FAM150B," respectively) were discovered as physiological ligands of Alk and Ltk. Here, we employ zebrafish as a model system to explore the physiological function and to characterize in vivo links between Alk and Ltk with their ligands. Unlike the two ligands encoded by mammalian genomes, the zebrafish genome contains three genes: ,, and Our experiments demonstrate that these ligands play an important role in zebrafish pigment development. Deficiency in, , and results in strong impairment in iridophore patterning of embryonic and adult zebrafish that is phenocopied in zebrafish deficient in Ltk. We show that and are essential for embryonic iridophore development and adult body coloration. In contrast, and are essential for iridophore formation in the adult eye. Importantly, these processes are entirely mediated by Ltk and not by Alk. These experiments establish a physiological link between augmentor ligands and Ltk and demonstrate that particular augmentors activate Ltk in a tissue-specific context to induce iridophore differentiation from neural crest-derived cells and pigment progenitor cells.

Anaplastic lymphoma kinase (ALK) is one of the few remaining "orphan" receptor tyrosine kinases (RTKs) in which the ligands are unknown. Ligand-mediated activation of RTKs is important throughout development. ALK is particularly relevant to the development of the nervous system. Increased activation of RTKs by mutation, genetic amplification, or signals from the stroma contributes to disease progression and acquired drug resistance in cancer. Aberrant activation of ALK occurs in subsets of lung adenocarcinoma, neuroblastoma, and other cancers. We found that heparin is a ligand that binds specifically to the ALK extracellular domain. Whereas heparins with short chain lengths bound to ALK in a monovalent manner and did not activate the receptor, longer heparin chains induced ALK dimerization and activation in cultured neuroblastoma cells. Heparin lacking N- and O-linked sulfate groups or other glycosaminoglycans with sulfation patterns different than heparin failed to activate ALK. Moreover, antibodies that bound to the extracellular domain of ALK interfered with heparin binding and prevented heparin-mediated activation of ALK. Thus, heparin and perhaps related glycosaminoglycans function as ligands for ALK, revealing a potential mechanism for the regulation of ALK activity in vivo and suggesting an approach for developing ALK-targeted therapies for cancer.

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. Highresolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V L ) and variable heavy (V H ) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereoand chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.catalytic antibodies | crystal structure | insecticide | organophosphate hydrolysis

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