scholarly journals Biochemical and NMR characterization of the interactions of Vav2–SH2 domain with lipids and the EphA2 juxtamembrane region on membrane

2020 ◽  
Vol 477 (19) ◽  
pp. 3791-3801
Author(s):  
Liang Ge ◽  
Bo Wu ◽  
Youjia Zhang ◽  
Jiarong Wang ◽  
Hongxin Zhao ◽  
...  
Keyword(s):  
Lipid Membrane ◽  
Lipid Binding ◽  
Sh2 Domain ◽  
Nucleotide Exchange ◽  
Surface Receptors ◽  
Guanine Nucleotide Exchange ◽  
Juxtamembrane Region ◽  
Nucleotide Exchange Factor ◽  
Nmr Characterization ◽  
Wide Range

Vav2 is a ubiquitous guanine nucleotide exchange factor (GEF) for Rho family GTPases that is involved in regulating a wide range of biological processes. It interacts with several tyrosine-phosphorylated cell surface receptors, including the Eph family receptors, through its SH2 domain. The interaction of Vav2 with EphA2 is crucial for EphA2-mediated tumor angiogenesis. Here we show that Vav2–SH2 domain is a lipid-binding module that can recognize PI(4,5)P2 and PI(3,4,5)P3 lipids weakly but specifically. The specific lipid-binding site in Vav2–SH2 domain was identified by NMR chemical shift perturbation experiments using the head groups of PI(4,5)P2 and PI(3,4,5)P3, both of which bind to Vav2–SH2 with millimolar binding affinities. In addition, the interaction between Vav2–SH2 and the phosphorylated juxtamembrane region (JM) of EphA2 (Y594 phosphorylated) was investigated using NMR techniques. Furthermore, by using a nickel–lipid containing peptide-based nanodiscs system, we studied the binding of Vav2–SH2 to the phosphorylated JM region of EphA2 on lipid membrane and uncovered a role of membrane environment in modulating this protein–protein recognition.

scholarly journals Induction of cell retraction by the combined actions of Abl–CrkII and Rho–ROCK1 signaling

2008 ◽  
Vol 183 (4) ◽  
pp. 711-723 ◽  
Author(s):  
XiaoDong Huang ◽  
Diana Wu ◽  
Hua Jin ◽  
Dwayne Stupack ◽  
Jean Y.J. Wang
Keyword(s):  
Nucleotide Exchange ◽  
Downstream Target ◽  
Abl Tyrosine Kinase ◽  
Guanine Nucleotide Exchange ◽  
Cell Matrix ◽  
Nucleotide Exchange Factor ◽  
Wide Range ◽  
Important Regulator ◽  
Guanosine Triphosphatase ◽  
Cell Retraction

Dynamic modulation of cell adhesion is integral to a wide range of biological processes. The small guanosine triphosphatase (GTPase) Rap1 is an important regulator of cell–cell and cell–matrix adhesions. We show here that induced expression of activated Abl tyrosine kinase reduces Rap1-GTP levels through phosphorylation of Tyr221 of CrkII, which disrupts interaction of CrkII with C3G, a guanine nucleotide exchange factor for Rap1. Abl-dependent down-regulation of Rap1-GTP causes cell rounding and detachment only when the Rho–ROCK1 pathway is also activated, for example, by lysophosphatidic acid (LPA). During ephrin-A1–induced retraction of PC3 prostate cancer cells, we show that endogenous Abl is activated and disrupts the CrkII–C3G complex to reduce Rap1-GTP. Interestingly, ephrin-A1–induced PC3 cell retraction also requires LPA, which stimulates Rho to a much higher level than that is activated by ephrin-A1. Our results establish Rap1 as another downstream target of the Abl–CrkII signaling module and show that Abl–CrkII collaborates with Rho–ROCK1 to stimulate cell retraction.

scholarly journals Coordination of Cytokinesis and Cell Separation by Endosomal Targeting of a Cdc42-specific Guanine Nucleotide Exchange Factor in Ustilago maydis

2009 ◽  
Vol 20 (3) ◽  
pp. 1081-1088 ◽  
Author(s):  
Kay Oliver Schink ◽  
Michael Bölker
Keyword(s):  
Cell Separation ◽  
Guanine Nucleotide Exchange Factor ◽  
Ustilago Maydis ◽  
Lipid Binding ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Fyve Domain ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The small GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in most eukaryotic cells. In Ustilago maydis, Cdc42 and the guanine nucleotide exchange factor (GEF) Don1 regulate cytokinesis and cell separation. Don1 belongs to the FGD1 family of Cdc42-specific GEFs that are characterized by a C-terminal lipid-binding FYVE domain. Although the FGD1/frabin family of Rho-GEFs is evolutionary conserved from fungi to mammals the role of the FYVE domain for its biological function is unknown. Here, we show that the FYVE domain is specific for phosphatidylinositol-3-phosphate (PtdIns(3)P) and targets Don1 to endosomal vesicles. During cytokinesis asymmetric accumulation of Don1-containing vesicles occurs at the site of septation. We could show that FYVE-dependent localization is critical for the function of Don1 at normal expression levels but can be compensated for by overexpression of Don1 lacking a functional FYVE domain. Our results demonstrate that endosomal compartmentalization of a Cdc42-specific exchange factor is involved in the coordination of cytokinesis and cell separation.

scholarly journals Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells

2017 ◽  
Vol 28 (10) ◽  
pp. 1347-1360 ◽  
Author(s):  
Brian J. Rosenberg ◽  
Hava Gil-Henn ◽  
Christopher C. Mader ◽  
Tiffany Halo ◽  
Taofei Yin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Invasive Breast Cancer ◽  
Breast Cancer Cells ◽  
Sh2 Domain ◽  
Guanine Nucleotide ◽  
Matrix Degradation ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Breast carcinoma cells use specialized, actin-rich protrusions called invadopodia to degrade and invade through the extracellular matrix. Phosphorylation of the actin nucleation–promoting factor and actin-stabilizing protein cortactin downstream of the epidermal growth factor receptor–Src-Arg kinase cascade is known to be a critical trigger for invadopodium maturation and subsequent cell invasion in breast cancer cells. The functions of cortactin phosphorylation in this process, however, are not completely understood. We identify the Rho-family guanine nucleotide exchange factor Vav2 in a comprehensive screen for human SH2 domains that bind selectively to phosphorylated cortactin. We demonstrate that the Vav2 SH2 domain binds selectively to phosphotyrosine-containing peptides corresponding to cortactin tyrosines Y421 and Y466 but not to Y482. Mutation of the Vav2 SH2 domain disrupts its recruitment to invadopodia, and an SH2-domain mutant form of Vav2 cannot support efficient matrix degradation in invasive MDA-MB-231 breast cancer cells. We show that Vav2 function is required for promoting invadopodium maturation and consequent actin polymerization, matrix degradation, and invasive migratory behavior. Using biochemical assays and a novel Rac3 biosensor, we show that Vav2 promotes Rac3 activation at invadopodia. Rac3 knockdown reduces matrix degradation by invadopodia, whereas a constitutively active Rac3 can rescue the deficits in invadopodium function in Vav2-knockdown cells. Together these data indicate that phosphorylated cortactin recruits Vav2 to activate Rac3 and promote invadopodial maturation in invasive breast cancer cells.

scholarly journals The Rab32/BLOC-3 dependent pathway mediates host- defence against different pathogens in human macrophages

2019 ◽  
Author(s):  
Massimiliano Baldassarre ◽  
Virtu Solano-Collado ◽  
Arda Balci ◽  
Rosa A. Colamarino ◽  
Ivy M Dambuza ◽  
...  
Keyword(s):  
Oxidative Burst ◽  
Mammalian Species ◽  
Host Defence ◽  
Intracellular Pathogens ◽  
Nucleotide Exchange ◽  
Human Macrophages ◽  
Guanine Nucleotide Exchange ◽  
Trafficking Pathway ◽  
Nucleotide Exchange Factor ◽  
Wide Range

ABSTRACTMacrophages provide a first line of defence against microorganisms, and while some mechanisms to kill pathogens such as the oxidative burst are well described, others are still undefined or unknown. Here we report that the Rab32 GTPase and its guanine nucleotide exchange factor BLOC-3 are central components of a trafficking pathway that controls both bacterial and fungal intracellular pathogens. This broad host-defence mechanism is active in both human and murine macrophages and is independent of well known antimicrobial mechanisms such as the NADPH-dependent oxidative burst, production of nitric oxide and antimicrobial peptides. To survive in human macrophages, Salmonella Typhi actively counteracts the Rab32/BLOC-3 pathway through its Salmonella pathogenicity island-1-encoded type III secretion system. These findings demonstrate that the Rab32/BLOC-3 pathway is a novel and universal host-defence pathway and protects mammalian species from a wide range of intracellular pathogens.

scholarly journals GrpL, a Grb2-related Adaptor Protein, Interacts with SLP-76 to Regulate Nuclear Factor of Activated T Cell Activation

1999 ◽  
Vol 189 (8) ◽  
pp. 1243-1253 ◽  
Author(s):  
Che-Leung Law ◽  
Maria K. Ewings ◽  
Preet M. Chaudhary ◽  
Sasha A. Solow ◽  
Theodore J. Yun ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Nuclear Factor ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain–containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL). Expression of GrpL is restricted to hematopoietic tissues, and it is distinguished from Grb2 by having a proline-rich region. GrpL can be coimmunoprecipitated with SLP-76 but not with Sos1 or Sos2 from Jurkat cell lysates. In contrast, Grb2 can be coimmunoprecipitated with Sos1 and Sos2 but not with SLP-76. Moreover, tyrosine-phosphorylated LAT/pp36/38 in detergent lysates prepared from anti-CD3 stimulated T cells associated with Grb2 but not GrpL. These data reveal the presence of distinct complexes involving GrpL and Grb2 in T cells. A functional role of the GrpL–SLP-76 complex is suggested by the ability of GrpL to act alone or in concert with SLP-76 to augment NF-AT activation in Jurkat T cells.

scholarly journals The Src Homology 2 Domain of Vav Is Required for Its Compartmentation to the Plasma Membrane and Activation of C-Jun Nh2-Terminal Kinase 1

2000 ◽  
Vol 191 (1) ◽  
pp. 47-60 ◽  
Author(s):  
Ramachandran Arudchandran ◽  
Martin J. Brown ◽  
Matthew J. Peirce ◽  
James S. Song ◽  
Juan Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Src Homology 2 ◽  
Nucleotide Exchange Factor ◽  
Downstream Effectors ◽  
Src Homology ◽  
Src Homology 2 Domain

Vav is a hematopoietic cell–specific guanine nucleotide exchange factor (GEF) whose activation is mediated by receptor engagement. The relationship of Vav localization to its function is presently unclear. We found that Vav redistributes to the plasma membrane in response to Fc∈ receptor I (Fc∈RI) engagement. The redistribution of Vav was mediated by its Src homology 2 (SH2) domain and required Syk activity. The Fc∈RI and Vav were found to colocalize and were recruited to glycosphingolipid-enriched microdomains (GEMs). The scaffold protein, linker for activation of T cells (LAT), and Rac1 (a target of Vav activity) were constitutively present in GEMs. Expression of an SH2 domain–containing COOH-terminal fragment of Vav inhibited Vav phosphorylation and movement to the GEMs but had no effect on the tyrosine phosphorylation of the adaptor protein, SLP-76 (SH2 domain–containing leukocyte protein of 76 kD), and LAT. However, assembly of the multiprotein complex containing these proteins was inhibited. In addition, Fc∈RI-dependent activation of c-Jun NH2-terminal kinase 1 (JNK1) was also inhibited. Thus, Vav localization to the plasma membrane is mediated by its SH2 domain and may serve to regulate downstream effectors like JNK1.

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