Adopting ALK and LTK

COMMENTARY COMMENTARY Adopting ALK and LTK Greg Lemke1 Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037 RTKs and Their Ligands Aberrant, uncontrolled activation of receptor tyrosine kinases (RTKs) is the “original sin” of many human cancers, in that this activation can both initiate and drive the evolution of tumors (1). Mutation and overexpression of the multiple members of the epidermal growth factor/ErbB receptor family, to cite only a single example, constitute cancer drivers that have been clinically targeted by a panoply of biologic and small-molecule inhibitors for more than a decade (2, 3). Given the consequences of their activation, RTKs are always tightly regulated: their steady-state enzymatic (tyrosine kinase) activity is very low in the absence of activation, which is normally triggered by the binding of a protein ligand to the RTK extracellular domain (4). This aspect has made the identification of RTK ligands an especially important goal, and studies over the last three decades have uncovered the ligands for nearly all of the 58 RTKs encoded in the human genome. Two closely related RTKs— the anaplastic lymphoma kinase (ALK) and the leukocyte tyrosine kinase (LTK)—have been notable holdouts, in that they have remained receptor “orphans” without a ligand. This is no longer the case. In PNAS, Reshetnyak et al. (5) identify “family with sequence similarity” (FAM) 150A and 150B— rechristened augmentor-β and augmentor-α, respectively, by the authors—as protein ligands for ALK and LTK. These two RTKs share distinctive structural features, including highly conserved tyrosine kinase domains and unusual glycine-rich regions in their ligand-binding extracellular domains (ectodomains) that exhibit 55% amino acid identity between the receptors. ALK has been very widely studied in cancer (6), particularly in nonsmall-cell lung carcinoma, neuroblastoma, and anaplastic largecell lymphoma, with which it was first associated (7). ALK-driven cancers have been seen to arise from gene fusion events between the ALK kinase domain and various protein-coding domains, by mutations in fulllength ALK, or via overexpression of the ALK protein. Crizotinib, ceritinib, and other smallmolecule ALK inhibitors are now used as therapies for nonsmall-cell lung carcinoma www.pnas.org/cgi/doi/10.1073/pnas.1521923113 (8). This clinical activity notwithstanding, the biological roles that ALK plays in either vertebrate development or mature physiology are not well understood. ALK knockout mice do not display strong phenotypes, although defects in mature brain (cortical and hippocampal) function have been reported (9). LTK is murkier still: experiments in zebrafish Reshetnyak et al. identify “family with sequence similarity” (FAM) 150A and 150B— rechristened augmentor-β and augmentor-α, respectively, by the authors—as protein ligands for ALK and LTK. are consistent with a role in neural crest cell development (10), and genome-wide association studies have linked polymorphisms in the human LTK gene to lupus (11), but its role in cancer remains unclear. ”De-Orphaning“ LTK and ALK The first identification of the FAM 150A/B proteins as LTK ligands was made last year by Zhang et al. in the Williams group (12), who used a proteome screening assay to identify FAM150A as a protein that bound to the extracellular domain of LTK and activated the endogenous receptor expressed by SKN-SH neuroblastoma cells. Following up on this work, Reshetnyak et al. in the Schlessinger laboratory now identify FAM150A (augmentor-β or AUG-β, in their new nomenclature) as a single-specificity ligand for LTK, and FAM150B (AUG-α) as a dual-specificity ligand that binds and activates both ALK and LTK (5). The new work was greatly facilitated by the ability of Reshetnyak et al. to produce and purify relatively large amounts of recombinant AUG-α and AUG-β, which was not possible in the earlier study. Abundant ligand expression was achieved only through the clever coexpression of recombinant Ig Fc fusion proteins for both AUG-α/β and LTK/ALK extracellular domains in the same HEK293 cells. Protein A-based chromatographic purification of the ligand– ectodomain complexes from conditioned medium followed by proteolytic cleavage to remove the Fc domain resulted in a highly pure ligand that could be used for extensive cell-based activation and binding assays. Reshetnyak et al.’s (5) conclusions with respect to receptor specificity for AUG-α and AUG-β are based on both differential receptor activation (stimulation of receptor autophosphorylation) and differential receptor binding. AUG-α was seen to stimulate the autophosphorylation of both ALK and LTK, individually expressed in NIH 3T3 cells, at high picomolar concentrations. (The preparation of pure recombinant ligands allowed for precise quantification.) In contrast, AUG-β was an equivalently strong activator of LTK—but a very weak (albeit detectable) activator of ALK—in the same cells. Similarly, in surface plasmon resonance assays of the binding of AUG-α and AUG-β to immobilized LTK or ALK Fc fusion proteins, AUG-α bound equally well to both receptor ectodomains [equilibrium dissociation constants (KDs) of 11.4 and 7.1 nM], whereas AUG-β displayed a ∼20-fold binding preference for LTK over ALK (KDs of 3.7 versus 74.3 nM). Related experiments, with similar but not identical conclusions, were recently published by Guan et al. of the Palmer group (13). These investigators also identified the FAM150A/B proteins as ligands for ALK and LTK, but concluded that both FAM150A and FAM150B are activating ligands for ALK. The two sets of analyses used different cells for assay and ligand/receptor expression, and in most of the experiments of Guan et al. the expression levels of the recombinant ligands and receptors were unknown. (Ligands were assayed from conditioned media, or were coexpressed with receptors in cell lines at unmeasured levels.) Guan et al. used surface plasmon resonance methods similar to those of Reshetnyak et al. (5) to quantify FAM150A (AUG-β) binding to the ALK ectodomain, but measured a somewhat lower KD (∼20 nM) for binding. Author contributions: G.L. wrote the paper. The author declares no conflict of interest. See companion article on page 15862. 1 Email: lemke@salk.edu. PNAS | December 29, 2015 | vol. 112 | no. 52 | 15783–15784 Guan et al. (13) also coexpressed human ALK with either FAM150A or FAM150B in vivo, in the developing Drosophila eye. Expression of FAM150A, FAM150B, or human (wild-type) ALK alone yielded no perturbation of fly eye development, but expression of either FAM150A or FAM150B together with human ALK resulted in a rough eye phenotype (13). Again, however, the expression levels of the ligands and the receptors were uncontrolled, and it is possible that these levels were anomalously high. An additional unresolved issue with respect to ALK activation specifically relates to the role of heparin and heparan sulfate proteoglycans (HSPGs) in the modulation of ALK signaling. In studies published earlier this year, Murray et al., also in the Schlessinger group, identified heparin as an activating ligand for ALK in NB-1 neuroblastoma cells (14). These investigators found that acidic sulfated glycosaminoglycan polymers bound to the very basic amino terminus of ALK, and strongly potentiated ALK autophosphorylation in NB-1 cells. This was of particular interest because HSPGs are well-known modulators of FGF binding and signaling through FGF receptors (15). Heparin activation of ALK raised the possibility that HSPGs were interacting with an at-the-time unknown protein ligand (FAM150A or B?) for this receptor (16). 15784 | www.pnas.org/cgi/doi/10.1073/pnas.1521923113 When Reshetnyak et al. (5) examined this issue, they found that heparin potentiated ALK signaling in NB-1 cells when coapplied together with a very low concentration (0.064 nM) of AUG-α, but that at higher concentrations this potentiation was overshadowed by the effect of AUG-α alone; and similar activation of ALK by heparin was seen neither in NIH 3T3 cells stably expressing ALK, nor by the Palmer group (13) (who found that heparin indeed binds to FAM150A) in IMR-32 cells. The exact position of HSPGs in the hierarchy of ALK signaling, therefore, awaits further functional, biochemical, and structural analyses. Although there is still work to be done, the studies of Zhang et al. (12), Guan et al. (13), and Reshetnyak et al. (5) unambiguously position AUG-α/β (FAM150B/A) as protein ligands for the ALK/LTK family. 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