scholarly journals The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signaling hubs

2021 ◽  
Author(s):  
Lung-Yu Liang ◽  
Michael Roy ◽  
Christopher Horne ◽  
Jarrod J Sandow ◽  
Minglyanna Surudoi ◽  
...  
Keyword(s):  
Cell Communication ◽  
Biochemical Properties ◽  
Eph Receptors ◽  
Eph Receptor ◽  
Sh2 Domains ◽  
Downstream Signaling ◽  
Juxtamembrane Region ◽  
Intracellular Signals ◽  
High Flexibility ◽  
Catalytic Functions

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell-cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically-dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane region, pseudokinase and SAM domains. Using small-angle X-ray scattering and crosslinking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosines in the juxtamembrane region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signaling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically-targeted to counter oncogenic signalling.

scholarly journals Intramolecular Regulatory Switch in ZAP-70: Analogy with Receptor Tyrosine Kinases

2005 ◽  
Vol 25 (12) ◽  
pp. 4924-4933 ◽  
Author(s):  
Tomas Brdicka ◽  
Theresa A. Kadlecek ◽  
Jeroen P. Roose ◽  
Alexander W. Pastuszak ◽  
Arthur Weiss
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Catalytic Domain ◽  
Receptor Tyrosine Kinases ◽  
Kinase Domain ◽  
Sh2 Domains ◽  
Downstream Signaling ◽  
Juxtamembrane Region ◽  
Activated T Cell ◽  
Regulatory Effects

ABSTRACT ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.

scholarly journals EPH tyrosine kinase receptors regulate epithelial morphogenesis and phosphorylate the PAR-3 scaffold protein to modulate downstream signaling networks

2020 ◽  
Author(s):  
Sara L. Banerjee ◽  
Noémie Lavoie ◽  
Kévin Jacquet ◽  
Frédéric Lessard ◽  
Josée N. Lavoie ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Cell Communication ◽  
Effector Proteins ◽  
Signaling Networks ◽  
Eph Receptors ◽  
Loss Of Function ◽  
Downstream Signaling ◽  
Cellular Phenotypes ◽  
Regulated Functions

SUMMARYThe EPH family is the largest among receptor tyrosine kinases (RTKs) in humans. In contrast to other RTKs, EPH receptors (EPHRs) cognate ligands, ephrins, are tethered to the cell surface. This results in EPHRs-ephrin signaling being mainly involved in short-range cell-cell communication events that regulate cell adhesion, migration and tissue boundary formation. Although EPHRs functions have been broadly studied, the molecular mechanisms by which they mediate these processes are far from being understood. To address this question, we sought to identify new downstream effector proteins for EPHRs and to determine their requirement for EPHR-regulated functions. To unravel EPHR-associated signaling complexes under native conditions, we applied a mass spectrometry-based approach, namely BioID proximity labeling. We obtained a composite proximity network from EPHA4, -B2, -B3 and -B4 receptors that comprises 395 proteins, most of which were not previously linked to EPH signaling. A gene ontology and pathway term analysis of the most common candidates highlighted cell polarity as a novel function associated with EPHR activity. We found that EPHA1 and EPHB4 expression is restricted to the basal and lateral membrane domains in polarized Caco-2 3D spheroidal cell cultures. We further discovered that their depletion impairs spheroid morphogenesis. In parallel, we examined the contribution of a number of candidates, selected from EPHR proximity networks, via loss-of-function in an EPHR-dependent cell segregation assay. We showed that depletion of the signaling scaffold PAR-3 blocks cell sorting. We also delineated a signalling complex involving C-terminal SRC kinase (CSK), whose recruitment to PAR-3 complexes is dependent on EPHR signals. Our work sheds light on EPHR signaling networks and provides a better understanding of the mechanisms by which EPHRs signal at the membrane to contribute to the establishment of cellular phenotypes.

scholarly journals Roles of Eph receptors and ephrins in segmental patterning

2000 ◽  
Vol 355 (1399) ◽  
pp. 993-1002 ◽  
Author(s):  
Qiling Xu ◽  
Georg Mellitzer ◽  
David G. Wilkinson
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinases ◽  
Cell Communication ◽  
Cell Contact ◽  
Eph Receptors ◽  
Biochemical Basis ◽  
Eph Receptor ◽  
Mediate Cell ◽  
Glycosylphosphatidyl Inositol

Eph receptor tyrosine kinases and their membrane–bound ligands, ephrins, have key roles in patterning and morphogenesis. Interactions between these molecules are promiscuous, but largely fall into two groups: EphA receptors bind to glycosylphosphatidyl inositol–anchored ephrin–A ligands, and EphB receptors bind to transmembrane ephrin–B proteins. Ephrin–B proteins transduce signals, such that bidirectional signalling can occur upon interaction with the Eph receptor. In many tissues, there are complementary and overlapping expression domains of interacting Eph receptors and ephrins. An important role of Eph receptors and ephrins is to mediate cell contact–dependent repulsion, and this has been implicated in the pathfinding of axons and neural crest cells, and the restriction of cell intermingling between hindbrain segments. Studies in an in vitro system show that bidirectional activation is required to prevent intermingling between cell populations, whereas unidirectional activation can restrict cell communication via gap junctions. Recent work indicates that Eph receptors can also upregulate cell adhesion, but the biochemical basis of repulsion versus adhesion responses is unclear. Eph receptors and ephrins have thus emerged as key regulators that, in parallel with cell adhesion molecules, underlie the establishment and maintenance of patterns of cellular organization.

scholarly journals The Shb scaffold binds the Nck adaptor protein, p120 RasGAP, and Chimaerins and thereby facilitates heterotypic cell segregation by the receptor EphB2

2020 ◽  
Vol 295 (12) ◽  
pp. 3932-3944 ◽  
Author(s):  
Melany J. Wagner ◽  
Marilyn S. Hsiung ◽  
Gerald D. Gish ◽  
Rick D. Bagshaw ◽  
Sasha A. Doodnauth ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Cell Movement ◽  
Adaptor Protein ◽  
Receptor Activation ◽  
Sh2 Domain ◽  
Tumor Formation ◽  
Eph Receptors ◽  
Eph Receptor ◽  
Varied Composition ◽  
Cell Segregation

Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)–mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain–containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and β-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2–ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.

scholarly journals Phosphorylation of Tyrosine Residues in the Kinase Domain and Juxtamembrane Region Regulates the Biological and Catalytic Activities of Eph Receptors

2000 ◽  
Vol 20 (13) ◽  
pp. 4791-4805 ◽  
Author(s):  
Kathleen L. Binns ◽  
Paul P. Taylor ◽  
Frank Sicheri ◽  
Tony Pawson ◽  
Sacha J. Holland
Keyword(s):  
Tyrosine Kinases ◽  
Biological Activities ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Eph Receptors ◽  
Amino Acid Homology ◽  
Tyrosine Residues ◽  
Juxtamembrane Region ◽  
Activation Segment

ABSTRACT Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

Adaptor Protein Lnk Binds to PDGFRA, PDGFRB and FIP1L1-PDGFRA, but Not to the TEL-PDGFRB Fusion Protein.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2213-2213
Author(s):  
Saskia Gueller ◽  
Sigal Gery ◽  
H. Phillip Koeffler
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Pleckstrin Homology Domain ◽  
Wild Type ◽  
Nih3t3 Cells ◽  
Sh2 Domains ◽  
Juxtamembrane Region

Abstract PDGFRA and PDGFRB (platelet derived growth factor receptors alpha and beta) are frequently expressed on malignant hematopoietic cells and regulate various cellular responses such as development, proliferation, differentiation, cell survival and cellular transformation. Stimulation by either autocrine loops or constitutional activation by chromosomal translocation (i.e. chronic myelomonocytic leukemia [CMML, TEL-PDGFRB] or chronic eosinophilic leukemia [CEL, FIP1L1-PDGFRA]) makes them important factors in development of hematopoietic disorders. Normally, interaction with the ligand PDGF, induces dimerization of two distinct receptor subunits, resulting in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. We hypothesized that one such protein may be the adaptor Lnk, a negative regulator of several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain, a pleckstrin homology domain (PH) and a dimerization domain (DD). The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. We investigated the ability of Lnk to bind to PDGFRA, PDGFRB, FIP1L1-PDGFRA and TEL-PDGFRB. To determine the domain of Lnk that is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including: a mutation in the SH2 domain (R392E); deletion of the SH2 domain; deletion of the PH and SH2 domains and a construct only containing the DD domain. 293T cells were co-transfected with cDNAs encoding either PDGFRA, PDGFRB or one of the translocation products and either wild-type or mutant Lnk. Whole cell lysates were used to perform immunoprecipitation with either V5-tag or PDGFR antibodies. Binding of Lnk and PDGFR was detected by Western blot probed with PDGFR or V5-tag antibodies. NIH3T3 cells were transfected either with empty vector or Lnk cDNA, transfectants were selected for 5 days with G418, serum starved for 16 hours and induced with PDGF for 10 minutes. Phosphorylation of downstream targets of PDGFRA and PDGFRB was detected by Western blot. Our data showed that Lnk bound to PDGFRA and PDGFRB only after exposure of the cells to PDGF and to the FIP1L1-PDGFRA fusion protein independent of PDGF exposure. Mutation or deletion of the Lnk SH2 domain abolished binding completely in PDGFRA and FIP1L1-PDGFRA, but just partly in PDGFRB. Expression of Lnk in NIH3T3 cells inhibited phosphorylation of ERK after treatment with PDGF. In other experiments, we determined that Lnk bound the juxtamembrane region of this class of receptors. Interestingly, the TEL-PDGFRB fusion protein was unable to bind Lnk, although its breakpoint in PDGFRB is distal to the juxtamembrane domain and the whole intracellular region of PDGFRB is included in the fusion protein. Further exploration of the mechanisms by which Lnk affects wild-type or PDGFR fusion product will provide insight into the molecular pathophysiology of myeloid disorders and could help develop new treatments.

Identification of Tyrosine Residues of Importance for Survival Signaling through the Scaffolding Protein Gab2 in Both Wild-Type FLT3 and the FLT3-ITD.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1622-1622
Author(s):  
Kristina Masson ◽  
Tao Liu ◽  
Jianmin Sun ◽  
Lars Ronnstrand
Keyword(s):  
Scaffolding Protein ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Internal Tandem Duplication ◽  
Tyrosine Residues ◽  
Downstream Signaling ◽  
Juxtamembrane Region ◽  
Future Potential ◽  
Survival Signaling

Abstract The receptor tyrosine kinase FLT3 is normally expressed in hematopoietic progenitor cells and has been implicated as a major cause of transformation in acute myeloid leukemia, where it in approximately 30% of cases is mutated and constitutively active. This is in most cases due to duplication of a DNA sequence coding for amino acids in the juxtamembrane region of FLT3, commonly referred to as ITD (Internal Tandem Duplication). In this study we have identified several novel in vivo tyrosine phosphorylation sites that are phosphorylated in wild-type FLT3 upon ligand stimulation and that are constitutively phosphorylated in the FLT3-ITD. We were able to demonstrate that these phosphorylation sites are critical for full phosphorylation of the scaffolding protein Gab2 both in wild-type FLT3 and FLT3-ITD. Y-to-F mutants of either wild-type FLT3 or FLT3-ITD, lacking these tyrosine residues, fail to phosphorylate Gab2 and demonstrate a considerable reduction in phosphorylation of Akt and Erk. Furthermore, FL-dependent survival and proliferation of wild-type FLT3 expressing Ba/F3 cells as well as FL-independent survival and proliferation of Ba/F3 cells transfected with FLT3-ITD was dramatically reduced by mutation of these tyrosine residues. In the case of the FLT3-ITD, this was shown to correlate with strongly reduced STAT5 phosphorylation. To verify the importance of Gab2 in FLT3-ITD signaling, we used siRNA technology to knock down the expression of Gab2 in the human AML cell line MV4-11 that is known to express FLT3-ITD. Knockdown of Gab2 expression led to a dramatic reduction in the phosphorylation of Akt, Erk and Stat5. To summarize, we have identified novel phosphorylation sites in FLT3 and how they link to downstream signaling of survival and proliferation. These findings not only reveal novel phosphorylation sites in FLT3 but also contribute to the understanding of the molecular mechanism by which FLT3-ITD functions in pathological conditions. Future studies are aiming at elucidating the mechanism by which Gab2 mediates phosphorylation and activation of STAT5, which could be a future potential target for therapy in AML with FLT3-ITD.

scholarly journals Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors

2011 ◽  
Vol 195 (6) ◽  
pp. 1033-1045 ◽  
Author(s):  
Peter W. Janes ◽  
Bettina Griesshaber ◽  
Lakmali Atapattu ◽  
Eva Nievergall ◽  
Linda L. Hii ◽  
...  
Keyword(s):  
Cellular Responses ◽  
Eph Receptors ◽  
Wild Type ◽  
Eph Receptor ◽  
Receptor Complexes ◽  
Ephb Receptor ◽  
Receptor Profile ◽  
Ephrin Ligands ◽  
The Individual ◽  
Cross Inhibition

Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph–Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters.

Dps biomineralizing proteins: multifunctional architects of nature

2012 ◽  
Vol 445 (3) ◽  
pp. 297-311 ◽  
Author(s):  
Kornelius Zeth
Keyword(s):  
Iron Uptake ◽  
Iron Oxidation ◽  
Hollow Spheres ◽  
Cell Communication ◽  
Memory Storage ◽  
Oxygen Species ◽  
Inner Surface ◽  
Technical Production ◽  
Catalytic Functions ◽  
Ros Reactive Oxygen Species

Dps proteins are the structural relatives of bacterioferritins and ferritins ubiquitously present in the bacterial and archaeal kingdoms. The ball-shaped enzymes play important roles in the detoxification of ROS (reactive oxygen species), in iron scavenging to prevent Fenton reactions and in the mechanical protection of DNA. Detoxification of ROS and iron chaperoning represent the most archetypical functions of dodecameric Dps enzymes. Recent crystallographic studies of these dodecameric complexes have unravelled species-dependent mechanisms of iron uptake into the hollow spheres. Subsequent functions in iron oxidation at ferroxidase centres are highly conserved among bacteria. Final nucleation of iron as iron oxide nanoparticles has been demonstrated to originate at acidic residues located on the inner surface. Some Dps enzymes are also implicated in newly observed catalytic functions related to the formation of molecules playing roles in bacterium–host cell communication. Most recently, Dps complexes are attracting attention in semiconductor science as biomimetic tools for the technical production of the smallest metal-based quantum nanodots used in nanotechnological approaches, such as memory storage or solar cell development.

scholarly journals PTP1B regulates Eph receptor function and trafficking

2010 ◽  
Vol 191 (6) ◽  
pp. 1189-1203 ◽  
Author(s):  
Eva Nievergall ◽  
Peter W. Janes ◽  
Carolin Stegmayer ◽  
Mary E. Vail ◽  
Fawaz G. Haj ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatases ◽  
Surface Concentration ◽  
Receptor Internalization ◽  
Fluorescence Lifetime Imaging ◽  
Dependent Manner ◽  
Cellular Interactions ◽  
Eph Receptors ◽  
Eph Receptor ◽  
Lifetime Imaging ◽  
Underlying Mechanisms

Eph receptors orchestrate cell positioning during normal and oncogenic development. Their function is spatially and temporally controlled by protein tyrosine phosphatases (PTPs), but the underlying mechanisms are unclear and the identity of most regulatory PTPs are unknown. We demonstrate here that PTP1B governs signaling and biological activity of EphA3. Changes in PTP1B expression significantly affect duration and amplitude of EphA3 phosphorylation and biological function, whereas confocal fluorescence lifetime imaging microscopy (FLIM) reveals direct interactions between PTP1B and EphA3 before ligand-stimulated receptor internalization and, subsequently, on endosomes. Moreover, overexpression of wild-type (w/t) PTP1B and the [D-A] substrate–trapping mutant decelerate ephrin-induced EphA3 trafficking in a dose-dependent manner, which reveals its role in controlling EphA3 cell surface concentration. Furthermore, we provide evidence that in areas of Eph/ephrin-mediated cell–cell contacts, the EphA3–PTP1B interaction can occur directly at the plasma membrane. Our studies for the first time provide molecular, mechanistic, and functional insights into the role of PTP1B controlling Eph/ephrin-facilitated cellular interactions.

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