The Transmembrane Domain of PDGFR-β Plays An Important Role in ETV6-PDGFR-β Activation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5320-5320
Author(s):  
Federica Toffalini ◽  
Jean-Baptiste Demoulin
Keyword(s):  
Tyrosine Kinases ◽  
Transmembrane Domain ◽  
Myeloproliferative Neoplasms ◽  
Chromosomal Translocations ◽  
Wild Type ◽  
Sam Domain ◽  
Helix Loop Helix ◽  
Hybrid Proteins ◽  
Tm Domain ◽  
Myelomonocytic Leukemia

Abstract The ETV6-PDGFRβ hybrid protein (EPβ) is found in a subset of patients with chronic myelomonocytic leukemia (CMML) associated with eosinophilia. EPβ is the archetype of a larger group of hybrid receptors that are produced by chromosomal translocations of PDGFR genes and cause atypical myeloproliferative neoplasms. In EPβ, the N-terminal portion of the ETV6 transcription factor replaces the PDGFRβ ligand-binding domain and induces the activation of the chimera through its pointed domain (PNT domain, also called helix-loop-helix or SAM domain). This domain mediates ligand-independent EPβ oligomerization, resulting in constitutive tyrosine kinase activity. In addition, we showed that EPβ and other hybrid receptor tyrosine kinases are much more stable than wild-type receptors and that the deletion of the PNT domain induced EPβ protein degradation, suggesting a link between the clustering and the stabilization of the EPβ protein. The PDGFRβ transmembrane domain (TM domain) is retained in EPβ and in most PDGFRβ hybrid proteins that have been described. We observed that the deletion of the TM domain (EPβ-ΔTM mutant) strongly impaired the ability of EPβ to sustain growth factor-independent proliferation of Ba/F3 and 32D cells. The phosphorylation of the mutant protein was also markedly reduced. We confirmed that EPβ is not inserted in membranes but resides in the cytosol, indicating that the PDGFRβ TM domain does not act as a transmembrane domain in EPβ but has a completely different function. The EPβ-ΔTM mutant retained the ability to self-associate in co-immunoprecipitation experiments, but showed a decreased level of polymerization when using cross-linking agents, suggesting that this domain is required for optimal clustering of EPβ. In line with our findings on the PNT domain, the EPβ-ΔTM protein was less stable and, as a result, was expressed at a lower level. In conclusion, we demonstrate that the TM domain plays a role in EPβ activation by promoting the clustering of the protein and by preventing its degradation in cooperation with the PNT domain.

scholarly journals Ectopic EphA4 Receptor Induces Posterior Protrusions via FGF Signaling in Xenopus Embryos

2004 ◽  
Vol 15 (4) ◽  
pp. 1647-1655 ◽  
Author(s):  
Eui Kyun Park ◽  
Neil Warner ◽  
Yong-Sik Bong ◽  
David Stapleton ◽  
Ryu Maeda ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Sh2 Domain ◽  
Binding Motif ◽  
Eph Receptors ◽  
Fgf Signaling ◽  
Wild Type ◽  
Sam Domain ◽  
Xenopus Embryos

The Eph family of receptor tyrosine kinases regulates numerous biological processes. To examine the biochemical and developmental contributions of specific structural motifs within Eph receptors, wild-type or mutant forms of the EphA4 receptor were ectopically expressed in developing Xenopus embryos. Wild-type EphA4 and a mutant lacking both the SAM domain and PDZ binding motif were constitutively tyrosine phosphorylated in vivo and catalytically active in vitro. EphA4 induced loss of cell adhesion, ventro-lateral protrusions, and severely expanded posterior structures in Xenopus embryos. Moreover, mutation of a conserved SAM domain tyrosine to phenylalanine (Y928F) enhanced the ability of EphA4 to induce these phenotypes, suggesting that the SAM domain may negatively regulate some aspects of EphA4 activity in Xenopus. Analysis of double mutants revealed that the Y928F EphA4 phenotypes were dependent on kinase activity; juxtamembrane sites of tyrosine phosphorylation and SH2 domain-binding were required for cell dissociation, but not for posterior protrusions. The induction of protrusions and expansion of posterior structures is similar to phenotypic effects observed in Xenopus embryos expressing activated FGFR1. Furthermore, the budding ectopic protrusions induced by EphA4 express FGF-8, FGFR1, and FGFR4a. In addition, antisense morpholino oligonucleotide-mediated loss of FGF-8 expression in vivo substantially reduced the phenotypic effects in EphA4Y928F expressing embryos, suggesting a connection between Eph and FGF signaling.

scholarly journals Voltage-Dependent Inactivation of MscS Occurs Independently of the Positively Charged Residues in the Transmembrane Domain

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Takeshi Nomura ◽  
Masahiro Sokabe ◽  
Kenjiro Yoshimura
Keyword(s):  
Voltage Dependence ◽  
Transmembrane Domain ◽  
Wild Type ◽  
Dependent Inactivation ◽  
Mechanical Threshold ◽  
Voltage Dependent ◽  
Arginine Residues ◽  
Tm Domain ◽  
Small Conductance ◽  
Positively Charged

MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and plays a major role in avoiding cell lysis upon rapid osmotic downshock. The gating of MscS is modulated by voltage, but little is known about how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-54, and Arg-74) in the transmembrane (TM) domain are possible to respond to voltage judging from the MscS structure. To examine whether these residues are involved in the voltage dependence of MscS, we neutralized the charge of each residue by substituting with asparagine (R46N, R54N, and R74N). Mechanical threshold for the opening of the expressed wild-type MscS and asparagine mutants did not change with voltage in the range from-40 to +100 mV. By contrast, inactivation process of wild-type MscS was strongly affected by voltage. The wild-type MscS inactivated at +60 to +80 mV but not at-60 to +40 mV. The voltage dependence of the inactivation rate of all mutants tested, that is, R46N, R54N, R74N, and R46N/R74N MscS, was almost indistinguishable from that of the wild-type MscS. These findings indicate that the voltage dependence of the inactivation occurs independently of the positive charges of R46, R54, and R74.

scholarly journals Mutations of E3 Ubiquitin Ligase Cbl Family Members Constitute a Novel Common Pathogenic Lesion in Myeloid Malignancies

2009 ◽  
Vol 27 (36) ◽  
pp. 6109-6116 ◽  
Author(s):  
Hideki Makishima ◽  
Heather Cazzolli ◽  
Hadrian Szpurka ◽  
Andrew Dunbar ◽  
Ramon Tiu ◽  
...  
Keyword(s):  
Clinical Features ◽  
Ubiquitin Ligase ◽  
Tyrosine Kinases ◽  
Myeloproliferative Neoplasms ◽  
Uniparental Disomy ◽  
E3 Ubiquitin Ligase ◽  
Myelogenous Leukemia ◽  
Juvenile Myelomonocytic Leukemia ◽  
Aberrant Expression ◽  
Myelomonocytic Leukemia

Purpose Acquired somatic uniparental disomy (UPD) is commonly observed in myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), or secondary acute myelogenous leukemia (sAML) and may point toward genes harboring mutations. Recurrent UPD11q led to identification of homozygous mutations in c-Cbl, an E3 ubiquitin ligase involved in attenuation of proliferative signals transduced by activated receptor tyrosine kinases. We examined the role and frequency of Cbl gene family mutations in MPN and related conditions. Methods We applied high-density SNP-A karyotyping to identify loss of heterozygosity of 11q in 442 patients with MDS, MDS/MPN, MPN, sAML evolved from these conditions, and primary AML. We sequenced c-Cbl, Cbl-b, and Cbl-c in patients with or without corresponding UPD or deletions and correlated mutational status with clinical features and outcomes. Results We identified c-Cbl mutations in 5% and 9% of patients with chronic myelomonocytic leukemia (CMML) and sAML, and also in CML blast crisis and juvenile myelomonocytic leukemia (JMML). Most mutations were homozygous and affected c-Cbl; mutations in Cbl-b were also found in patients with similar clinical features. Patients with Cbl family mutations showed poor prognosis, with a median survival of 5 months. Pathomorphologic features included monocytosis, monocytoid blasts, aberrant expression of phosphoSTAT5, and c-kit overexpression. Serial studies showed acquisition of c-Cbl mutations during malignant evolution. Conclusion Mutations in the Cbl family RING finger domain or linker sequence constitute important pathogenic lesions associated with not only preleukemic CMML, JMML, and other MPN, but also progression to AML, suggesting that impairment of degradation of activated tyrosine kinases constitutes an important cancer mechanism.

A Conserved Transmembrane Domain Interface Regulates Integrin Function

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2858-2858 ◽  
Author(s):  
Bryan W Berger ◽  
Lisa M. Span ◽  
Daniel W Kulp ◽  
Paul C. Billings ◽  
William F. DeGrado ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Transmembrane Domain ◽  
Dominant Negative ◽  
The Other ◽  
Type I ◽  
Wild Type ◽  
Suspension Cells ◽  
Α Subunit ◽  
Tm Domain ◽  
Domain Separation

Abstract Integrins are a superfamily of transmembrane (TM) α/β heterodimers that mediate fundamental cellular adhesive functions. Platelet integrins, for example, mediate stable platelet adhesion to collagen and fibronectin and the formation of stable platelet aggregates. Integrins reside on cell surfaces in an equilibrium between inactive and active conformations. An essential feature of this equilibrium is interaction of the integrin α and β subunit TM domains. Thus, when integrins are inactive, the α and β TM domains are in proximity, but they separate when integrins assume an active conformation. Moreover, inducing TM domain separation alone is sufficient to cause integrin activation. Previously, we reported that the TM domains of the platelet integrin αIIbβ3 interact both heteromerically and homomerically and that the strength of their heteromeric interaction is necessarily weak to allow regulated TM domain separation. To address whether these observations can be extended to the other members of the integrin superfamily, we focused initially on αvβ3, α2β1 and α5β1, integrins present in platelets, using a dominant-negative ToxR-based assay. ToxR is a single-pass TM transcriptional factor from V. cholera that activates the cholera toxin (ctx) promoter when it dimerizes in the inner membrane of E. coli. By co-expressing wild-type ToxR with either wild-type ToxR or an R96K ToxR mutant that can dimerize but is unable to activate the ctx promoter, we can measure the homomeric and heteromeric interaction of each integrin TM domain. Using alanine and leucine scanning mutagenesis, we found that like αIIb, homo-oligomerization of other integrin α subunit TM domains is preferred over hetero-oligomerization, and that the relative strength of homo-oligomerization correlates with the presence of a canonical small residue-xxx-small residue motif followed one turn of the TM helix by a leucine (G, A, S-xxx-G-xxx-L). This motif also mediates the hetero-oligomerization of these TM domains with either β3 or β1. By contrast, a different motif (V-xxx-I-xxx-G) mediates the heteromeric interaction of both β3 and β1 with their complementary α subunits. Mutations that disrupt either the αIIb or β3 interaction motif induce constitutive αIIbβ3 activation. To determine if this is also the case for β1-containing integrins, we introduced disruptive interfacial mutations into the full-length integrins and expressed the mutants in either the β1-deficient Jurkat A1 cells or in HEK293 suspension cells. We found that the β1 mutations V716A, I720A and G724L caused a substantial increase in the static adhesion of A1 cells to laminin, fibronectin, the α4β1-specific peptide H1, as well as type I, II and type IV collagen, whereas mutation of the canonical G-xxx-G motif did not. On the other hand, an increase in binding to type I collagen and fibronectin was observed for mutations of the interfacial α2 residues S1009, G1013, and L1017 and the interfacial α5 residues A964, G968, and L972, respectively. Thus, our studies indicate that β1 and β3 integrins employ a novel, specific, and conserved reciprocating ‘large-small’ TM packing interface that interacts less strongly than the canonical small-residue-xxx-small residue motif. It is also noteworthy that this interface is present in all integrins except β4 and is overrepresented in databases of TM helix-helix interaction as well. Accordingly, it is likely that this type of interface evolved to mediate TM domain interactions that are capable of regulation.

An Update on JAK Inhibitors

2019 ◽  
Vol 26 (10) ◽  
pp. 1806-1832 ◽  
Author(s):  
Francesca Musumeci ◽  
Chiara Greco ◽  
Ilaria Giacchello ◽  
Anna Lucia Fallacara ◽  
Munjed M. Ibrahim ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Tyrosine Kinases ◽  
Chemical Structure ◽  
Receptor Tyrosine Kinases ◽  
Myeloproliferative Neoplasms ◽  
Pharmaceutical Companies ◽  
Jak Inhibitors ◽  
Pyrimidine Derivatives ◽  
Janus Kinases ◽  
Structure Activity

Janus kinases (JAKs) are a family of non-receptor tyrosine kinases, composed by four members, JAK1, JAK2, JAK3 and TYK2. JAKs are involved in different inflammatory and autoimmune diseases, as well as in malignancies, through the activation of the JAK/STAT signalling pathway. Furthermore, the V617F mutation in JAK2 was identified in patients affected by myeloproliferative neoplasms. This knowledge prompted researchers from academia and pharmaceutical companies to investigate this field in order to discover small molecule JAK inhibitors. These efforts recently afforded to the market approval of four JAK inhibitors. Despite the fact that all these drugs are pyrrolo[2,3-d]pyrimidine derivatives, many compounds endowed with different heterocyclic scaffolds have been reported in the literature as selective or multi-JAK inhibitors, and a number of them is currently being evaluated in clinical trials. In this review we will report many representative compounds that have been published in articles or patents in the last five years (period 2013-2017). The inhibitors will be classified on the basis of their chemical structure, focusing, when possible, on their structure activity relationships, selectivity and biological activity. For every class of derivatives, compounds disclosed before 2013 that have entered clinical trials will also be briefly reported, to underline the importance of a particular chemical scaffold in the search for new inhibitors.

scholarly journals High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells

2021 ◽  
Vol 11 (7) ◽  
Author(s):  
Ruochen Jia ◽  
Leon Kutzner ◽  
Anna Koren ◽  
Kathrin Runggatscher ◽  
Peter Májek ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Synthetic Lethality ◽  
Myeloproliferative Neoplasms ◽  
Hematopoietic Cells ◽  
Replication Stress ◽  
Phase Cell ◽  
Nuclear Staining ◽  
Wild Type ◽  
High Throughput Drug Screening

AbstractMutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.

scholarly journals Ectopic Overexpression of Histone H3K4 Methyltransferase CsSDG36 from Tea Plant Decreases Hyperosmotic Stress Tolerance in Arabidopsis thaliana

2021 ◽  
Vol 22 (10) ◽  
pp. 5064
Author(s):  
Qinghua Chen ◽  
Linghui Guo ◽  
Yanwen Yuan ◽  
Shuangling Hu ◽  
Fei Guo ◽  
...  
Keyword(s):  
Drought Stress ◽  
Transmembrane Domain ◽  
Tea Plant ◽  
Microtubule Assembly ◽  
Pcr Analysis ◽  
Drought Response ◽  
Stomatal Development ◽  
Wild Type ◽  
Over Expression ◽  
Histone H3k4

Histone methylation plays an important regulatory role in the drought response of many plants, but its regulatory mechanism in the drought response of the tea plant remains poorly understood. Here, drought stress was shown to induce lower relative water content and significantly downregulate the methylations of histone H3K4 in the tea plant. Based on our previous analysis of the SET Domain Group (SDG) gene family, the full-length coding sequence (CDS) of CsSDG36 was cloned from the tea cultivar ‘Fuding Dabaicha’. Bioinformatics analysis showed that the open reading frame (ORF) of the CsSDG36 gene was 3138 bp, encoding 1045 amino acids and containing the conserved structural domains of PWWP, PHD, SET and PostSET. The CsSDG36 protein showed a close relationship to AtATX4 of the TRX subfamily, with a molecular weight of 118,249.89 Da, and a theoretical isoelectric point of 8.87, belonging to a hydrophilic protein without a transmembrane domain, probably located on the nucleus. The expression of CsSDG36 was not detected in the wild type, while it was clearly detected in the over-expression lines of Arabidopsis. Compared with the wild type, the over-expression lines exhibited lower hyperosmotic resistance by accelerating plant water loss, increasing reactive oxygen species (ROS) pressure, and increasing leaf stomatal density. RNA-seq analysis suggested that the CsSDG36 overexpression caused the differential expression of genes related to chromatin assembly, microtubule assembly, and leaf stomatal development pathways. qRT-PCR analysis revealed the significant down-regulation of stomatal development-related genes (BASL, SBT1.2(SDD1), EPF2, TCX3, CHAL, TMM, SPCH, ERL1, and EPFL9) in the overexpression lines. This study provides a novel sight on the function of histone methyltransferase CsSDG36 under drought stress.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

scholarly journals A single amino acid substitution (N297A) in the conserved NPXXY sequence of the human N-formyl peptide receptor results in inhibition of desensitization and endocytosis, and a dose-dependent shift in p42/44 mitogen-activated protein kinase activation and chemotaxis

2000 ◽  
Vol 352 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Jeannie M. GRIPENTROG ◽  
Algirdas J. JESAITIS ◽  
Heini M. MIETTINEN
Keyword(s):  
Protein Kinase ◽  
Transmembrane Domain ◽  
Mitogen Activated Protein Kinase ◽  
Formyl Peptide Receptor ◽  
Wild Type ◽  
Peptide Receptor ◽  
Formyl Peptide ◽  
Mitogen Activated Protein ◽  
Dose Dependent Increase ◽  
Dose Dependent

The formyl peptide receptor (FPR) is a G-protein-coupled receptor (GPCR) that mediates chemotaxis and stimulates the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase pathway. We have examined the functional effects of substitutions of a conserved aspartic acid residue in the second transmembrane domain (D71A) and of residues in the conserved NPXXY motif in the seventh transmembrane domain (N297A and Y301A). These mutated receptors, expressed in Chinese hamster ovary (CHO) cells, bind ligand with affinities similar to wild-type FPR, but the D71A mutant is uncoupled from G-protein [Miettinen, Mills, Gripentrog, Dratz, Granger and Jesaitis (1997) J. Immunol 159, 4045–4054]. In the present study, we show that both the D71A and N297A mutations resulted in defective endocytosis. The N297A substitution also prevented desensitization, as determined by intracellular calcium mobilization by sequential stimulation with ligand. In chemotaxis assays, the N297A mutation resulted in cell migration towards gradients of up to 100nM N-formyl-methionyl-leucyl-phenylalanine (fMLF), whereas cells expressing the wild-type FPR and the Y301A mutant were no longer chemotactically responsive at 10–100nM fMLF. Maximal activation of p42/44 MAPK occurred in CHO cells expressing wild-type FPR at 10nM–100nM fMLF, whereas cells expressing the N297A mutant showed a dose-dependent increase in the amount of phosphorylated p42/44 MAPK up to 1–10µM fMLF. Since the MAPK kinase inhibitor PD98059 blocked fMLF-induced chemotaxis, our results suggest that the dose-dependent increase in p42/44 MAPK activation may correlate with the increased chemotactic migration of N297A transfectants at 10nM–100nM fMLF.

scholarly journals Conformational clamping by a membrane ligand activates the EphA2 receptor

2021 ◽  
Author(s):  
Justin M Westerfield ◽  
Amita Sahoo ◽  
Daiane S Alves ◽  
Brayan Grau ◽  
Alayna Cameron ◽  
...  
Keyword(s):  
Drug Target ◽  
Transmembrane Domain ◽  
Binding Mode ◽  
Membrane Receptors ◽  
Binding Motif ◽  
Active Conformation ◽  
Dynamic Simulations ◽  
Membrane Peptide ◽  
Migration Assays ◽  
Tm Domain

The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migration assays that identified a new potent peptide variant. We also performed a mutational screen that determined the helical interface that mediates dimerization of the TM domain of EphA2 in cells. These results, together with molecular dynamic simulations, allowed to elucidate the molecular mechanism that TYPE7 uses to activate EphA2, where the membrane peptide acts as a molecular clamp that wraps around the TM dimer of the receptor. We propose that this binding mode stabilizes the active conformation of EphA2. Our data, additionally, provide clues into the properties that TM ligands need to have in order to achieve activation of membrane receptors.

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