Rotational Coupling of the Transmembrane and Kinase Domains of the Neu Receptor Tyrosine Kinase

Bell, Charlotte A.; Tynan, John A.; Hart, Kristen C.; Meyer, April N.; Robertson, Scott C.; Donoghue, Daniel J.

doi:10.1091/mbc.11.10.3589

Cited by 135 publications

(145 citation statements)

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“…Single and multiple tyrosine mutations in the TACC3 region (Y798F, Y853F, Y867F, Y878F) were introduced by PCR-based site-directed mutagenesis. DNA constructs were subcloned into pLXSN vector (10) for focus, proliferation, and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. All clones were confirmed by DNA sequencing.…”

Section: Dna Constructsmentioning

confidence: 99%

“…Bradford assay or Lowry assay was used to measure total protein concentration. Antibodies were added to lysates for overnight incubation at 4 C with rocking, followed by immunoprecipitation, as described previously (10). Samples were separated by 10% or 12.5% SDS-PAGE and transferred to Immobilon-P membranes (Millipore).…”

Section: Transfection Immunoprecipitation and Immunoblot Analysismentioning

confidence: 99%

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Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

Nelson

Meyer

Siari

et al. 2016

Molecular Cancer Research

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Fibroblast growth factor receptors (FGFR) are critical for cell proliferation and differentiation. Mutation and/or translocation of FGFRs lead to aberrant signaling that often results in developmental syndromes or cancer growth. As sequencing of human tumors becomes more frequent, so does the detection of FGFR translocations and fusion proteins. The research conducted in this article examines a frequently identified fusion protein between FGFR3 and transforming acidic coiled-coil containing protein 3 (TACC3), frequently identified in glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, and cervical cancer. Using titanium dioxide-based phosphopeptide enrichment (TiO 2 )-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), it was demonstrated that the fused coiled-coil TACC3 domain results in constitutive phosphorylation of key activating FGFR3 tyrosine residues. The presence of the TACC coiled-coil domain leads to increased and altered levels of FGFR3 activation, fusion protein phosphorylation, MAPK pathway activation, nuclear localization, cellular transformation, and IL3-independent proliferation. Introduction of K508R FGFR3 kinase-dead mutation abrogates these effects, except for nuclear localization which is due solely to the TACC3 domain.Implications: These results demonstrate that FGFR3 kinase activity is essential for the oncogenic effects of the FGFR3-TACC3 fusion protein and could serve as a therapeutic target, but that phosphorylated tyrosine residues within the TACC3-derived portion are not critical for activity.

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Section: Dna Constructsmentioning

confidence: 99%

Section: Transfection Immunoprecipitation and Immunoblot Analysismentioning

confidence: 99%

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

Nelson

Meyer

Siari

et al. 2016

Molecular Cancer Research

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“…Bell et al (62) have observed that the activation of an RTK occurs only for a specific TM dimer interface. The rotation of the dimer interface leads to periodic oscillations in kinase activity.…”

Section: Structural Link Between the Tm And The Catalytic Domainsmentioning

confidence: 99%

Role of Receptor Tyrosine Kinase Transmembrane Domains in Cell Signaling and Human Pathologies

2006

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Receptor tyrosine kinases (RTKs) conduct biochemical signals via lateral dimerization in thwe plasma membrane, and their transmembrane (TM) domains play an important role in the dimerization process. Here we present two models of RTK-mediated signaling, and we discuss the role of the TM domains within the framework of these two models. We summarize findings of single amino acid mutations in RTK TM domains that induce unregulated signaling and, as a consequence, pathological phenotypes. We review current knowledge of pathology induction mechanisms due to these mutations, focusing on the structural and thermodynamic basis of pathogenic dimer stabilization. RECEPTOR TYROSINE KINASES: ROLE IN BIOLOGY AND PATHOLOGYReceptor tyrosine kinases (RTKs) are single-pass membrane proteins with an extracellular ligand-binding domain and an intracellular kinase domain. Members of this large group of membrane proteins have been classified based on their structural and ligand-affinity properties (1). The RTK family includes several sub-families, including the epidermal growth factor receptors (EGFRs or ErbBs), the fibroblast growth factor receptors (FGFRs), the insulin and the insulin-like growth factor receptors (IR and IGFR), the platelet-derived growth factor receptors (PDGFRs), the vascular endothelial growth factor receptors (VEGFRs), the hepatocyte growth factor receptors (HGFRs), and the nerve growth factor receptors (NGFRs) (2).RTKs conduct biochemical signals via lateral dimerization in the plasma membrane (3). All the above RTKs, with the exception of the insulin and the insulin-like growth factor receptors, exist in a monomer-dimer equilibrium. The dimer, which is stabilized upon ligand binding, is the signaling competent structure. It is believed that the contact between the two cytoplasmic domains in the dimer stimulates catalytic activity, and results in the intermolecular autophosphorylation of the receptors. This activates the catalytic domains and triggers signaling cascades that lead to the phosphorylation of cytoplasmic substrates.RTK-mediated signals play key roles in the regulation of various cellular processes, such as control of cell growth, differentiation, metabolism, and migration (3). The essential and * kh@jhu.edu, tel. 410-516-8939, fax 410-516-5293. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2015 January 21. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript diverse roles of RTKs are evident from the various developmental abnormalities and cancers that occur due to gain-of-function RTK signaling (4;5). In humans, signaling irregularities can be the result of (1) the generation of oncogenic fusion proteins (chromosomal translocation), (2) overactivation through mutations or deletions, and (3) overexpression as a result of gene amplification.Below, we provide a short overview of some RTKs that are critical to cell life, and of their role in disease.EGFRs-The subfamily of EGFRs consists of four members: EGFR (6), also designated a...

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“…of at least three separate experiments. Oncogenic Neu (NeuNT), activated by the Val664Glu mutation, served as the positive control (set at 100%) (Bell et al, 2000). The transforming activities of ptc1 and ptc1-Q688X constructs are presented as percentages with respect to the positive control.…”

Section: Focus Assaysmentioning

confidence: 99%

Constitutive activation of the shh–ptc1 pathway by a patched1 mutation identified in BCC

2004

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Mutations in the transmembrane receptor patched1 (ptc1) are responsible for the majority of basal cell carcinoma (BCC) cases. Many of these mutations, including ptc1-Q688X, result in premature truncation of the ptc1 protein. ptc1-Q688X has been identified in patients with both BCC and nevoid basal cell carcinoma syndrome, an inheritable disorder causing a predisposition to cancer susceptibility. Here we describe a mechanism by which ptc1-Q688X causes constitutive cellular signaling. Cells expressing ptc1-Q688X demonstrate an increase in cell cycle progression and induce cell transformation. The ptc1-Q688X mutant enhances Gli1 activity, a downstream reporter of sonic hedgehog (shh)-ptc1 signaling, independent of shh stimulation. In contrast to wild-type ptc1, ptc1-Q688X fails to associate with endogenous cyclin B1. Expression of nuclear-targeted cyclin B1 derivatives promotes Gli1-dependent transcription, which correlates temporally with cyclin B1-cdk1 kinase activity. Coexpression of wild-type ptc1 with a nuclear-targeted cyclin B1 derivative, mutated to mimic constitutive phosphorylation, dramatically decreases Gli1 activity. In addition, the coexpression of this constitutively nuclear cyclin B1 derivative with ptc1-Q688X substantially enhances foci formation. These studies therefore describe a molecular mechanism for the aberrant activity of ptc1-Q688X that includes the premature activation of the transcription factor Gli1. Oncogene (2005) 24, 902-915.

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Rotational Coupling of the Transmembrane and Kinase Domains of the Neu Receptor Tyrosine Kinase

Cited by 135 publications

References 51 publications

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

Role of Receptor Tyrosine Kinase Transmembrane Domains in Cell Signaling and Human Pathologies

Constitutive activation of the shh–ptc1 pathway by a patched1 mutation identified in BCC

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