3′UTRs Regulate Mouse Ntrk2 mRNA Distribution in Cortical Neurons

Abstract There are two major isoforms of NTRK2 (neurotrophic receptor tyrosine kinase 2, or TrkB), full-length isoform with tyrosine kinase (TK) domain intact (+) and spliced isoform without tyrosine kinase domain (TK(−)). Within each isoform, there exist subtypes with minor modifications of the protein sequences. In human, the NTRK2 mRNA transcripts encoding TK(+) have same 3′UTRs, while the transcripts encoding subtypes of NTRK2 TK(−) have two completely different 3′UTRs. In mouse, the mRNA transcripts encoding same NTRK2 protein sequence for either TK(+) or TK(−) have long or short 3′UTRs, respectively. The physiological functions of these different 3′UTRs are still unknown. Pilocarpine stimulation increased Ntrk2 mRNA levels in soma, while the increase in synaptosome was smaller. FISH results further showed that mouse Ntrk2 transcripts with different 3′UTRs were distributed differently in cultured cortical neurons. The transcripts with long 3′UTR were distributed more in apical dendrites compared with transcripts with short 3′UTR. Our results provide evidence of non-coding 3′UTR function in regulating mRNA distribution in neurons.

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CSIG-31. ALTERNATIVE RECEPTOR TYROSINE KINASE SIGNALING AS A RESISTANCE MECHANISM TO ERK INHIBITION IN HIGH-GRADE GLIOMAS

Neuro-Oncology ◽

10.1093/neuonc/noz175.201 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi50-vi51

Author(s):

Ann-Catherine Stanton ◽

Robert Koncar ◽

Brian Golbourn ◽

Michelle Wassell ◽

Nishant Agrawal ◽

...

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Protein Level ◽

Resistance Mechanisms ◽

Kinase Domain ◽

High Grade ◽

Transactivation Domain ◽

Tyrosine Kinase 2 ◽

High Grade Gliomas

Abstract Pediatric High-Grade Gliomas (PHGG), which include Diffuse Midline Gliomas (DMG), are a leading cause of brain tumor death in children. Our recent work has identified extracellular signal-regulated kinase 5 (ERK5) as a critical mediator of cell survival in PHGG. Suppression of ERK5 genetically or pharmacologically leads to decreased cell proliferation and increased apoptosis both in vitro and in vivo in multiple PHGG and H3K27M mutant DMG cell lines. Mechanistically, we show that ERK5 directly stabilizes the proto-oncogene MYC at the protein level, providing rationale to clinically target ERK5. ERK5 contains both a kinase domain (KD) and a transactivation domain (TAD), unlike all other ERKs. Unexpectedly, we found that our ERK5 depleted cells could be partially rescued by an ERK5 kinase domain dead (ERK5-KDD) but TAD intact construct. Additionally, persistent ERK5 depletion does not result in complete growth inhibition and therefore we set out to determine potential adaptation or resistance mechanisms in response to ERK5 loss. To address this, we performed RNA sequencing of DMG cells, comparing control cells to ERK5 knockdown cells, and performed gene-ontology (GO) pathway analysis to identify transcriptional changes that occur in response to ERK5 depletion. We identified 105 differentially expressed genes, and GO analysis identified alternative receptor tyrosine kinase (RTK) gene-expression as one of the top biological processes upregulated in response to ERK5 loss. We validated our top targets at the RNA and the protein level. Our top targets were Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4) and Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), both clinically actionable targets. Our future work will focus on functional validation of these RTKs as potential resistance mechanisms to ERK5 loss. Identification of resistance mechanisms to ERK5 loss will have both biological and translational relevance and may lead to effective therapeutic combinations.

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STRUCTURAL CHARACTERISATION OF SUBSTRATE BINDING TO THE HUMAN INSULIN RECEPTOR TYROSINE KINASE DOMAIN

Biochemical Society Transactions ◽

10.1042/bst020269s ◽

1992 ◽

Vol 20 (3) ◽

pp. 269S-269S ◽

Cited By ~ 3

Author(s):

Yuan Gao ◽

Noeleen Keane ◽

Barry A. Levine ◽

Riki Alejos ◽

Leland Ellis

Keyword(s):

Tyrosine Kinase ◽

Insulin Receptor ◽

Human Insulin ◽

Receptor Tyrosine Kinase ◽

Substrate Binding ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Insulin Receptor Tyrosine Kinase ◽

Structural Characterisation ◽

Human Insulin Receptor

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Structure of the FGF Receptor Tyrosine Kinase Domain Reveals a Novel Autoinhibitory Mechanism

Cell ◽

10.1016/s0092-8674(00)80131-2 ◽

1996 ◽

Vol 86 (4) ◽

pp. 577-587 ◽

Cited By ~ 239

Author(s):

Moosa Mohammadi ◽

Joseph Schlessinger ◽

Stevan R Hubbard

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Fgf Receptor

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Mutations in the Receptor Tyrosine Kinase Pathway Are Associated with Clinical Outcome in Patients with Acute Myeloblastic Leukemia Harboring t(8;21)(q22;q22).

Blood ◽

10.1182/blood.v104.11.3000.3000 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3000-3000

Author(s):

Tomoko Nanri ◽

Naofumi Matsuno ◽

Toshiro Kawakita ◽

Hitoshi Suzushima ◽

Fumio Kawano ◽

...

Keyword(s):

Clinical Outcome ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Disease Free Survival ◽

Kinase Domain ◽

Acute Myeloblastic Leukemia ◽

Tyrosine Kinase Domain ◽

Juxtamembrane Domain ◽

Ras Gene ◽

Hematopoietic Stem

Abstract AML1-MTG8 generated by t(8;21)(q22;q22) contributes to leukemic transformation but additional events are required for full leukemogenesis. We examined whether mutations in the receptor tyrosine kinase (RTK) pathway could be the genetic events that cause acute myeloblastic leukemia (AML) harboring t(8;21). Mutations in the second tyrosine kinase domain, juxtamembrane domain and exon 8 of the C-KIT gene were observed in 7, 1 and 3 of 37 AML patients with t(8;21), respectively. Three patients showed an internal tandem duplication in the juxtamembrane domain of the FLT3 gene. One patient had a mutation in the K-Ras gene at codon 12. As the occurrence of these mutations was mutually exclusive, a total of 15 (41%) patients showed mutations in the RTK pathway. These results suggest that AML1-MTG8 predisposes cells to the acquisition of activating mutations in the RTK pathway as an additional event leading to the development of AML. Ten of 15 patients with mutations in the RTK pathway relapsed, compared with only 3 of 19 patients lacking such mutations (p=0.0042). Furthermore, the 6-year disease-free survival (DFS) in patients with mutations was 12% compared to 55% in those without mutations (p=0.0344). When patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT) were censored at the date of the HSCT, patients with mutations had a 6-year DFS of 0% versus 60% for the patients without mutations (p=0.0096) These observations indicate that RTK mutations are associated with the clinical outcome in t(8;21) AML.

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No Evidence for Constitutively Activated FLT3 in Juvenile Myelo-Monocytic Leukemia.

Blood ◽

10.1182/blood.v106.11.4915.4915 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4915-4915

Author(s):

Andrica C.H. de Vries ◽

Ronald W. Stam ◽

Pauline Schneider ◽

Charlotte M. Niemeyer ◽

Elisabeth R. van Wering ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Kinase Domain ◽

Leukemic Cells ◽

Tyrosine Kinase Domain ◽

Mrna Levels ◽

Monocytic Leukemia ◽

Real Time Quantitative Pcr ◽

Flt3 Inhibitor ◽

Flt3 Mutations

Abstract Activating FLT3 mutations have been identified as prognostic factors in multiple myeloid malignancies. Recent studies have demonstrated that ligand-independent activation of FLT3 can also result from overexpression of wild-type FLT3. In addition, ligand-dependent activation has been observed in leukemic cells co-expressing FLT3 ligand (FLT3L), resulting in autocrine FLT3 signaling which is independent of FLT3 mutations. In Juvenile Myelo-Monocytic Leukemia (JMML), FLT3 internal tandem duplications (FLT3/ITDs) mutations affecting the tyrosine kinase domain (TKD) are rare. However, no data are yet available on the frequency of expression levels of FLT3 and FLT3L in JMML. If activated FLT3 occurs in JMML, one could imagine that these patients might benefit from treatment with small molecule FLT3 inhibitors, especially as to date the curative treatment of JMML is limited to allogeneous bone marrow transplantation. In 36 JMML patients FLT3 and FLT3L mRNA levels were assessed using real-time quantitative PCR (Taqman). Furthermore these samples were screened for the presence of activating FLT3/ITDs and FLT3/TKD mutations. MTT assays were performed to assess the response of JMML cells to the known FLT3 inhibitor PKC412 (Novartis). FLT3 appeared to be expressed only at basal levels and FLT3L expression was very low. In none of the 36 JMML samples FLT3/ITDs or TDK mutations were found, consistent with the observation that PKC412 was not cytotoxic in JMML samples (n=12), in contrast to leukemic cells of children with ALL which carried an activated FLT3. These data suggest that constitutively activated FLT3 does not occur in JMML. Therefore targeting FLT3 by tyrosine kinase inhibitors like PKC412 is unlikely to be effective in JMML.

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Antibodies to insulin receptor tyrosine kinase stimulate its activity towards exogenous substrates without inducing receptor autophosphorylation

Biochemical Journal ◽

10.1042/bj2600749 ◽

1989 ◽

Vol 260 (3) ◽

pp. 749-756 ◽

Cited By ~ 11

Author(s):

V Baron ◽

N Gautier ◽

N Rochet ◽

R Ballotti ◽

B Rossi ◽

...

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Insulin Receptor ◽

Receptor Tyrosine Kinase ◽

Kinase Activity ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Insulin Receptor Tyrosine Kinase ◽

Substrate Phosphorylation ◽

Exogenous Substrates

Anti-peptide antibodies directed against a highly-conserved sequence of the insulin receptor tyrosine kinase domain have been used to study the relationship between this specific region and kinase activation. Antibodies have been prepared by the injection into a rabbit of a synthetic peptide (P2) corresponding to residues 1110-1125 of the proreceptor. The peptide exhibits 88-95% sequence similarity with the corresponding sequence in the v-ros protein and in receptors for epidermal growth factor and for insulin-like growth factor 1. Two antibodies with different specificities could be separated from total antiserum obtained after immunization with P2. One antibody [anti-(P-Tyr)] cross-reacted with phosphotyrosine and immunoprecipitated solely autophosphorylated receptors. This antibody was shown to increase or decrease the receptor tyrosine kinase activity depending on its concentration. In all circumstances receptor autophosphorylation and substrate phosphorylation were modulated in a parallel fashion. The second antibody (anti-P2) failed to immunoprecipitate the insulin receptor, but was found to interact with both the peptide and the receptor by e.l.i.s.a. assay. Using a tyrosine co-polymer we found that anti-P2 activated the insulin receptor kinase leading to substrate phosphorylation at a level similar to that observed with insulin. This effect was additive to the hormonal effect. In contrast, receptor autophosphorylation was not modified by the anti-peptide. The differential effect of this anti-peptide further supports the idea that receptor autophosphorylation and kinase activity towards exogenous substrates might be independently regulated. Finally, our data suggest that conformational changes in the receptor tyrosine kinase domain may be sufficient for activation of its enzymic activity.

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