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

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.

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Interplay of Eph-Ephrin Signalling and Cadherin Function in Cell Segregation and Boundary Formation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.784039 ◽

2021 ◽

Vol 9 ◽

Author(s):

David G. Wilkinson

Keyword(s):

Receptor Activation ◽

Craniofacial Development ◽

Cell Populations ◽

Eph Receptors ◽

Eph Receptor ◽

Cortical Tension ◽

Differential Adhesion ◽

Distinct Cell ◽

The Difference ◽

Cell Segregation

The segregation of distinct cell populations to form sharp boundaries is crucial for stabilising tissue organisation, for example during hindbrain segmentation in craniofacial development. Two types of mechanisms have been found to underlie cell segregation: differential adhesion mediated by cadherins, and Eph receptor and ephrin signalling at the heterotypic interface which regulates cell adhesion, cortical tension and repulsion. An interplay occurs between these mechanisms since cadherins have been found to contribute to Eph-ephrin-mediated cell segregation. This may reflect that Eph receptor activation acts through multiple pathways to decrease cadherin-mediated adhesion which can drive cell segregation. However, Eph receptors mainly drive cell segregation through increased heterotypic tension or repulsion. Cadherins contribute to cell segregation by antagonising homotypic tension within each cell population. This suppression of homotypic tension increases the difference with heterotypic tension triggered by Eph receptor activation, and it is this differential tension that drives cell segregation and border sharpening.

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EphB2 and EphB4 receptors forward signaling promotes SDF-1–induced endothelial cell chemotaxis and branching remodeling

Blood ◽

10.1182/blood-2006-05-023341 ◽

2006 ◽

Vol 108 (9) ◽

pp. 2914-2922 ◽

Cited By ~ 62

Author(s):

Ombretta Salvucci ◽

Maria de la Luz Sierra ◽

Jose A. Martina ◽

Peter J. McCormick ◽

Giovanna Tosato

Keyword(s):

Extracellular Matrix ◽

Endothelial Cell ◽

Tyrosine Kinases ◽

Molecular Mechanisms ◽

Cell Movement ◽

Cell Contact ◽

Eph Receptor ◽

Cell Clustering ◽

Chemokine Ligand ◽

Ephrin Ligands

Abstract The complex molecular mechanisms that drive endothelial cell movement and the formation of new vessels are poorly understood and require further investigation. Eph receptor tyrosine kinases and their membrane-anchored ephrin ligands regulate cell movements mostly by cell–cell contact, whereas the G-protein–coupled receptor CXCR4 and its unique SDF-1 chemokine ligand regulate cell movement mostly through soluble gradients. By using biochemical and functional approaches, we investigated how ephrinB and SDF-1 orchestrate endothelial cell movement and morphogenesis into capillary-like structures. We describe how endogenous EphB2 and EphB4 signaling are required for the formation of extracellular matrix–dependent capillary-like structures in primary human endothelial cells. We further demonstrate that EphB2 and EphB4 activation enhance SDF-1–induced signaling and chemotaxis that are also required for extracellular matrix–dependent endothelial cell clustering. These results support a model in which SDF-1 gradients first promote endothelial cell clustering and then EphB2 and EphB4 critically contribute to subsequent cell movement and alignment into cord-like structures. This study reveals a requirement for endogenous Eph signaling in endothelial cell morphogenic processes, uncovers a novel link between EphB forward signaling and SDF-1–induced signaling, and demonstrates a mechanism for cooperative regulation of endothelial cell movement.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e03-09-0674 ◽

2004 ◽

Vol 15 (4) ◽

pp. 1647-1655 ◽

Cited By ~ 25

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.

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The Cytokine Signal Inhibitor Lnk Promotes αIIbβ3 Integrin Outside-In Signaling through β3 Tyrosine Phosphorylation in Platelets.

Blood ◽

10.1182/blood.v110.11.3651.3651 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3651-3651

Author(s):

Koji Eto ◽

Hitoshi Takizawa ◽

Satoshi Takaki ◽

Hidekazu Nishikii ◽

Atsushi Oda ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Adaptor Protein ◽

Sh2 Domain ◽

Cytokine Signaling ◽

Adapter Protein ◽

Clot Retraction ◽

Hematopoietic Stem ◽

Kinase Activation ◽

C Terminus

Abstract Lnk is an SH2 domain-containing adapter protein that inhibits cytokine signaling. Lnk−/− mice exhibit a marked increase in numbers of hematopoietic stem cells, megakaryocytes and platelets, presumably due to the lack of negative regulation in thrombopoietin-mediated signals by Lnk. We previously reported that Lnk might play an unanticipated role in platelet integrin αIIbβ3 outside-in signaling. Lnk−/− platelets exhibited defects in full spreading on fibrinogen, clot retraction and formation of thrombi on collagen under flow conditions while they showed normal inside-out signaling (Blood, 106 (11):115a, 2005). However the mechanism(s) in which Lnk participates in αIIbβ3 outside-in signaling had not been elucidated. Here we report that in normal platelets Lnk forms a complex with c-Src, Syk, Fyn and adhesion and degranulation promoting adaptor protein (ADAP) but not SLP-76 in a manner dependent on αIIbβ3 ligation and Src kinase activation. c-Src-, but not Syk-, mediated tyrosine phosphorylation of C-terminus in Lnk appeared to be indispensable for the complex formation and Lnk-mediated function. Furthermore we have shown that Lnk is required for the association of Fyn to αIIbβ3 and for β3 subunit tyrosine phosphorylation while activation of non-receptor tyrosine kinases (c-Src and Syk) in proximity to αIIbβ3 is independent of Lnk. Thus, these results provide new insights into Lnk function and the mechanism by which Lnk contributes to integrin signaling in the adhesion responses of platelets.

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Adaptor Protein Lnk Binds to JAK3 and Negatively Regulates a Mutant Activating Allele Associated with Megakaryocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.5044.5044 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5044-5044

Author(s):

Maya Koren-Michowitz ◽

Sigal Gery ◽

Daniel Nowak ◽

Phillip H Koeffler

Keyword(s):

Tyrosine Kinases ◽

Receptor Tyrosine Kinases ◽

Conflicts Of Interest ◽

Severe Combined Immunodeficiency ◽

Leukemia Cell ◽

Adaptor Protein ◽

Sh2 Domain ◽

Leukemic Cells ◽

Leukemia Cell Line ◽

Ph Domains

Abstract Abstract 5044 The adaptor protein Lnk is known to associate with hematopoietic cytokine receptors such as cKIT, MPL and PDGFR, as well as, non-receptor tyrosine kinases such as JAK2, and is considered to have an inhibitory effect on these signaling pathways. JAK3 is expressed mainly in the hematopoietic system and its absence is associated with autosomal recessive severe combined immunodeficiency (SCID). Recently, activating mutations of JAK3 were described in transient myeloproliferative disorder (TMD) and acute megakaryocytic leukemia (AMKL) in Down syndrome (DS) patients as well as adult non-DS AMKL. JAK3 mutations were also rarely described in solid tumors and B-ALL. The 50% homology between JAK3 and JAK2 has led us to study the association between Lnk and JAK3.293T cells were co-transfected with cDNAs encoding either wild-type (WT) JAK3 or JAK3 harboring an activating A572V mutation (JAK3 A572V), as well as the WT V5-tagged Lnk. Whole cell lysates were used for immunoprecipitation with either V5-tag or JAK3 antibodies. Binding of Lnk and JAK3 was detected by Western blot probed with JAK3 or V5-tag antibodies. To determine which domain of Lnk 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 (del SH2) and deletion of the PH and SH2 (del SH2/PH) domains. Our results show that WT Lnk binds to WT JAK3, as well as JAK3 A572V. The R392E and del SH2 Lnk mutants retained JAK3 binding capacity while deletion of both SH2 and PH domains of Lnk abolished JAK3 binding. In order to study the biological effect of Lnk binding to JAK3, we infected CMK cells, a megakaryocytic leukemia cell line harboring JAK3 A572V, with a bicistronic retroviral MSCV-IRES-GFP (MIG) WT Lnk vector. Effect on growth was assessed in GFP positive sorted cells by cell count and colony formation in methylcellulose. CMK cells infected with MIG WT Lnk grew slower in liquid culture and had decreased clonogenic growth in soft agar culture compared to cells infected with MIG vector alone. In summary, we show for the first time that Lnk can bind to WT and mutant JAK3 and slow the growth of leukemic cells harboring an activating JAK3 mutation. Developing a small molecule mimetic of Lnk may have a therapeutic role in the treatment of hematopoietic malignancies associated with a variety of activated tyrosine kinase receptors and non-receptor tyrosine kinases including JAK3, as well as secondary signaling proteins. Disclosures No relevant conflicts of interest to declare.

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Tyrosine 842 Residue in the Activation Loop of FLT3-ITD Is Indespensible for Oncogenic Transformation

Blood ◽

10.1182/blood.v128.22.1561.1561 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1561-1561

Author(s):

Julhash U. Kazi ◽

Lars Rönnstrand

Keyword(s):

Tyrosine Kinases ◽

Receptor Tyrosine Kinases ◽

Conflicts Of Interest ◽

Point Mutations ◽

Cellular Transformation ◽

Receptor Activation ◽

Tumor Formation ◽

Activation Loop ◽

Tyrosine Residues

Abstract The receptor tyrosine kinase FLT3 is mutated in around 30% of acute myeloid leukemia patients. The so-called internal tandem duplication (ITD) mutation in the juxtamembrane domain is the most common type of mutation in FLT3. Other oncogenic mutations include point mutations in the kinase domain. One of the hallmark of receptor activation is phosphorylation on several tyrosine residues in the receptor. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. The tyrosine residue Y842, which is located in the activation loop of FLT3, and is well conserved in all receptor tyrosine kinases. It has been demonstrated that tyrosine phosphorylation of the activation loop is essential for the activity of some but not all receptor tyrosine kinases. In this report we show that phosphorylation on Y842 residue is dispensable for receptor activity and stability. However, Y842 plays critical roles in oncogenic FLT3-ITD mediated transformation. We have shown that a Y-to-F mutation (Y842F) results in reduced cell viability and proliferation. This mutation also led to dramatic decrease in in vitro colony formation in semi-solid medium. Furthermore, cells carrying the Y842F mutant of FLT3-ITD displayed significant delay in tumor formation in nude mice. Gene expression analysis, using microarrays, showed that mutation of Y842 causes suppression of anti-apoptotic genes. Finally, we showed that phosphorylated Y842 is a binding site for SHP2 and mutation of this site results in impaired activation of signaling through the RAS/ERK pathway. Collectively, our data suggest that Y842 in FLT3 plays an important role in FLT3-mediated RAS/ERK signaling and cellular transformation. Disclosures No relevant conflicts of interest to declare.

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Regulation of FcεRI-mediated degranulation by an adaptor protein 3BP2 in rat basophilic leukemia RBL-2H3 cells

Blood ◽

10.1182/blood-2001-12-0340 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2138-2144 ◽

Cited By ~ 40

Author(s):

Kiyonao Sada ◽

S. M. Shahjahan Miah ◽

Koichiro Maeno ◽

Shinkou Kyo ◽

Xiujuan Qu ◽

...

Keyword(s):

Mast Cells ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Calcium Influx ◽

Adaptor Protein ◽

Cellular Protein ◽

Sh2 Domain ◽

External Sources ◽

High Affinity Ige Receptor ◽

Basophilic Leukemia

Abstract Aggregation of high-affinity IgE receptor FcεRI induces sequential activation of nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, leading to degranulation in mast cells. A hematopoietic cell–specific adaptor protein, 3BP2, that was originally identified as an Abl SH3-binding protein was rapidly tyrosine phosphorylated by the aggregation of FcεRI on rat basophilic leukemia RBL-2H3 cells. Tyrosine phosphorylation of 3BP2 did not depend on calcium influx from external sources. To examine the role of 3BP2 in mast cells, we overexpressed the SH2 domain of 3BP2 in the RBL-2H3 cells. Overexpression of 3BP2-SH2 domain resulted in a suppression of antigen-induced degranulation as assessed by β-hexosaminidase release. Even though overall tyrosine phosphorylation of cellular protein was not altered, antigen-mediated tyrosine phosphorylation of phospholipase C-γ (PLC-γ) and calcium mobilization were significantly suppressed in the cells overexpressing the 3BP2-SH2 domain. Furthermore, antigen stimulation induced the association of 3BP2-SH2 domain with LAT and other signaling molecule complexes in the RBL-2H3 cells. FcεRI-mediated phosphorylation of JNK and ERK was not affected by the overexpression of 3BP2-SH2 domain. These data indicate that 3BP2 functions to positively regulate the FcεRI-mediated tyrosine phosphorylation of PLC-γ and thereby the signals leading to degranulation.

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Dancing with the dead: Eph receptors and their kinase-null partnersThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o10-145 ◽

2011 ◽

Vol 89 (2) ◽

pp. 115-129 ◽

Cited By ~ 30

Author(s):

Luke Truitt ◽

Andrew Freywald

Keyword(s):

Membrane Proteins ◽

Tyrosine Kinases ◽

Molecular Mechanisms ◽

Receptor Tyrosine Kinases ◽

Peer Review Process ◽

Eph Receptors ◽

Eph Receptor ◽

Biological Responses ◽

Wide Range ◽

Multicellular Organisms

Eph receptor tyrosine kinases and their ligands, ephrins, are membrane proteins coordinating a wide range of biological functions both in developing embryos and in adult multicellular organisms. Numerous studies have implicated Eph receptors in the induction of opposing responses, including cell adhesion or repulsion, support or inhibition of cell proliferation and cell migration, and progression or suppression of multiple malignancies. Similar to other receptor tyrosine kinases, Eph receptors rely on their ability to catalyze tyrosine phosphorylation for signal transduction. Interestingly, however, Eph receptors also actively utilize three kinase-deficient receptor tyrosine kinases, EphB6, EphA10, and Ryk, in their signaling network. The accumulating evidence suggests that the unusual flexibility of the Eph family, allowing it to initiate antagonistic responses, might be partially explained by the influence of the kinase-dead participants and that the exact outcome of an Eph-mediated action is likely to be defined by the balance between the signaling of catalytically potent and catalytically null receptors. We discuss in this minireview the emerging functions of the kinase-dead EphB6, EphA10, and Ryk receptors both in normal biological responses and in malignancy, and analyze currently available information related to the molecular mechanisms of their action in the context of the Eph family.

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Grb2 monomer–dimer equilibrium determines normal versus oncogenic function

Nature Communications ◽

10.1038/ncomms8354 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 16

Author(s):

Zamal Ahmed ◽

Zahra Timsah ◽

Kin M. Suen ◽

Nathan P. Cook ◽

Gilbert R. Lee ◽

...

Keyword(s):

Map Kinase ◽

Cancer Progression ◽

Protein Interactions ◽

Tyrosine Kinases ◽

Growth Factor Receptor ◽

Adaptor Protein ◽

Sh2 Domain ◽

Breast Cancers ◽

Mitogen Activated Protein ◽

Self Association

Abstract The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

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Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation

Molecular and Cellular Biology ◽

10.1128/mcb.20.5.1537-1545.2000 ◽

2000 ◽

Vol 20 (5) ◽

pp. 1537-1545 ◽

Cited By ~ 96

Author(s):

Elena Becker ◽

Uyen Huynh-Do ◽

Sacha Holland ◽

Tony Pawson ◽

Tom O. Daniel ◽

...

Keyword(s):

Tyrosine Kinases ◽

Kinase Activity ◽

Sh2 Domain ◽

Mitogen Activated Protein Kinase ◽

Eph Receptors ◽

Tyrosine Kinase Signaling ◽

Amino Terminal ◽

Biological Responses ◽

Mitogen Activated Protein ◽

Tyrosine Phosphorylated Protein

ABSTRACT The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62 dok , RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62 dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

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