scholarly journals Autoinhibition of the Kit Receptor Tyrosine Kinase by the Cytosolic Juxtamembrane Region

2003 ◽  
Vol 23 (9) ◽  
pp. 3067-3078 ◽  
Author(s):  
Perry M. Chan ◽  
Subburaj Ilangumaran ◽  
Jose La Rose ◽  
Avijit Chakrabartty ◽  
Robert Rottapel
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Regulatory Function ◽  
Wild Type ◽  
Juxtamembrane Domain ◽  
Juxtamembrane Region ◽  
Phosphorylated Form ◽  
Oncogenic Mutations ◽  
Kit Receptor ◽  
Oncogenic Form

ABSTRACT Genetic studies have implicated the cytosolic juxtamembrane region of the Kit receptor tyrosine kinase as an autoinhibitory regulatory domain. Mutations in the juxtamembrane domain are associated with cancers, such as gastrointestinal stromal tumors and mastocytosis, and result in constitutive activation of Kit. Here we elucidate the biochemical mechanism of this regulation. A synthetic peptide encompassing the juxtamembrane region demonstrates cooperative thermal denaturation, suggesting that it folds as an autonomous domain. The juxtamembrane peptide directly interacted with the N-terminal ATP-binding lobe of the kinase domain. A mutation in the juxtamembrane region corresponding to an oncogenic form of Kit or a tyrosine-phosphorylated form of the juxtamembrane peptide disrupted the stability of this domain and its interaction with the N-terminal kinase lobe. Kinetic analysis of the Kit kinase harboring oncogenic mutations in the juxtamembrane region displayed faster activation times than the wild-type kinase. Addition of exogenous wild-type juxtamembrane peptide to active forms of Kit inhibited its kinase activity in trans, whereas the mutant peptide and a phosphorylated form of the wild-type peptide were less effective inhibitors. Lastly, expression of the Kit juxtamembrane peptide in cells which harbor an oncogenic form of Kit inhibited cell growth in a Kit-specific manner. Together, these results show the Kit kinase is autoinhibited through an intramolecular interaction with the juxtamembrane domain, and tyrosine phosphorylation and oncogenic mutations relieved the regulatory function of the juxtamembrane domain.

PKCδ plays opposite roles in growth mediated by wild-type Kit and an oncogenic Kit mutant

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 1923-1929 ◽  
Author(s):  
Tanya Jelacic ◽  
Diana Linnekin
Keyword(s):  
Receptor Tyrosine Kinase ◽  
Catalytic Domain ◽  
Dominant Negative ◽  
Wild Type ◽  
Aspartic Acid Residue ◽  
Kit Receptor ◽  
Murine Mast Cell ◽  
Oncogenic Form ◽  
In Cells

AbstractThe Kit receptor tyrosine kinase is critical for normal hematopoiesis. Mutation of the aspartic acid residue encoded by codon 816 of human c-kit or codon 814 of the murine gene results in an oncogenic form of Kit. Here we investigate the role of protein kinase Cδ (PKCδ) in responses mediated by wild-type murine Kit and the D814Y mutant in a murine mast cell-like line. PKCδ is activated after wild-type (WT) Kit binds stem cell factor (SCF), is constitutively active in cells expressing the Kit catalytic domain mutant, and coprecipitates with both forms of Kit. Inhibition of PKCδ had opposite effects on growth mediated by wild-type and mutant Kit. Both rottlerin and a dominant-negative PKCδ construct inhibited the growth of cells expressing mutant Kit, while SCF-induced growth of cells expressing wild-type Kit was not inhibited. Further, overexpression of PKCδ inhibited growth of cells expressing wild-type Kit and enhanced growth of cells expressing the Kit mutant. These data demonstrate that PKCδ contributes to factor-independent growth of cells expressing the D814Y mutant, but negatively regulates SCF-induced growth of cells expressing wild-type Kit. This is the first demonstration that PKCδ has different functions in cells expressing normal versus oncogenic forms of a receptor.

scholarly journals Substrate phosphorylation specificity of the human c-kit receptor tyrosine kinase.

1991 ◽  
Vol 266 (30) ◽  
pp. 19908-19916 ◽  
Author(s):  
R. Herbst ◽  
R. Lammers ◽  
J. Schlessinger ◽  
A. Ullrich
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Kit Receptor ◽  
Substrate Phosphorylation

scholarly journals Expression of KIT Receptor Tyrosine Kinase in Endothelial Cells of Juvenile Brain Tumors

2009 ◽  
Vol 20 (4) ◽  
pp. 763-770 ◽  
Author(s):  
Marjut Puputti ◽  
Olli Tynninen ◽  
Paula Pernilä ◽  
Marko Salmi ◽  
Sirpa Jalkanen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Brain Tumors ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Kit Receptor

scholarly journals Distinct role for c-kit receptor tyrosine kinase and SgIGSF adhesion molecule in attachment of mast cells to fibroblasts

2005 ◽  
Vol 85 (3) ◽  
pp. 426-435 ◽  
Author(s):  
Yu-ichiro Koma ◽  
Akihiko Ito ◽  
Kenji Watabe ◽  
Tatsumi Hirata ◽  
Masao Mizuki ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tyrosine Kinase ◽  
Adhesion Molecule ◽  
Receptor Tyrosine Kinase ◽  
Kit Receptor ◽  
Distinct Role

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 ◽  
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.

Identification of Two Src Recruitment Sites in the Juxtamembrane Region of Flt3 with Opposing Effects on Flt3-Ligand-Induced Signaling.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2289-2289
Author(s):  
Lars Ronnstrand ◽  
Elke Heiss ◽  
Christina Sundberg ◽  
Kristina Masson ◽  
Malin Pedersen ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Src Family Kinases ◽  
Phosphorylation Sites ◽  
Flt3 Ligand ◽  
Erk Activation ◽  
Juxtamembrane Region ◽  
Signal Relay

Abstract Early signal relay steps upon ligand-binding to the receptor tyrosine kinase Flt3, i.e. sites of Flt3-autophosphorylation and subsequent docking partners, are mainly unresolved. Here we demonstrate for the first time identification of ligand-induced in vivo phosphorylation sites in Flt3. By immunoprecipitation of specific tryptic peptides contained in the juxtamembrane region of human Flt3 and subsequent radiosequencing we identified the tyrosine residues 572, 589, 591 and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we examined Flt3-ligand-mediated responses in WT-Flt3, Y589F-Flt3 and Y599F-Flt3 expressing 32D cells. Compared to WT-Flt3-32D cells, 32D-Y589F-Flt3 showed upon ligand-stimulation enhanced Erk activation as well as proliferation/survival whereas 32D-Y599F-Flt3 cells displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for multiple signal relay molecules including Src family kinases. Consistently, 32D-Y589F-Flt3 and 32D-Y599F-Flt3 showed decreased FL-triggered Src activation, impaired phosphorylation of the adapter molecules Cbl and ShcA and deficient receptor ubiquitination and degradation. Interference with the Src-dependent negative regulation of Flt3 signaling may account for the enhanced mitogenic response of Y589F-Flt3. pY599 was additionally found to interact with the protein tyrosine phosphatase Shp2. As Y599F-Flt3-32D lacked ligand-induced Shp2 phosphorylation and since silencing of Shp2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3-phenotype we hypothesize that recruitment of Shp2 to pY599 contributes to FL-mediated Erk activation and proliferation. To summarize, our work presents novel insights in Flt3-mediated signal transduction. We have identified the in vivo autophosphorylation sites of the juxtamembrane region of Flt3, revealed Src family kinases and Shp2 as binding partners of pY589 and/or pY599, respectively, as well as their potential impact on FL-mediated signaling in Flt3-32D cells. Future work will now focus on elucidation of additional and possibly novel interaction partners of the found phosphorylation sites by employing an unbiased proteomics approach. With this gained knowledge it will be of interest to see whether ITDs differing in the nature of the duplicated tyrosines also confer distinct signaling behavior. If so, these tyrosines might serve as a diagnostic marker and point towards a successful combinatorial therapy consisting of a receptor tyrosine kinase inhibitor and an inhibitor for the specifically affected signal transduction pathway.

The Novel Inhibitor PLX3397 Effectively Inhibits FLT3-Mutant AML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3632-3632 ◽  
Author(s):  
Elizabeth Burton ◽  
Bernice Wong ◽  
Jiazhong Zhang ◽  
Brian West ◽  
Gideon Bollag ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Tumor Regression ◽  
Wild Type ◽  
Gut Peptides ◽  
Cellular Assays ◽  
Juxtamembrane Region ◽  
Clear Dose Response ◽  
Major Subset ◽  
Subcutaneous Xenograft

Abstract Abstract 3632 Mutations of FLT3 (FMS-like tyrosine kinase 3) are frequently present in acute myeloid leukemias (AML) and associated with poor prognosis. Such mutations alter the conformation and constitutively activate the FLT3 tyrosine kinase. PLX3397 is a novel, orally active, selective small molecule inhibitor of the FLT3-mutant as well as FMS and KIT kinases. The high selectivity of PLX3397 has been demonstrated in biochemical assays against a panel of over 200 recombinant kinases. FLT3, FMS and KIT are significantly inhibited at low nanomolar concentrations. The majority of kinases screened were not inhibited by PLX3397 (IC50=>10 μM) and the only other kinase with significant sub-micromolar inhibition was KDR. PLX3397 exhibits favorable pharmaceutical properties and demonstrated minimal off-target activity when tested in a broad array of 71 targets in 8 families (Neurotransmitter-related, Steroids, Ion Channels, Nitric Oxide, Prostaglandins, Growth Factors, Brain/Gut Peptides, and Enzymes). In cellular assays PLX3397 effectively inhibited ligand-stimulated autophosphorylation of the endogenous receptor tyrosine kinases FMS (IC50=20 nM) and KIT (IC50=120 nM), but not ligand-stimulated wild-type FLT3 (IC50=1.7 μM). In SEMK2 cells, which over-express wild type FLT3, PLX3397 inhibited FLT3 autophosphorylation with an IC50 of 240 nM. The phosphorylation of the activated FLT3-ITD protein in MV-4-11 and MOLM-14 cells harboring mutations (FLT3-ITD) of the juxtamembrane region of FLT3 was efficiently inhibited (IC50 26 nM and 30 nM respectively), indicating a possible application in this major subset of FLT3 mutated AML. In parallel with inhibition of FLT3-mutant autophosphorylation, the phosphorylation of known downstream effectors (STAT5, AKT, MEK, and ERK) is also inhibited. However, no inhibition was observed for KDR phosphorylation. PLX3397 potently inhibited of mutant-FLT3 driven proliferation in both MV-4-11 and MOLM-14 AML cells in culture (IC50=100–200 nM), but was less effective against HL60 cells expressing wild-type FLT3. MV-4-11 cells grown as subcutaneous xenograft tumors in mice were also highly sensitive to PLX3397 dosed orally at 10 or 30 mg/kg qd, with tumor regression observed at the higher dose. Primary samples collected from AML patients at relapse were tested in culture. While no significant effects were seen in FLT3 wild type samples below 1 μM, a clear dose response to PLX3397 was observed in samples with FLT3-ITD mutations. Because the estimated protein binding of PLX3397 in plasma is >99%, we also tested the drug against the MV-4-11 cell line in plasma and found the IC50 for inhibition of autophosphorylation to be 2.8 μM. In humans trough levels of PLX3397 (10 μM) capable of inhibiting FLT3-mutants can be safely achieved. A Phase I/II study is planned to evaluate the safety and explore the efficacy of PLX3397 in patients with FLT3-mutant- AML. Disclosures: Burton: Plexxikon Inc.: Employment. Wong:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. West:Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Habets:Plexxikon Inc.: Employment. Nguyen:Plexxikon Inc.: Employment. Levis:Plexxikon Inc.: Honoraria.

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