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...

The far-red light (FR) photoreceptor phytochrome A (phyA) contains no DNA binding domain but associates with the CHALCONE SYNTHASE promoter through its chaperone FAR-RED ELONGATED HYPOCOTYL1 and transcription factors. Here, we performed a genome-wide identification of phyA targets using a combination of phyA chromatin immunoprecipitation and RNA sequencing methods in Arabidopsis thaliana. Our results indicate that phyA signaling widely affects gene promoters involved in multiple FR-modulated aspects of plant growth. Furthermore, we observed an enrichment of hormone-and stressresponsive elements in the phyA direct target promoters, indicating that a much broader than expected range of transcription factors is involved in the phyA signaling pathway. To verify our hypothesis that phyA regulates genes other than light-responsive ones through the interaction with corresponding transcription factors, we examined the action of phyA on one of its direct target genes, NAC019, which encodes an abscisic acid-dependent transcription factor. The phyA signaling cascade not only targets two G-boxes on the NAC019 promoter for subsequent transcriptional regulation but also positively coordinates with the abscisic acid signaling response for root elongation inhibition under FR. Our study provides new insight into how plants rapidly fine-tune their growth strategy upon changes in the light environment by escorting photoreceptors to the promoters of hormone-or stress-responsive genes for individualized modulation.

Emerging plants have to adapt to a high ratio of far-red light (FR)/red light (R) light in the canopy before they reach the R-enriched direct sunlight. Phytochrome A (phyA) is the single dominant photoreceptor in young Arabidopsis thaliana seedlings that initiates photomorphogenesis in response to a FR-enriched environment and transduces increasing R signals to early responsive genes. To date, how phyA differentially transmits FR and R signals to downstream genes remains obscure.Here, we present a phyA pathway in which FAR-RED ELONGATED HYPOCOTYL1 (FHY1), an essential partner of phyA, directly guides phyA to target gene promoters and coactivates transcription. Furthermore, we identified two phosphorylation sites on FHY1, Ser-39 and Thr-61, whose phosphorylation by phyA under R inhibits phyA signaling at each step of its pathway. Deregulation of FHY1 phosphorylation renders seedlings colorblind to FR and R. Finally, we show that the weaker phyA response resulting from FHY1 phosphorylation ensures the seedling deetiolation process in response to a R-enriched light condition. Collectively, our results reveal FHY1 phosphorylation as a key mechanism for FR/R spectrum-specific responses in plants and an essential event for plant adaption to changing light conditions in nature.

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.

Adipocyte function is crucial for the control of whole body energy homeostasis. Pathway analysis of differentiating 3T3-L1 adipocytes reveals that major metabolic pathways induced during differentiation involve mitochondrial function. However, it is not clear why differentiated white adipocytes require enhanced respiratory chain activity relative to pre-adipocytes. To address this question, we used small interference RNA to interfere with the induction of the transcription factor Tfam, which is highly induced between days 2 and 4 of differentiation and is crucial for replication of mitochondrial DNA. Interference with Tfam resulted in cells with decreased respiratory chain capacity, reflected by decreased basal oxygen consumption, and decreased mitochondrial ATP synthesis, but no difference in many other adipocyte functions or expression levels of adipose-specific genes. However, insulin-stimulated GLUT4 translocation to the cell surface and subsequent glucose transport are impaired in Tfam knockdown cells. Paradoxically, insulin-stimulated Akt phosphorylation is significantly enhanced in these cells. These studies reveal independent links between mitochondrial function, insulin signaling, and glucose transport, in which impaired respiratory chain activity enhances insulin signaling to Akt phosphorylation, but impairs GLUT4 translocation. These results indicate that mitochondrial respiratory chain dysfunction in adipocytes can cause impaired insulin responsiveness of GLUT4 translocation by a mechanism downstream of the Akt protein kinase.A large body of evidence has pointed to a close relationship between ectopic fat accumulation in tissues such as muscle and liver and the development of insulin resistance (1-3). The primary defense against such ectopic lipid accumulation is a well functioning adipose tissue, capable of sequestering excess calories in the form of stored triglycerides (4). In addition to this crucial role, adipose tissue is an endocrine organ that controls whole body energy homeostasis by secreting multiple cytokines that signal to other tissues (5, 6). The central role of adipose tissue in energy homeostasis is underscored by recent findings indicating that adipose tissue is a primary locus for the alterations induced by caloric restriction that accompany longevity (7,8). Thus, the cell biological mechanism involved in optimal adipose tissue development and function are crucial for the control of whole organism energy homeostasis and the determination of life span.Adipocyte differentiation is accompanied by an expansion of mitochondrial mass (9, 10), but the functional role of the relatively high levels of mitochondria in white adipocytes compared with those in adipose stroma and other tissues is not clear. High mitochondria levels may be required for the support of adipocyte-specific ATP-requiring processes (11), or to support metabolic functions such as glyceroneogenesis, which is required for triglyceride deposition (12, 13). White adipocyte mitochondria levels in rodents and humans change ma...

The unfolded protein response (UPR) is a homeostatic signaling mechanism that balances the protein folding capacity of the endoplasmic reticulum (ER) with the secretory protein load of the cell. ER protein folding capacity is dependent on the abundance of chaperones, which is increased in response to UPR signaling, and on a sufficient ATP supply for their activity. An essential branch of the UPR entails the splicing of XBP1 mRNA to form the XBP1 transcription factor. XBP1 has been shown to be required during adipocyte differentiation, enabling mature adipocytes to secrete adiponectin, and during differentiation of B cells into antibody-secreting plasma cells. Here we find that adenylate kinase 2 (AK2), a mitochondrial enzyme that regulates adenine nucleotide interconversion within the intermembrane space, is markedly induced during adipocyte and B cell differentiation. Depletion of AK2 by RNAi impairs adiponectin secretion in 3T3-L1 adipocytes, IgM secretion in BCL1 cells, and the induction of the UPR during differentiation of both cell types. These results reveal a new mechanism by which mitochondria support ER function and suggest that specific mitochondrial defects may give rise to impaired UPR signaling. The requirement for AK2 for UPR induction may explain the pathogenesis of the profound hematopoietic defects of reticular dysgenesis, a disease associated with mutations of the AK2 gene in humans.Cellular homeostasis depends on the continuous synthesis of transmembrane and secretory proteins to maintain cellular integrity and an appropriate extracellular environment. These proteins are translocated into the endoplasmic reticulum (ER) 2 for post-translational processing, involving the function of numerous chaperones that catalyze ATP-dependent protein folding. When the need for endoplasmic reticulum folding capacity rises (ER stress), a transient inhibition of protein synthesis is triggered followed by an increase in chaperone levels. This coordinated response is known as the unfolded protein response (UPR). When the UPR fails to resolve ER stress, a global response is triggered that can lead to apoptosis and is linked to multiple human pathologies (1-5).The UPR is initiated through three known signaling pathways, the PERK, ATF6, and IRE1 pathways (2, 6, 7). PERK activation transiently decreases protein synthesis, and ATF6 is proteolytically cleaved to form a transcription factor that induces chaperone transcription. IRE1 is a bifunctional kinase/site-specific endoribonuclease that catalyzes XBP1 mRNA splicing, generating a stable mRNA product (sXBP1) from which the transcription factor XBP1 is translated. Like ATF6, XBP1 induces chaperone transcription. An important feature of IRE1 is that, unlike other autophosphorylating kinases, IRE1 is not activated by self-phosphorylation but rather by the occupancy of its ATP binding site (8).Given the requirement for ATP in chaperone function and IRE1 activation, the initiation and maintenance of the UPR are likely to be influenced by cellular energy levels. Several o...

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-β receptor expression and signaling, and blocking TGF-β receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-β signaling with BET bromodomain inhibition may offer new therapeutic benefits.bromodomain inhibitor | TRIM33 | JQ1 | drug resistance | TGF-β

Recent work has led to the identification of novel endocytic compartments with functional roles in both protein trafficking and growth factor signal transduction. The phosphatidylinositol 3-phosphate binding, FYVE domain-containing protein WDFY2 is localized to a distinct subset of early endosomes, which are localized close to the plasma membrane. Here, we find that the serine/threonine kinase Akt interacts with these endosomes in an isoform-specific manner. Using quantitative fluorescence microscopy we demonstrate specific co-localization of WDFY2 with endogenous Akt2, but not Akt1. Moreover, depletion of WDFY2 leads to impaired phosphorylation of Akt in response to insulin due to isoform specific reduction of Akt2, but not Akt1, protein levels, and to a marked reduction in the insulin-stimulated phosphorylation of numerous Akt substrates. This is accompanied by an impairment in insulin-stimulated glucose transport and, after prolonged silencing, a reduction in the level of expression of adipogenic genes. We propose that WDFY2-enriched endosomes serve as a scaffold that enables specificity of insulin signaling through Akt2.The early endocytic pathway is increasingly being recognized as a complex and heterogeneous membrane population in which distinct endosomal populations are specialized for the trafficking of different receptor types (1, 2). Complexity and specialization in the endosomal pathway are achieved by the action of small GTPases and by the generation of specific phosphoinositides on the endosomal surface. One of the best studied examples of this mechanism is the specific and temporal targeting of proteins containing FYVE domains to phosphatidylinositol 3-phosphate (3-6), which is present almost exclusively in endosomal membranes. The human genome encodes for Ͼ30 proteins that contain FYVE domains, several of which are highly conserved and which may contribute in different ways toward establishing the complexity and functionality of the endocytic pathway. We recently characterized one of these proteins, WDFY2, named for its content of WD40 motifs and a FYVE domain (7). In Caenorhabditis elegans, WDFY2 depletion impairs endocytosis in coelomocytes, and in mammalian cells it defines a distinct set of endosomes that lack the canonical markers EEA1 and Rab5 and are further distinguished by their close proximity to the plasma membrane (7,8).In addition to internalization, the endosomal pathway plays a critical role in modulating signal transduction. Growth factor receptors are internalized immediately following activation, and both their fate and their signaling functions are affected by their transit through the endocytic pathway (9 -13). Different receptors traffic through distinct early endosomal compartments (1, 2), and their signaling functions are modulated by the specific nature of the endosomes through which they traffic. For example, signaling by transforming growth factor ␤ is influenced by the endosomal localization of the SMAD-interacting protein SARA, which is found in endosomes containing the...