Tyrosine 842 Residue in the Activation Loop of FLT3-ITD Is Indespensible for Oncogenic Transformation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1561-1561
Author(s):  
Julhash U. Kazi ◽  
Lars Rönnstrand
Keyword(s):  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Conflicts Of Interest ◽  
Point Mutations ◽  
Cellular Transformation ◽  
Receptor Activation ◽  
Tumor Formation ◽  
Activation Loop ◽  
Tyrosine Residues

Abstract The receptor tyrosine kinase FLT3 is mutated in around 30% of acute myeloid leukemia patients. The so-called internal tandem duplication (ITD) mutation in the juxtamembrane domain is the most common type of mutation in FLT3. Other oncogenic mutations include point mutations in the kinase domain. One of the hallmark of receptor activation is phosphorylation on several tyrosine residues in the receptor. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. The tyrosine residue Y842, which is located in the activation loop of FLT3, and is well conserved in all receptor tyrosine kinases. It has been demonstrated that tyrosine phosphorylation of the activation loop is essential for the activity of some but not all receptor tyrosine kinases. In this report we show that phosphorylation on Y842 residue is dispensable for receptor activity and stability. However, Y842 plays critical roles in oncogenic FLT3-ITD mediated transformation. We have shown that a Y-to-F mutation (Y842F) results in reduced cell viability and proliferation. This mutation also led to dramatic decrease in in vitro colony formation in semi-solid medium. Furthermore, cells carrying the Y842F mutant of FLT3-ITD displayed significant delay in tumor formation in nude mice. Gene expression analysis, using microarrays, showed that mutation of Y842 causes suppression of anti-apoptotic genes. Finally, we showed that phosphorylated Y842 is a binding site for SHP2 and mutation of this site results in impaired activation of signaling through the RAS/ERK pathway. Collectively, our data suggest that Y842 in FLT3 plays an important role in FLT3-mediated RAS/ERK signaling and cellular transformation. Disclosures No relevant conflicts of interest to declare.

scholarly journals G-CSF receptor activation of the Src kinase Lyn is mediated by Gab2 recruitment of the Shp2 phosphatase

Blood ◽  
2011 ◽  
Vol 118 (4) ◽  
pp. 1077-1086 ◽  
Author(s):  
Muneyoshi Futami ◽  
Quan-sheng Zhu ◽  
Zakary L. Whichard ◽  
Ling Xia ◽  
Yuehai Ke ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Src Kinase ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor ◽  
In Cells

Abstract Src activation involves the coordinated regulation of positive and negative tyrosine phosphorylation sites. The mechanism whereby receptor tyrosine kinases, cytokine receptors, and integrins activate Src is not known. Here, we demonstrate that granulocyte colony-stimulating factor (G-CSF) activates Lyn, the predominant Src kinase in myeloid cells, through Gab2-mediated recruitment of Shp2. After G-CSF stimulation, Lyn dynamically associates with Gab2 in a spatiotemporal manner. The dephosphorylation of phospho-Lyn Tyr507 was abrogated in Shp2-deficient cells transfected with the G-CSF receptor but intact in cells expressing phosphatase-defective Shp2. Auto-phosphorylation of Lyn Tyr396 was impaired in cells treated with Gab2 siRNA. The constitutively activated Shp2E76A directed the dephosphorylation of phospho-Lyn Tyr507 in vitro. Tyr507 did not undergo dephosphorylation in G-CSF–stimulated cells expressing a mutant Gab2 unable to bind Shp2. We propose that Gab2 forms a complex with Lyn and after G-CSF stimulation, Gab2 recruits Shp2, which dephosphorylates phospho-Lyn Tyr507, leading to Lyn activation.

p60src and p72Syk Tyrosine Kinases Activate Both Lw and CD44 to Mediate Sickle Red Blood Cell Adhesion to Endothelium

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3223-3223
Author(s):  
Edward Chiou ◽  
Rahima Zennadi
Keyword(s):  
Shear Stress ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Conflicts Of Interest ◽  
Selective Inhibitor ◽  
Intermittent Flow ◽  
Αvβ3 Integrin ◽  
Src Tyrosine Kinase

Abstract Abstract 3223 The vaso-occlusive process in patients with sickle cell disease is complex and involves interactions between hemoglobin S red blood cells (SSRBCs) and vascular endothelial cells (ECs). However, the pathophysiologic triggers inducing SSRBC adhesion and vaso-occlusion are poorly understood. Elucidation of these mechanisms at the molecular level would allow development of new preventive and treatment strategies to abrogate vaso-occlusive events. Because non-receptor tyrosine kinases in non-erythroid cells are known to mediate cell-cell interactions, we have now investigated the role of non-receptor tyrosine kinases in modulating SSRBC adherence to cultured ECs, identified the kinases involved, and defined both the receptors on activated SSRBCs and the ligands on ECs involved in these interactions. Less than 20% of non-treated SSRBCs were able to adhere to non-stimulated ECs in vitro in intermittent flow conditions at a shear stress of 2 dynes/cm2. However, treatment of SSRBCs with sodium orthovanadate (Na3VO4), a potent broad spectrum inhibitor of protein tyrosine phosphatases, was able to significantly up-regulate RBC adhesion to cultured ECs by 4.6±1-fold over baseline adhesion in vitro. Na3VO4 in contrast, completely failed to increase adhesion of normal RBCs to cultured ECs. The increased SSRBC adhesion induced by Na3VO4 was significantly inhibited with piceatannol (p<0.001), which inhibits non-receptor tyrosine kinases p72Syk and p56Lck, and PP1 (p<0.001), an src-selective tyrosine p56Lck, p59Fyn and p60src kinase inhibitor. However, genistein, a broad range inhibitor of tyrosine kinases, and damnacanthal, a highly potent and selective inhibitor of p56Lck, completely failed to inhibit the effect of Na3VO4 on SSRBC adhesion. In addition, phenylarsine oxide, which specifically activates p56Lck tyrosine kinase, did not affect SSRBC adhesion to ECs. Together, these data suggest that SSRBC adhesion to ECs can be upregulated via activation of at least p72Syk and p60src tyrosine kinases, but not via the src-tyrosine kinase p56Lck. We further confirmed that increased SSRBC adhesion by Na3VO4 treatment is indeed a result of the activation of p72Syk and p60src tyrosine kinases. Slight basal phosphorylation of p72Syk and p60src was detected in all SSRBC samples tested. However, Na3VO4-treatment of SSRBCs significantly enhanced phosphorylation of both p60src and p72Syk kinases over basal phosphorylation (p<0.05), and this effect induced by Na3VO4 was completely blocked with the src-selective inhibitor PP1 or PP2, and piceatannol, respectively, suggesting that p72Syk and p60src undergo enhanced activation and are involved in up-regulation of SSRBC adhesion to endothelium. Moreover, at a shear stress of 2 dynes/cm2, both anti-LW (ICAM-4) and anti-CD44 antibodies individually inhibited adhesion of activated SSRBCs to ECs, by up to 83% and 78%, respectively. Recombinant soluble LW (srLW) and CD44 (srCD44) proteins also completely abolished adhesion of activated SSRBCs to ECs, identifying LW and CD44 as the RBC receptors involved in this interaction. The EC ligands for activated SSRBCs were also identified using antibody inhibition studies, as the αvβ3 integrin, a ligand previously shown to be important to SSRBC adhesion to activated ECs in vivo, and the endothelial CD44. These data demonstrate that activation of p72Syk and p60src-dependent pathways can act to activate LW- and CD44-mediated SSRBC adhesion to endothelial αvβ3 integrin and CD44, respectively, suggesting that this mechanism may initiate or exacerbate vaso-occlusion by increasing SSRBC adhesion to the endothelium. RBC CD44 is also the first adhesion molecule shown to be involved in SSRBC adhesion to endothelium. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Loss of Src-like Adaptor Protein 2 Expression Increases the Transforming Potential of Oncogenic FLT3-ITD

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5106-5106
Author(s):  
Sausan A. Moharram ◽  
Julhash U. Kazi ◽  
Lars Rönnstrand
Keyword(s):  
Conflicts Of Interest ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Tumor Formation ◽  
Signaling Proteins ◽  
Short Term Treatment ◽  
Tyrosine Residues ◽  
Alternative Approach

Abstract The receptor tyrosine kinase FLT3 is found to be a mutated oncogene in hematological malignancies including acute myeloid leukemia (AML). FLT3 inhibitors in combination with chemotherapy display promising results in a clinical setting, but patients relapse after short-term treatment due to the development of resistant disease. Therefore, targeting signaling proteins downstream of FLT3 can be an alternative approach for the treatment of patients carrying mutant FLT3. Activated FLT3 is constitutively phosphorylated on several tyrosine residues. These tyrosine residues facilitate association of SH2 domain-containing signaling proteins. By using a panel of SH2 domain-containing proteins we identified SLAP2 as a potent interaction partner of FLT3. The interaction in between FLT3 and SLAP2 occurs when FLT3 is activated and an intact SH2 domain of SLAP2 is required for the interaction. SLAP2 associates with FLT3 mainly through its SRC binding sites and expression of SLAP2 inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation in vitro, and tumor formation in vivo. By analysis of patient expression data, we found that loss of SLAP2 expression correlates with poor prognosis of AML patients carrying FLT3-ITD. SLAP2 inhibits FLT3-mediated downstream signaling such as activation of AKT, ERK, p38 and STAT5. Inhibition is partially mediated through ubiquitination-mediated degradation of FLT3. Taken together our current study demonstrates that SLAP2 is an important regulator of FLT3-mediated oncogenic signaling and thus modulation of the SLAP2 expression levels can be an alternative approach for the treatment of FLT3-ITD positive malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Signaling Pathways Regulating TC21-induced Tumorigenesis

2007 ◽  
Vol 282 (38) ◽  
pp. 27713-27720 ◽  
Author(s):  
Mete Erdogan ◽  
Ambra Pozzi ◽  
Neil Bhowmick ◽  
Harold L Moses ◽  
Roy Zent
Keyword(s):  
P38 Mapk ◽  
Cancer Progression ◽  
Transforming Growth Factor ◽  
Cellular Transformation ◽  
Tumor Formation ◽  
Transformed Cells ◽  
Growth Inhibitory ◽  
Phosphoinositide 3 Kinase

TC21(R-Ras2), a Ras-related GTPase with transforming potential similar to H-, K- and N-Ras, is implicated in the pathogenesis of human cancers. Transforming growth factor β (TGF-β), a cytokine that plays a significant role in modulating tumorigenesis, normally prevents uncontrolled cell proliferation but paradoxically induces proliferation in H-Ras-transformed cancer cells. Although TC21 activates some pathways that mediate cellular transformation by the classical Ras proteins, the mechanisms through which TC21 induces tumor formation and how TGF-β regulates TC21 transformed cells is not known. To better understand the role of TC21 in cancer progression, we overexpressed an activated G23V mutant of TC21 in a nontumorigenic murine mammary epithelial (EpH4) cell line. Mutant TC21-expressing cells were significantly more oncogenic than cells expressing activated G12V H-Ras both in vivo and in vitro. TC21-induced transformation and proliferation required activation of p38 MAPK, mTOR (the mammalian target of rapamycin), and phosphoinositide 3-kinase but not Akt/PKB. Transformation by TC21 rendered EpH4 cells insensitive to the growth inhibitory effects of TGF-β, and the soft agar growth of these cells was increased upon TGF-β stimulation. Despite losing responsiveness to TGF-β-mediated growth inhibition, both Smad-dependent and independent pathways remained intact in TC21-transformed cells. Thus, overexpression of active TC21 in EpH4 cells induces tumorigenicity through the phosphoinositide 3-kinase, p38 MAPK, and mTOR pathways, and these cells lose their sensitivity to the normal growth inhibitory role of TGF-β.

scholarly journals In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases

Cell ◽  
1995 ◽  
Vol 82 (3) ◽  
pp. 359-370 ◽  
Author(s):  
Uwe Drescher ◽  
Claus Kremoser ◽  
Claudia Handwerker ◽  
Jürgen Löschinger ◽  
Masaharu Noda ◽  
...  
Keyword(s):  
Ganglion Cell ◽  
Retinal Ganglion Cell ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Retinal Ganglion ◽  
Eph Receptor

Eph/Ephrin signalling in skeletal muscle development and regeneration

2014 ◽  
Author(s):  
◽  
Danny A. Stark
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cells ◽  
Tyrosine Kinases ◽  
Muscle Development ◽  
Receptor Tyrosine Kinases ◽  
Repulsive Interaction ◽  
Type I ◽  
Ephrin Ligands

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Skeletal muscle can be isolated into 642 individual muscles and makes up to one third to one half of the mass of the human body. Each of these muscles is specified and patterned prenatally and after birth they will increase in size and take on characteristics suited to each muscle's unique function. To make the muscles functional, each muscle cell must be innervated by a motor neuron, which will also affect the characteristics of the mature muscle. In a healthy adult, muscles will maintain their specialized pattern and function during physiological homeostasis, and will also recapitulate them if the integrity or health of the muscle is disrupted. This repair and regeneration is dependent satellite cells, the skeletal muscle stem cells. In this dissertation, we study a family of receptor tyrosine kinases, Ephs, and their juxtacrine ephrin ligands in the context of skeletal muscle specification and regeneration. First, using a classical ephrin 'stripe' assay to test for contact-mediated repulsion, we found that satellite cells respond to a subset of ephrins with repulsive motility in vitro and that these forward signals through Ephs also promote patterning of differentiating myotubes parallel to ephrin stripes. This pattering can be replicated in a heterologous in vivo system (the hindbrain of the developing quail, where neural crest cells migrate in streams to the branchial arches, and in the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite). Second, we present evidence that specific pairwise interactions between Eph receptor tyrosine kinases and ephrin ligands are required to ensure appropriate muscle innervation when it is originally set during postnatal development and when it is recapitulated after muscle or nerve trauma during adulthood. We show expression of a single ephrin, ephrin-A3, exclusively on type I (slow) myofibers shortly after birth, while its receptor EphA8 is only localized to fast motor endplates, suggesting a functional repulsive interaction for motor axon guidance and/or synaptogenesis. Adult EFNA3-/- mutant mice show a significant loss of slow myofibers, while misexpression of ephrin-A3 on fast myofibers results in a switch from a fast fiber type to slow in the context of sciatic nerve injury and regrowth. Third, we show that EphA7 is expressed on satellite cell derived myocytes in vitro, and marks both myocytes and regenerating myofibers in vivo. In the EPHA7 knockout mouse, we find a regeneration defect in a barium chloride injury model starting 3 days post injection in vivo, and that cultured mutant satellite cells are slow to differentiate and divide. Finally, we present other potential Ephs and ephrins that may affect skeletal muscle, such as EphB1 that is expressed on all MyHC-IIb fibers and a subset of MyHC-IIx fibers, and we show a multitude of Ephs and ephrins at the neuromuscular junction that appear to localize on specific myofibers and at different areas of the synapse. We propose that Eph/ephrin signaling, though well studied in development, continues to be important in regulating post natal development, regeneration, and homeostasis of skeletal muscle.

scholarly journals Helicobacter Pylori Targets the EPHA2 Receptor Tyrosine Kinase in Gastric Cells Modulating Key Cellular Functions

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 513 ◽  
Author(s):  
Marina Leite ◽  
Miguel S. Marques ◽  
Joana Melo ◽  
Marta T. Pinto ◽  
Bruno Cavadas ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Receptor Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Receptor Activation ◽  
Degradation Pathway ◽  
Mrna Levels ◽  
H Pylori

Helicobacter pylori, a stomach-colonizing Gram-negative bacterium, is the main etiological factor of various gastroduodenal diseases, including gastric adenocarcinoma. By establishing a life-long infection of the gastric mucosa, H. pylori continuously activates host-signaling pathways, in particular those associated with receptor tyrosine kinases. Using two different gastric epithelial cell lines, we show that H. pylori targets the receptor tyrosine kinase EPHA2. For long periods of time post-infection, H. pylori induces EPHA2 protein downregulation without affecting its mRNA levels, an effect preceded by receptor activation via phosphorylation. EPHA2 receptor downregulation occurs via the lysosomal degradation pathway and is independent of the H. pylori virulence factors CagA, VacA, and T4SS. Using small interfering RNA, we show that EPHA2 knockdown affects cell–cell and cell–matrix adhesion, invasion, and angiogenesis, which are critical cellular processes in early gastric lesions and carcinogenesis mediated by the bacteria. This work contributes to the unraveling of the underlying mechanisms of H. pylori–host interactions and associated diseases. Additionally, it raises awareness for potential interference between H. pylori infection and the efficacy of gastric cancer therapies targeting receptors tyrosine kinases, given that infection affects the steady-state levels and dynamics of some receptor tyrosine kinases (RTKs) and their signaling pathways.

scholarly journals TrkB deubiquitylation by USP8 regulates receptor levels and BDNF-dependent neuronal differentiation

2020 ◽  
Vol 133 (24) ◽  
pp. jcs247841 ◽  
Author(s):  
Carlos Martín-Rodríguez ◽  
Minseok Song ◽  
Begoña Anta ◽  
Francisco J. González-Calvo ◽  
Rubén Deogracias ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Neurotrophic Factor ◽  
Tyrosine Kinases ◽  
Hippocampal Neurons ◽  
Receptor Tyrosine Kinases ◽  
Neurotrophin Receptor ◽  
C Terminus ◽  
Carboxyl Terminal

ABSTRACTUbiquitylation of receptor tyrosine kinases (RTKs) regulates both the levels and functions of these receptors. The neurotrophin receptor TrkB (also known as NTRK2), a RTK, is ubiquitylated upon activation by brain-derived neurotrophic factor (BDNF) binding. Although TrkB ubiquitylation has been demonstrated, there is a lack of knowledge regarding the precise repertoire of proteins that regulates TrkB ubiquitylation. Here, we provide mechanistic evidence indicating that ubiquitin carboxyl-terminal hydrolase 8 (USP8) modulates BDNF- and TrkB-dependent neuronal differentiation. USP8 binds to the C-terminus of TrkB using its microtubule-interacting domain (MIT). Immunopurified USP8 deubiquitylates TrkB in vitro, whereas knockdown of USP8 results in enhanced ubiquitylation of TrkB upon BDNF treatment in neurons. As a consequence of USP8 depletion, TrkB levels and its activation are reduced. Moreover, USP8 protein regulates the differentiation and correct BDNF-dependent dendritic formation of hippocampal neurons in vitro and in vivo. We conclude that USP8 positively regulates the levels and activation of TrkB, modulating BDNF-dependent neuronal differentiation.This article has an associated First Person interview with the first author of the paper.

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

2018 ◽  
Vol 11 (553) ◽  
pp. eaat5916 ◽  
Author(s):  
Kexin Shen ◽  
Jamie A. Moroco ◽  
Ravi K. Patel ◽  
Haibin Shi ◽  
John R. Engen ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Colony Formation ◽  
Family Members ◽  
Cellular Transformation ◽  
Soft Agar ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Mass Spectrometry Data ◽  
Activation Loop ◽  
Exchange Mass Spectrometry

Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2–mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.

scholarly journals Receptor Tyrosine Kinases and Their Signaling Pathways as Therapeutic Targets of Curcumin in Cancer

2021 ◽  
Vol 12 ◽  
Author(s):  
Sareshma Sudhesh Dev ◽  
Syafiq Asnawi Zainal Abidin ◽  
Reyhaneh Farghadani ◽  
Iekhsan Othman ◽  
Rakesh Naidu
Keyword(s):  
Signaling Pathways ◽  
Cancer Progression ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Surface Proteins ◽  
Downstream Signaling ◽  
Anti Cancer ◽  
Rtk Inhibitors

Receptor tyrosine kinases (RTKs) are transmembrane cell-surface proteins that act as signal transducers. They regulate essential cellular processes like proliferation, apoptosis, differentiation and metabolism. RTK alteration occurs in a broad spectrum of cancers, emphasising its crucial role in cancer progression and as a suitable therapeutic target. The use of small molecule RTK inhibitors however, has been crippled by the emergence of resistance, highlighting the need for a pleiotropic anti-cancer agent that can replace or be used in combination with existing pharmacological agents to enhance treatment efficacy. Curcumin is an attractive therapeutic agent mainly due to its potent anti-cancer effects, extensive range of targets and minimal toxicity. Out of the numerous documented targets of curcumin, RTKs appear to be one of the main nodes of curcumin-mediated inhibition. Many studies have found that curcumin influences RTK activation and their downstream signaling pathways resulting in increased apoptosis, decreased proliferation and decreased migration in cancer both in vitro and in vivo. This review focused on how curcumin exhibits anti-cancer effects through inhibition of RTKs and downstream signaling pathways like the MAPK, PI3K/Akt, JAK/STAT, and NF-κB pathways. Combination studies of curcumin and RTK inhibitors were also analysed with emphasis on their common molecular targets.

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