EGFRvIV: a previously uncharacterized oncogenic mutant reveals a kinase autoinhibitory mechanism

Tumor cells often subvert normal regulatory mechanisms of signal transduction. This study shows this principle by studying yet uncharacterized mutants of the epidermal growth factor receptor (EGFR) previously identified in glioblastoma multiforme, which is the most aggressive brain tumor in adults. Unlike the well-characterized EGFRvIII mutant form, which lacks a portion of the ligand-binding cleft within the extracellular domain, EGFRvIVa and EGFRvIVb lack internal segments distal to the intracellular tyrosine kinase domain. By constructing the mutants and by ectopic expression in naive cells, we show that both mutants confer an oncogenic potential in vitro, as well as tumorigenic growth in animals. The underlying mechanisms entail constitutive receptor dimerization and basal activation of the kinase domain, likely through a mechanism that relieves a restraining molecular fold, along with stabilization due to association with HSP90. Phosphoproteomic analyses delineated the signaling pathways preferentially engaged by EGFRvIVb-identified unique substrates. This information, along with remarkable sensitivities to tyrosine kinase blockers and to a chaperone inhibitor, proposes strategies for pharmacological interception in brain tumors harboring EGFRvIV mutations.Goldhirsh FoundationNational Cancer Institute (U.S.) (CA118705)National Cancer Institute (U.S.) (CA141556)National Cancer Institute (U.S.) (U54-CA112967

Co-Conserved Features Associated with cis Regulation of ErbB Tyrosine Kinases

BACKGROUND: The epidermal growth factor receptor kinases, or ErbB kinases, belong to a large sub-group of receptor tyrosine kinases (RTKs), which share a conserved catalytic core. The catalytic core of ErbB kinases have functionally diverged from other RTKs in that they are activated by a unique allosteric mechanism that involves specific interactions between the kinase core and the flanking Juxtamembrane (JM) and COOH-terminal tail (C-terminal tail). Although extensive studies on ErbB and related tyrosine kinases have provided important insights into the structural basis for ErbB kinase functional divergence, the sequence features that contribute to the unique regulation of ErbB kinases have not been systematically explored. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we use a Bayesian approach to identify the selective sequence constraints that most distinguish ErbB kinases from other receptor tyrosine kinases. We find that strong ErbB kinase-specific constraints are imposed on residues that tether the JM and C-terminal tail to key functional regions of the kinase core. A conserved RIxKExE motif in the JM-kinase linker region and a glutamine in the inter-lobe linker are identified as two of the most distinguishing features of the ErbB family. While the RIxKExE motif tethers the C-terminal tail to the N-lobe of the kinase domain, the glutamine tethers the C-terminal tail to hinge regions critical for inter-lobe movement. Comparison of the active and inactive crystal structures of ErbB kinases indicates that the identified residues are conformationally malleable and can potentially contribute to the cis regulation of the kinase core by the JM and C-terminal tail. ErbB3, and EGFR orthologs in sponges and parasitic worms, diverge from some of the canonical ErbB features, providing insights into sub-family and lineage-specific functional specialization. CONCLUSION/SIGNIFICANCE: Our analysis pinpoints key residues for mutational analysis, and provides new clues to cancer mutations that alter the canonical modes of ErbB kinase regulation

Hereditary cutaneomucosal venous malformations are caused by TIE2 mutations with widely variable hyper-phosphorylating effects

Mutations in the angiopoietin receptor TIE2/TEK have been identified as the cause for autosomal dominantly inherited cutaneomucosal venous malformation (VMCM). Thus far, two specific germline substitutions (R849W and Y897S), located in the kinase domain of TIE2, have been reported in five families. The mutations result in a fourfold increase in ligand-independent phosphorylation of the receptor. Here, we report 12 new families with TEK mutations. Although the phenotype is primarily characterized by small multifocal cutaneous vascular malformations, many affected members also have mucosal lesions. In addition, cardiac malformations are observed in some families. Six of the identified mutations are new, with three located in the tyrosine kinase domain, two in the kinase insert domain, and another in the carboxy terminal tail. The remaining six are R849W substitutions. Overexpression of the new mutants resulted in ligand-independent hyperphosphorylation of the receptor, suggesting this is a general feature of VMCM-causative TIE2 mutations. Moreover, variation in the level of activation demonstrates, to the best of our knowledge for the first time, that widely differing levels of chronic TIE2 hyperphosphorylation are tolerated in the heterozygous state, and are compatible with normal endothelial cell function except in the context of highly localized areas of lesion pathogenesis

Somatic mutations in the angiopoietin-receptor TIE2 can cause both solitary and multiple sporadic venous malformations

The vascular endothelial cell (EC)-specific receptor tyrosine kinase (RTK) TIE2 plays a crucial role in angiogenesis and cardiovascular development. In man, heterozygous TIE2 substitutions that induce in vitro ligand-independent hyperphosphorylation cause hereditary mucocutaneous venous malformations (VMCM, OMIM 600195), characterized by multifocal small bluish cutaneous and mucosal lesions, composed of enlarged, tortuous venous channels. We now identified a somatic, lesion-associated TIE2 “2nd-hit” alteration in one resected VMCM, from a patient (Sa-I.4) carrying the germline R849W. Unlike inherited intracellular TIE2 mutations, the somatic deletion mutant (“Del”) was not hyperphophorylated in vitro, nor did it exacerbate hyperphosphorylation of R849W in trans. In retrovirally transfected HUVECs, Del-TIE2 is retained in endoplasmic reticulum (ER), and shows no ability to respond to Ang1 by increased phosphorylation or translocation and clustering. Thus, the somatic deletion mutant acts as a null-allele, causing local loss-of-function of wild-type TIE2. We assessed for whether such localized, tissue-specific events play a role in the etiology of the far more common sporadic VM, characterized by extensive, unifocal lesions. Eight somatic TIE2 mutations were identified in lesions from 28 out of 57 patients (49.1%), not detected in their blood or in control tissues. They were all located within the first TK or KID of TIE2. The somatic mutations included a frequent L914F change, and a series of double-mutations that occurred in cis, all of which show varying degrees of ligand-independent hyperphosphorylation in vitro, with double-mutants showing even higher levels as compared to their constituent single-mutant forms. The most common of these mutants (germline R849W and somatic L914F), also profoundly affect receptor compartmentalization, and response to ligand. Like the wild-type, R849W shows an even cellular distribution, and is translocated to the cell-rear and retraction fibers in response to ligand; however, it shows significantly higher levels of activation than wild-type in these fibers. L914F, on the other hand, accumulates in an activated state in the Golgi and ER, and is incompletely translocated upon stimulation. Phosphorylation levels in L914F-HUVEC retraction-fibers is nevertheless significantly greater than with the wild-type. In conclusion, these data illustrate that a sporadic disease may be explained by somatic changes in a gene causing rare, inherited forms, and pinpoint TIE2 pathways as potential therapeutic targets for VM. ([email protected]

Chromatin status and transcription factor binding to gonadotropin promoters in gonadotrope cell lines

Abstract Background Proper expression of key reproductive hormones from gonadotrope cells of the pituitary is required for pubertal onset and reproduction. To further our understanding of the molecular events taking place during embryonic development, leading to expression of the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), we characterized chromatin structure changes, imparted mainly by histone modifications, in model gonadotrope cell lines. Methods We evaluated chromatin status and gene expression profiles by chromatin immunoprecipitation assays, DNase sensitivity assay, and RNA sequencing in three developmentally staged gonadotrope cell lines, αT1–1 (progenitor, expressing Cga), αT3–1 (immature, expressing Cga and Gnrhr), and LβT2 (mature, expressing Cga, Gnrhr, Lhb, and Fshb), to assess changes in chromatin status and transcription factor access of gonadotrope-specific genes. Results We found the common mRNA α-subunit of LH and FSH, called Cga, to have an open chromatin conformation in all three cell lines. In contrast, chromatin status of Gnrhr is open only in αT3–1 and LβT2 cells. Lhb begins to open in LβT2 cells and was further opened by activin treatment. Histone H3 modifications associated with active chromatin were high on Gnrhr in αT3–1 and LβT2, and Lhb in LβT2 cells, while H3 modifications associated with repressed chromatin were low on Gnrhr, Lhb, and Fshb in LβT2 cells. Finally, chromatin status correlates with the progressive access of LHX3 to Cga and Gnrhr, followed by PITX1 binding to the Lhb promoter. Conclusion Our data show the gonadotrope-specific genes Cga, Gnrhr, Lhb, and Fshb are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications