Regulation of Receptor Tyrosine Kinase Ligand Processing

A primary mode of regulating receptor tyrosine kinase (RTK) signaling is to control access of ligand to its receptor. Many RTK ligands are synthesized as transmembrane proteins. Frequently, the active ligand must be released from the membrane by proteolysis before signaling can occur. Here, we discuss RTK ligand shedding and describe the proteases that catalyze it in flies and mammals. We focus principally on the control of EGF receptor ligand shedding, but also refer to ligands of other RTKs. Two prominent themes emerge. First, control by regulated trafficking and cellular compartmentalization of the proteases and their ligand substrates plays a key role in shedding. Second, many external signals converge on the shedding proteases and their control machinery. Proteases therefore act as regulatory hubs that integrate information that the cell receives and translate it into precise outgoing signals. The activation of signaling by proteases is therefore an essential element of the cellular communication machinery.Cells must talk to one another. This principle applies throughout the tree of life: from unicellular bacteria, to the trillions of cells that coordinate to make a mammal. Communication between cells requires dedicated machinery, capable of relaying information across membranes. Transmembrane proteins are therefore essential for signaling. Understanding how this is regulated is paramount. In mammals, receptor tyrosine kinases (RTKs) and their ligands are important examples of such machinery (Schlessinger 2000), controlling many biological processes including development, immunity, tissue repair, and metabolic homeostasis (Ullrich and Schlessinger 1990). They are transmembrane proteins with an extracellular ligand-binding motif and an intracellular kinase domain. As discussed in other chapters, a common mode of RTK activation involves receptor dimerization induced by ligand binding (Lemmon and Schlessinger 2010).Regulated access of ligand to receptor, over distance and time, is key to controlling signaling. Ligands are frequently synthesized as transmembrane forms; when they remain membrane-tethered and cannot diffuse, the range over which they can operate is limited to adjacent cells (Massague and Pandiella 1993; Singh and Harris 2005). Other ligands are soluble secretory proteins. This enables paracrine and endocrine signaling—communication between nonadjacent cells. A more complex mode of signaling exploits the characteristics of both of the above. Ligand is synthesized as a transmembrane precursor, which is then shed from the cell surface by proteolysis. This adds an additional and stringent regulatory step to a signaling network (Massague and Pandiella 1993).This chapter will focus on RTK ligand cleavage and its regulation. We shall highlight how shedding is often critical for signaling, and describe the protease families that catalyze ligand release in flies and mammals. An emergent theme is that regulated trafficking and compartmentalization of ligand and protease modulate signaling. Another theme will be the range of stimuli that impinge on shedding.The epidermal growth factor receptor (EGFR) is an excellent model RTK to illustrate the regulation of ligand proteolysis because the requirement for ligand cleavage in signaling is well established, and the major physiological sheddases have been identified (Blobel 2005). Where warranted, physiological evidence for the role of ligand shedding in the regulation of other RTKs will also be discussed. Whereas we shall deal mostly with ADAM proteases (“a disintegrin and metalloprotease”), which represent the canonical mammalian RTK ligand shedding machinery, the rhomboid family of intramembrane proteases will also be discussed.     

Gas6 and the Receptor Tyrosine Kinase Axl in Clear Cell Renal Cell Carcinoma

Background

The molecular biology of renal cell carcinoma (RCC) is complex and not fully understood. We have recently found that the expression of the receptor tyrosine kinase Axl in the RCC tumors independently correlates with survival of the patients.

Principal Findings

Here, we have investigated the role of Axl and its ligand Gas6, the vitamin-K dependent protein product of the growth arrest-specific gene 6, in clear cell RCC (ccRCC) derived cells. The Axl protein was highly expressed in ccRCC cells deficient in functional von Hippel-Lindau (VHL) protein, a tumor suppressor gene often inactivated in ccRCC. VHL reconstituted cells expressed decreased levels of Axl protein, but not Axl mRNA, suggesting VHL to regulate Axl expression. Gas6-mediated activation of Axl in ccRCC cells resulted in Axl phosphorylation, receptor down-regulation, decreased cell-viability and migratory capacity. No effects of the Gas6/Axl system could be detected on invasion. Moreover, in ccRCC tumor tissues, Axl was phosphorylated and Gas6 γ-carboxylated, suggesting these molecules to be active in vivo.

Significance

These results provide novel information regarding the complex function of the Gas6/Axl system in ccRCC.     

K-562细胞胰岛素受体酪氨酸蛋白激酶活性及内源性底物的研究

本文对丁酸钠诱导分化的人白血病细胞株K-562细胞的胰岛素受体酪氨酸蛋白激酶性及细胞内源性底物进行了研究。结果表明,诱导分化后的细胞酪氨酸蛋白激活酶活性降低,胰岛素受体数量减少,酪氨酸蛋白激酶的一底物蛋白在分化后消失。该研究结果及其在该方面的进一步研究有可能为白血病发病机制的阐明以及为临床治疗白血病开辟靳途径提供一些有价值的线索。     

受体酪氨酸激酶Axl对胶质母细胞瘤细胞增殖、凋亡和侵袭的影响

摘要 目的：研究受体酪氨酸激酶Axl在胶质母细胞瘤组织和细胞系U-118MG细胞中的表达情况及其对U-118MG细胞增殖、凋亡、侵袭的影响。方法：收集2015年3月至2018年5月在本院进行手术切除并经病理分型证实的胶质母细胞瘤组织标本（n=30）,另取脑外伤手术中因作内减压而切除的正常脑组织作为对照（n=28）。采用荧光实时定量 (qRT-PCR)检测正常脑组织和胶质母细胞瘤肿瘤组织中Axl mRNA表达水平;采用Western blot检测人小神经胶质HM细胞、U-118MG细胞以及Axl-shRNA转染后U-118MG细胞中Axl蛋白表达水平;采用CCK-8检测Axl-shRNA转染后U-118MG细胞增殖能力;采用流式细胞术检测Axl-shRNA转染后U-118MG细胞凋亡水平;采用Transwell小室实验检测Axl-shRNA转染后U-118MG细胞的侵袭能力。结果：在胶质母细胞瘤组织中Axl mRNA表达水平显著高于正常脑组织（P<0.05）;U-118MG细胞Axl蛋白表达水平显著高于人小神经胶质细胞系HM细胞,差异有统计学意义（P<0.05）;转染Axl-shRNA后,U-118MG细胞中Axl蛋白表达水平显著降低（P<0.05）。与U-118MG细胞和转染control-shRNA细胞相比, 转染Axl-shRNA的U-118MG细胞增殖能力降低（P<0.05）,凋亡水平升高（P<0.05）,侵袭能力降低（P<0.05）。结论：在胶质母细胞瘤组织和U-118MG细胞中,Axl表达水平显著增高,并且Axl表达水平与U-118MG细胞增殖、凋亡及侵袭密切关联。     

Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

The receptor tyrosine kinase (RTK) ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375) including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.     

Prostaglandin E2 Induces Oncostatin M Expression in Human Chronic Wound Macrophages through Axl Receptor Tyrosine Kinase Pathway

Monocytes and macrophages (m) are plastic cells whose functions are governed by microenvironmental cues. Wound fluid bathing the wound tissue reflects the wound microenvironment. Current literature on wound inflammation is primarily based on the study of blood monocyte-derived macrophages, cells that have never been exposed to the wound microenvironment. We sought to compare pair-matched monocyte-derived macrophages with m isolated from chronic wounds of patients. Oncostatin M (OSM) was differentially overexpressed in pair-matched wound m. Both PGE(2) and its metabolite 13,14-dihydro-15-keto-PGE(2) (PGE-M) were abundant in wound fluid and induced OSM in wound-site m. Consistently, induction of OSM mRNA was observed in m isolated from PGE(2)-enriched polyvinyl alcohol sponges implanted in murine wounds. Treatment of human THP-1 cell-derived m with PGE(2) or PGE-M caused dose-dependent induction of OSM. Characterization of the signal transduction pathways demonstrated the involvement of EP4 receptor and cAMP signaling. In human m, PGE(2) phosphorylated Axl, a receptor tyrosine kinase (RTK). Axl phosphorylation was also induced by a cAMP analogue demonstrating interplay between the cAMP and RTK pathways. PGE(2)-dependent Axl phosphorylation led to AP-1 transactivation, which is directly implicated in inducible expression of OSM. Treatment of human m or mice excisional wounds with recombinant OSM resulted in an anti-inflammatory response as manifested by attenuated expression of endotoxin-induced TNF-α and IL-1β. OSM treatment also improved wound closure during the early inflammatory phase of healing. In summary, this work recognizes PGE(2) in the wound fluid as a potent inducer of m OSM, a cytokine with an anti-inflammatory role in cutaneous wound healing.     

Fms-Like Tyrosine Kinase 3 Ligand Controls Formation of Regulatory T Cells in Autoimmune Arthritis

Fms-like tyrosine kinase 3 ligand (Flt3L) is known as the primary differentiation and survival factor for dendritic cells (DCs). Furthermore, Flt3L is involved in the homeostatic feedback loop between DCs and regulatory T cell (Treg). We have previously shown that Flt3L accumulates in the synovial fluid in rheumatoid arthritis (RA) and that local exposure to Flt3L aggravates arthritis in mice, suggesting a possible involvement in RA pathogenesis. In the present study we investigated the role of Flt3L on DC populations, Tregs as well as inflammatory responses in experimental antigen-induced arthritis. Arthritis was induced in mBSA-immunized mice by local knee injection of mBSA and Flt3L was provided by daily intraperitoneal injections. Flow cytometry analysis of spleen and lymph nodes revealed an increased formation of DCs and subsequently Tregs in mice treated with Flt3L. Flt3L-treatment was also associated with a reduced production of mBSA specific antibodies and reduced levels of the pro-inflammatory cytokines IL-6 and TNF-α. Morphological evaluation of mBSA injected joints revealed reduced joint destruction in Flt3L treated mice. The role of DCs in mBSA arthritis was further challenged in an adoptive transfer experiment. Transfer of DCs in combination with T-cells from mBSA immunized mice, predisposed naïve recipients for arthritis and production of mBSA specific antibodies. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L facilitates formation of Treg cells and by this mechanism reduces severity of antigen-induced arthritis in mice. We suggest that high systemic levels of Flt3L have potential to modulate autoreactivity and autoimmunity.     

Slik and the Receptor Tyrosine Kinase Breathless Mediate Localized Activation of Moesin in Terminal Tracheal Cells

A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. Here we show that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. Our results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway.     

ErbB2受体酪氨酸激酶激酶区的表达与纯化

以GFP融合表达的形式在毕赤酵母中表达具有生物活性的受体酪氨酸激酶ErbB2的激酶区.构建受体酪氨酸激酶激酶区与GFP的融合表达载体pPIC3.5K,转化毕赤酵母GS115,通过组氨酸营养缺陷型筛选,G418高拷贝菌株筛选,以及摇瓶诱导表达筛选,选取较高水平表达菌株进行5升罐培养,以镍亲和层析手段纯化得到蛋白表达产物,进行SDS-PAGE分析和酶联免疫反应检测酶活.结果表明在毕赤酵母中成功诱导表达了约100kD的激酶融合蛋白并具有激酶活性.该研究为筛选ErbB2的抑制剂奠定了基础.     

RTKdb: database of Receptor Tyrosine Kinase

Receptor Tyrosine Kinases (RTK) are transmembrane receptors specifically found in metazoans. They represent an excellent model for studying evolution of cellular processes in metazoans because they encompass large families of modular proteins and belong to a major family of contingency generating molecules in eukaryotic cells: the protein kinases. Because tyrosine kinases have been under close scrutiny for many years in various species, they are associated with a wealth of information, mainly in mammals. Presently, most categories of RTK were identified in mammals, but in a near future other model species will be sequenced, and will bring us RTKs from other metazoan clades. Thus, collecting RTK sequences would provide a good starting point as a new model for comparative and evolutionary studies applying to multigene families. In this context, we are developing the Receptor Tyrosine Kinase database (RTKdb), which is the only database on tyrosine kinase receptors presently available. In this database, protein sequences from eight model metazoan species are organized under the format previously used for the HOVERGEN, HOBACGEN and NUREBASE systems. RTKdb can be accessed through the PBIL (P?le Bioinformatique Lyonnais) World Wide Web server at http://pbil.univ-lyon1.fr/RTKdb/, or through the FamFetch graphical user interface available at the same address.     

Ret：一种受体酪氨酸激酶及其基因突变与疾病

RET是一个在转化中发生重排的原癌基因,且因此行为而得名.它编码细胞膜受体酪氨酸激酶,初步研究表明它介导的信号转导途径较为独特.RET基因突变与人类4种癌症的发生相关：甲状腺乳头状腺癌存在RET基因与其他基因多种重排;多发性内分泌腺瘤2型,家族遗传甲状腺髓样癌等存在7个位点点突变;先天巨结肠疾病与RET基因缺失相关.因此近年来备受关注.对Ret蛋白的结构功能,RET基因突变对Ret蛋白功能的影响及与人类相关疾病的关系作一综述.     

Liver Kinase B1 Expression Promotes Phosphatase Activity and Abrogation of Receptor Tyrosine Kinase Phosphorylation in Human Cancer Cells

Aberrant receptor tyrosine kinase phosphorylation (pRTK) has been associated with diverse pathological conditions, including human neoplasms. In lung cancer, frequent liver kinase B1 (LKB1) mutations correlate with tumor progression, but potential links with pRTK remain unknown. Heightened and sustained receptor activation was demonstrated by LKB1-deficient A549 (lung) and HeLaS3 (cervical) cancer cell lines. Depletion (siRNA) of endogenous LKB1 expression in H1792 lung cancer cells also correlated with increased pRTK. However, ectopic LKB1 expression in A549 and HeLaS3 cell lines, as well as H1975 activating-EGF receptor mutant lung cancer cell resulted in dephosphorylation of several tumor-enhancing RTKs, including EGF receptor, ErbB2, hepatocyte growth factor receptor (c-Met), EphA2, rearranged during transfection (RET), and insulin-like growth factor I receptor. Receptor abrogation correlated with attenuation of phospho-Akt and increased apoptosis. Global phosphatase inhibition by orthovanadate or depletion of protein tyrosine phosphatases (PTPs) resulted in the recovery of receptor phosphorylation. Specifically, the activity of SHP-2, PTP-1β, and PTP-PEST was enhanced by LKB1-expressing cells. Our findings provide novel insight on how LKB1 loss of expression or function promotes aberrant RTK signaling and rapid growth of cancer cells.     

受体酪氨酸激酶对自噬的调控及其研究进展*

自噬是真核生物进化上保守的溶酶体降解的生物学过程,在维护细胞内的稳态、消除有害组分等方面起到了重要作用。受体酪氨酸激酶家族(receptor tyrosine kinase,RTKs)是一类激酶蛋白,在正常细胞和癌症细胞的运动和侵袭中起着重要作用。RTKs蛋白既能促进自噬,也能抑制自噬。研究显示,RTKs能够在肿瘤和相关疾病中发挥自噬作用,比如表皮生长因子受体(epidermal growth factor receptor,EGFR)可以抑制自噬,从而促进肿瘤生长、增殖;还能通过RTK/Ras/ERK信号通路诱导自噬,进而参与诸如细胞免疫反应之类的相关疾病。主要综述了RTKs对自噬的调控作用和相关研究成果,为靶点靶向疗法的理论依据提供了基础。     

The Receptor Tyrosine Kinase Met and Its Ligand Hepatocyte Growth Factor are Clustered at Excitatory Synapses and Can Enhance Clustering of Synaptic Proteins

The receptor protein tyrosine kinase Met and its ligand, hepatocyte growth factor, regulate cellular morphology, intercellular adhesion, and interactions among junctional proteins in numerous cell types. However, they have not been extensively studied in the central nervous system. We report that Met is clustered at excitatory synapses and that treatment of neurons with hepatocyte growth factor can enhance expression and clustering of synaptic proteins. We demonstrate that Met is present in clusters that strongly colocalize with the NR2B subunit of the N-methyl-D-aspartate receptor, PSD-95, and synapsin at excitatory synapses on hippocampal neurons in vitro. We also show that Met is clustered at the postsynaptic density of excitatory synapses in the CA1 region of the hippocampus with the use of immuno-electron microscopy. Hepatocyte growth factor also forms clusters that partially colocalize with PSD-95. Treatment of cultured neurons with exogenous hepatocyte growth factor increased expression of the NR2B subunit of the N-methyl-D-aspartate receptor, calcium/calmodulin-dependent protein kinase II, and the GluR1 subunit of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor. The size and number of clusters of these proteins were also increased at sites along dendrites in response to hepatocyte growth factor. These results suggest a novel role for Met and hepatocyte growth factor in regulating synapses.     

Overexpression of MERTK Receptor Tyrosine Kinase in Epithelial Cancer Cells Drives Efferocytosis in a Gain-of-Function Capacity

MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells.     

Receptor Tyrosine Kinase Signaling and Primordial Germ Cell Development

Primordial germ cells (PGCs) give rise to sperms and eggs. Their development is crucial to species propagation and has to be precisely controlled. Studies in several model organisms have identified many genes involved in the specification and guided migration of PGCs. However, the mechanisms governing the behaviors of this unique type of cells remain to be investigated. Interestingly, PGCs share certain cellular properties with metastasizing cancer cells including proliferation, invasion of other tissues, survival, and migration. Recently we have shown that in Drosophila the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development. In later stages, activation of both STAT and Ras, likely by other molecules, is required continuously for PGC migration. The requirement for RTK suggests molecular conservation between flies and mice in PGC development and also suggests that germ cells and cancer cells share certain intracellular signaling strategies.     

The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase

Unlike prototypical receptor tyrosine kinases (RTKs), which are single-chain polypeptides, the insulin receptor (InsR) is a preformed, covalently linked tetramer with two extracellular α subunits and two membrane-spanning, tyrosine kinase-containing β subunits. A single molecule of insulin binds asymmetrically to the ectodomain, triggering a conformational change that is transmitted to the cytoplasmic kinase domains, which facilitates their trans-phosphorylation. As in prototypical RTKs, tyrosine phosphorylation in the juxtamembrane region of InsR creates recruitment sites for downstream signaling proteins (IRS [InsR substrate] proteins, Shc) containing a phosphotyrosine-binding (PTB) domain, and tyrosine phosphorylation in the kinase activation loop stimulates InsR’s catalytic activity. For InsR, phosphorylation of the activation loop, which contains three tyrosine residues, also creates docking sites for adaptor proteins (Grb10/14, SH2B2) that possess specialized Src homology-2 (SH2) domains, which are dimeric and engage two phosphotyrosines in the activation loop.Insulin is a highly potent anabolic hormone that is critical for tissue development and for glucose homeostasis (Taniguchi et al. 2006). Released from the β cells of the pancreas, insulin regulates glucose output from the liver and glucose uptake into (primarily) skeletal muscle and adipose tissue. In addition, insulin promotes the synthesis and storage of carbohydrates, lipids, and protein. Insulin’s actions are mediated by the insulin receptor (InsR), a plasma membrane-resident glycoprotein and member of the receptor tyrosine kinase (RTK) family. Other members of the InsR subfamily of RTKs include the insulinlike growth factor-1 receptor (IGF1R) and insulin receptor-related receptor, the latter of which has no known ligand. As an RTK, InsR is ligand-activated through mechanisms that are both prototypical and atypical of RTKs. These mechanisms will be the focus of this article.     

Stat3 Activates the Receptor Tyrosine Kinase Like Orphan Receptor-1 Gene in Chronic Lymphocytic Leukemia Cells

Background

The receptor tyrosine kinase like orphan receptor (ROR)-1 gene is overexpressed in chronic lymphocytic leukemia (CLL). Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors γ-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1.

Methodology/Principal Findings

Because IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells.

Conclusion/Significance

Our data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells.     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.