The Endothelial Receptor Tyrosine Kinase Tie1 Activates Phosphatidylinositol 3-Kinase and Akt To Inhibit Apoptosis

ABSTRACT Tie1 is an orphan receptor tyrosine kinase that is expressed almost exclusively in endothelial cells and that is required for normal embryonic vascular development. Genetic studies suggest that Tie1 promotes endothelial cell survival, but other studies have suggested that the Tie1 kinase has little to no activity, and Tie1-mediated signaling pathways are unknown. To begin to study Tie1 signaling, a recombinant glutathione S-transferase (GST)-Tie1 kinase fusion protein was produced in insect cells and found to be autophosphorylated in vitro. GST-Tie1 but not a kinase-inactive mutant associated with a recombinant p85 SH2 domain protein in vitro, suggesting that Tie1 might signal through phosphatidylinositol (PI) 3-kinase. To study Tie1 signaling in a cellular context, a c-fms-Tie1 chimeric receptor (fTie1) was expressed in NIH 3T3 cells. Ligand stimulation of fTie1 resulted in Tie1 autophosphorylation and downstream activation of PI 3-kinase and Akt. Stimulation of fTie1-expressing cells potently inhibited UV irradiation-induced apoptosis in a PI 3-kinase-dependent manner. Moreover, both Akt phosphorylation and inhibition of apoptosis were abrogated by mutation of tyrosine 1113 to phenylalanine, suggesting that this residue is an important PI 3-kinase binding site. These findings are the first biochemical demonstration of a signal transduction pathway and corresponding cellular function for Tie1, and the antiapoptotic effect of Tie1 is consistent with the results of previous genetic studies.

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Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1998.274.4.e611 ◽

1998 ◽

Vol 274 (4) ◽

pp. E611-E617 ◽

Cited By ~ 46

Author(s):

Rae D. Record ◽

Larry L. Froelich ◽

Chris J. Vlahos ◽

Bonnie L. Blazer-Yost

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Sodium Transport ◽

Specific Inhibitor ◽

Dependent Manner ◽

Phosphatidylinositol 3 Kinase ◽

Insulin Receptor Tyrosine Kinase ◽

A6 Cells ◽

Phosphatidylinositol 3

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 ≈ 6 μM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

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Disorders of FZ-CRD; insights towards FZ-CRD folding and therapeutic landscape

Molecular Medicine ◽

10.1186/s10020-019-0129-7 ◽

2019 ◽

Vol 26 (1) ◽

Cited By ~ 2

Author(s):

Reham M. Milhem ◽

Bassam R. Ali

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Molecular Mechanisms ◽

Treatment Strategies ◽

Protein Glycosylation ◽

Orphan Receptor ◽

Misfolded Proteins ◽

Congenital Myasthenic Syndrome ◽

Missense Mutations

AbstractThe ER is hub for protein folding. Proteins that harbor a Frizzled cysteine-rich domain (FZ-CRD) possess 10 conserved cysteine motifs held by a unique disulfide bridge pattern which attains a correct fold in the ER. Little is known about implications of disease-causing missense mutations within FZ-CRD families. Mutations in FZ-CRD of Frizzled class receptor 4 (FZD4) and Muscle, skeletal, receptor tyrosine kinase (MuSK) and Receptor tyrosine kinase-like orphan receptor 2 (ROR2) cause Familial Exudative Vitreoretinopathy (FEVR), Congenital Myasthenic Syndrome (CMS), and Robinow Syndrome (RS) respectively. We highlight reported pathogenic inherited missense mutations in FZ-CRD of FZD4, MuSK and ROR2 which misfold, and traffic abnormally in the ER, with ER-associated degradation (ERAD) as a common pathogenic mechanism for disease. Our review shows that all studied FZ-CRD mutants of RS, FEVR and CMS result in misfolded proteins and/or partially misfolded proteins with an ERAD fate, thus we coin them as “disorders of FZ-CRD”. Abnormal trafficking was demonstrated in 17 of 29 mutants studied; 16 mutants were within and/or surrounding the FZ-CRD with two mutants distant from FZ-CRD. These ER-retained mutants were improperly N-glycosylated confirming ER-localization. FZD4 and MuSK mutants were tagged with polyubiquitin chains confirming targeting for proteasomal degradation. Investigating the cellular and molecular mechanisms of these mutations is important since misfolded protein and ER-targeted therapies are in development. The P344R-MuSK kinase mutant showed around 50% of its in-vitro autophosphorylation activity and P344R-MuSK increased two-fold on proteasome inhibition. M105T-FZD4, C204Y-FZD4, and P344R-MuSK mutants are thermosensitive and therefore, might benefit from extending the investigation to a larger number of chemical chaperones and/or proteasome inhibitors. Nonetheless, FZ-CRD ER-lipidation it less characterized in the literature and recent structural data sheds light on the importance of lipidation in protein glycosylation, proper folding, and ER trafficking. Current treatment strategies in-place for the conformational disease landscape is highlighted. From this review, we envision that disorders of FZ-CRD might be receptive to therapies that target FZ-CRD misfolding, regulation of fatty acids, and/or ER therapies; thus paving the way for a newly explored paradigm to treat different diseases with common defects.

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Insulin stimulates the tyrosine phosphorylation of caveolin.

The Journal of Cell Biology ◽

10.1083/jcb.129.6.1523 ◽

1995 ◽

Vol 129 (6) ◽

pp. 1523-1531 ◽

Cited By ~ 166

Author(s):

C C Mastick ◽

M J Brady ◽

A R Saltiel

Keyword(s):

Tyrosine Kinase ◽

Insulin Receptor ◽

Tyrosine Phosphorylation ◽

Insulin Dose ◽

Signal Transduction Pathway ◽

Kinase Assay ◽

Membrane Structures ◽

Insulin Dependent ◽

Stimulation Of

The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin-sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin-associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton-insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an approximately 60 kD protein that cross-reacts with an antibody against the Src-family kinase p59Fyn. Thus, the insulin-dependent tyrosine phosphorylation of caveolin represents a novel, insulin-specific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor.

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Angiopoietin-1-induced ubiquitylation of Tie2 by c-Cbl is required for internalization and degradation

Biochemical Journal ◽

10.1042/bj20091010 ◽

2009 ◽

Vol 423 (3) ◽

pp. 375-380 ◽

Cited By ~ 10

Author(s):

Christina Wehrle ◽

Paul Van Slyke ◽

Daniel J. Dumont

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Receptor Trafficking ◽

Dependent Manner ◽

Vascular Stability ◽

Epidermal Growth ◽

Viral Form ◽

Candidate Molecule ◽

Stimulation Of ◽

Angiopoietin 1

Tie2 [where ‘Tie’ is an acronym from tyrosine kinase with Ig and EGF (epidermal growth factor) homology domains] is a receptor tyrosine kinase expressed predominantly on the surface of endothelial cells. Activated by its ligands, the angiopoietins, Tie2 initiates signalling pathways that modulate vascular stability and angiogenesis. Deletion of either the Tie2 or Ang1 (angiopoietin-1) gene in mice results in lethal vascular defects, signifying their importance in vascular development. The mechanism employed by the Tie2 signalling machinery to attenuate or cause receptor trafficking is not well defined. Stimulation of Tie2-expressing cells with Ang1 results in its ubiquitylation, suggesting that this may provide the necessary signal for receptor turnover. Using a candidate molecule approach, we demonstrate that Tie2 co-immunoprecipitates with c-Cbl in an Ang1-dependent manner and its ubiquitylation can be inhibited by the dominant-interfering molecule v-Cbl (a viral form of c-Cbl that contains only the tyrosine kinase-binding domain region). Inhibition of the Tie2–Cbl interaction by overexpression of v-Cbl blocks ligand-induced Tie2 internalization and degradation. In summary, our results illustrate that c-Cbl interacts with the Tie2 signalling complex in a stimulation-dependent manner, and that this interaction is required for Tie2 ubiquitylation, internalization and degradation.

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A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4810 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4810-4818 ◽

Cited By ~ 34

Author(s):

K M Lai ◽

J P Olivier ◽

G D Gish ◽

M Henkemeyer ◽

J McGlade ◽

...

Keyword(s):

Tyrosine Kinase ◽

Binding Site ◽

Receptor Tyrosine Kinase ◽

Growth Factor Receptor ◽

Adaptor Protein ◽

Sh2 Domain ◽

Expression Library ◽

Close Relationship ◽

Ptb Domain

Antibodies to the human Shc adaptor protein were used to isolate a cDNA encoding a Drosophila Shc protein (dShc) by screening an expression library. The dshc gene, which maps to position 67B-C on the third chromosome, encodes a 45-kDa protein that is widely expressed throughout the Drosophila life cycle. In flies, the dShc protein physically associates with the activated Drosophila epidermal growth factor receptor homolog (DER) and is inducibly phosphorylated on tyrosine by DER. The 45-kDa dShc protein is closely related both in overall organization and in amino acid sequence (46% identity) to the 52-kDa mammalian Shc isoform. In addition to a C-terminal Src homology 2 (SH2) domain, dShc contains an N-terminal phosphotyrosine-binding (PTB) domain, which associates in vitro with the autophosphorylated DER receptor tyrosine kinase and with phosphopeptides containing an Asn-Pro-X-pTyr motif, where pTyr stands for phosphotyrosine. A potential binding site for the dShc PTB domain is located at Tyr-1228 of DER. These results indicate that the shc gene has been conserved in evolution, as have the binding properties of the Shc PTB and SH2 domains. Despite the close relationship between the Drosophila and mammalian Shc proteins, dShc lacks the high-affinity Grb2-binding site found in mammalian Shc, suggesting that Shc proteins may have functions in addition to regulation of the Ras pathway.

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Regulation of phospholipase C-γ1 by the actin-regulatory protein villin

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.3.c1046 ◽

2001 ◽

Vol 281 (3) ◽

pp. C1046-C1058 ◽

Cited By ~ 23

Author(s):

A. Panebra ◽

S.-X. Ma ◽

L.-W. Zhai ◽

X.-T. Wang ◽

S. G. Rhee ◽

...

Keyword(s):

Catalytic Activity ◽

Tyrosine Kinase ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Molecular Mechanisms ◽

Regulatory Protein ◽

Sh2 Domain ◽

Dependent Manner ◽

Kinase Gene

The actin-regulatory protein villin is tyrosine phosphorylated and associates with phospholipase C-γ1(PLC-γ1) in the brush border of intestinal epithelial cells. To study the mechanism of villin-associated PLC-γ1 activation, we reconstituted in vitro the tyrosine phosphorylation of villin and its association with PLC-γ1. Recombinant villin was phosphorylated in vitro by the nonreceptor tyrosine kinase c-src or by expression in the TKX1 competent cells that carry an inducible tyrosine kinase gene. Using in vitro binding assays, we demonstrated that tyrosine-phosphorylated villin associates with the COOH-terminal Src homology 2 (SH2) domain of PLC-γ1. The catalytic activity of PLC-γ1was inhibited by villin in a dose-dependent manner with half-maximal inhibition at a concentration of 12.4 μM. Villin inhibited PLC-γ1 activity by sequestering the substrate phosphatidylinositol 4,5-bisphosphate (PIP2), since increasing concentrations of PIP2 reversed the inhibitory effects of villin on PLC activity. The inhibition of PLC-γ1 activity by villin was reversed by the tyrosine phosphorylation of villin. Further, we demonstrated that tyrosine phosphorylation of villin abolished villin's ability to associate with PIP2. In conclusion, tyrosine-phosphorylated villin associates with the COOH-terminal SH2 domain of PLC-γ1and activates PLC-γ1 catalytic activity. Villin regulates PLC-γ1 activity by modifying its own ability to bind PIP2. This study provides biochemical proof of the functional relevance of tyrosine phosphorylation of villin and identifies the molecular mechanisms involved in the activation of PLC-γ1 by villin.

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Targeting RON receptor tyrosine kinase for treatment of advanced solid cancers: antibody–drug conjugates as lead drug candidates for clinical trials

Therapeutic Advances in Medical Oncology ◽

10.1177/1758835920920069 ◽

2020 ◽

Vol 12 ◽

pp. 175883592092006

Author(s):

Hang-Ping Yao ◽

Sreedhar Reddy Suthe ◽

Xiang-Min Tong ◽

Ming-Hai Wang

Keyword(s):

Clinical Trials ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Targeted Therapeutics ◽

Antibody Drug Conjugates ◽

Drug Candidates ◽

Drug Conjugates ◽

Ron Receptor ◽

Antibody Drug

The recepteur d’origine nantais (RON) receptor tyrosine kinase, belonging to the mesenchymal-to-epithelial transition proto-oncogene family, has been implicated in the pathogenesis of cancers derived from the colon, lung, breast, and pancreas. These findings lay the foundation for targeting RON for cancer treatment. However, development of RON-targeted therapeutics has not gained sufficient attention for the last decade. Although therapeutic monoclonal antibodies (TMABs) targeting RON have been validated in preclinical studies, results from clinical trials have met with limited success. This outcome diminishes pharmaceutical enthusiasm for further development of RON-targeted therapeutics. Recently, antibody–drug conjugates (ADCs) targeting RON have drawn special attention owing to their increased therapeutic activity. The rationale for developing anti-RON ADCs is based on the observation that cancer cells are not sufficiently addicted to RON signaling for survival. Thus, TMAB-mediated inhibition of RON signaling is ineffective for clinical application. In contrast, anti-RON ADCs combine a target-specific antibody with potent cytotoxins for cancer cell killing. This approach not only overcomes the shortcomings in TMAB-targeted therapies but also holds the promise for advancing anti-RON ADCs into clinical trials. In this review, we discuss the latest advancements in the development of anti-RON ADCs for targeted cancer therapy including drug conjugation profile, pharmacokinetic properties, cytotoxic effect in vitro, efficacy in tumor models, and toxicological activities in primates.

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Incerto-thalamic modulation of fear via GABA and dopamine

Neuropsychopharmacology ◽

10.1038/s41386-021-01006-5 ◽

2021 ◽

Author(s):

Archana Venkataraman ◽

Sarah C. Hunter ◽

Maria Dhinojwala ◽

Diana Ghebrezadik ◽

JiDong Guo ◽

...

Keyword(s):

Brain Regions ◽

Zona Incerta ◽

Dependent Manner ◽

Post Traumatic Stress ◽

Functional Roles ◽

Functional Connections ◽

Optogenetic Stimulation ◽

Fear Generalization ◽

Stimulation Of

AbstractFear generalization and deficits in extinction learning are debilitating dimensions of Post-Traumatic Stress Disorder (PTSD). Most understanding of the neurobiology underlying these dimensions comes from studies of cortical and limbic brain regions. While thalamic and subthalamic regions have been implicated in modulating fear, the potential for incerto-thalamic pathways to suppress fear generalization and rescue deficits in extinction recall remains unexplored. We first used patch-clamp electrophysiology to examine functional connections between the subthalamic zona incerta and thalamic reuniens (RE). Optogenetic stimulation of GABAergic ZI → RE cell terminals in vitro induced inhibitory post-synaptic currents (IPSCs) in the RE. We then combined high-intensity discriminative auditory fear conditioning with cell-type-specific and projection-specific optogenetics in mice to assess functional roles of GABAergic ZI → RE cell projections in modulating fear generalization and extinction recall. In addition, we used a similar approach to test the possibility of fear generalization and extinction recall being modulated by a smaller subset of GABAergic ZI → RE cells, the A13 dopaminergic cell population. Optogenetic stimulation of GABAergic ZI → RE cell terminals attenuated fear generalization and enhanced extinction recall. In contrast, optogenetic stimulation of dopaminergic ZI → RE cell terminals had no effect on fear generalization but enhanced extinction recall in a dopamine receptor D1-dependent manner. Our findings shed new light on the neuroanatomy and neurochemistry of ZI-located cells that contribute to adaptive fear by increasing the precision and extinction of learned associations. In so doing, these data reveal novel neuroanatomical substrates that could be therapeutically targeted for treatment of PTSD.

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Establishment of a New Device for Electrical Stimulation of Non-Degenerative Cartilage Cells In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22010394 ◽

2021 ◽

Vol 22 (1) ◽

pp. 394

Author(s):

Simone Krueger ◽

Alexander Riess ◽

Anika Jonitz-Heincke ◽

Alina Weizel ◽

Anika Seyfarth ◽

...

Keyword(s):

Electrical Stimulation ◽

Electric Fields ◽

Cartilage Tissue ◽

Type I ◽

Dependent Manner ◽

New Device ◽

Alternating Electric Fields ◽

Cartilage Cells ◽

Stimulation Of

In cell-based therapies for cartilage lesions, the main problem is still the formation of fibrous cartilage, caused by underlying de-differentiation processes ex vivo. Biophysical stimulation is a promising approach to optimize cell-based procedures and to adapt them more closely to physiological conditions. The occurrence of mechano-electrical transduction phenomena within cartilage tissue is physiological and based on streaming and diffusion potentials. The application of exogenous electric fields can be used to mimic endogenous fields and, thus, support the differentiation of chondrocytes in vitro. For this purpose, we have developed a new device for electrical stimulation of chondrocytes, which operates on the basis of capacitive coupling of alternating electric fields. The reusable and sterilizable stimulation device allows the simultaneous use of 12 cavities with independently applicable fields using only one main supply. The first parameter settings for the stimulation of human non-degenerative chondrocytes, seeded on collagen type I elastin-based scaffolds, were derived from numerical electric field simulations. Our first results suggest that applied alternating electric fields induce chondrogenic re-differentiation at the gene and especially at the protein level of human de-differentiated chondrocytes in a frequency-dependent manner. In future studies, further parameter optimizations will be performed to improve the differentiation capacity of human cartilage cells.

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