scholarly journals SH2-Containing Inositol 5′-Phosphatase SHIP2 Associates with the p130Cas Adapter Protein and Regulates Cellular Adhesion and Spreading

2001 ◽  
Vol 21 (4) ◽  
pp. 1416-1428 ◽  
Author(s):  
Nagendra Prasad ◽  
Robert S. Topping ◽  
Stuart J. Decker
Keyword(s):  
Catalytic Domain ◽  
Protein A ◽  
Cell Types ◽  
Cellular Adhesion ◽  
Sh2 Domain ◽  
Adapter Protein ◽  
Cytoskeleton Organization ◽  
Phosphorylated Form ◽  
Immunofluorescence Studies ◽  
Actin Cytoskeleton Organization

ABSTRACT In a previous study, we found that the SHIP2 protein became tyrosine phosphorylated and associated with the Shc adapter protein in response to the treatment of cells with growth factors and insulin (T. Habib, J. A. Hejna, R. E. Moses, and S. J. Decker, J. Biol. Chem. 273:18605–18609, 1998). We describe here a novel interaction between SHIP2 and the p130Cas adapter protein, a mediator of actin cytoskeleton organization. SHIP2 and p130Cas association was detected in anti-SHIP2 immunoprecipitates from several cell types. Reattachment of trypsinized cells stimulated tyrosine phosphorylation of SHIP2 and increased the formation of a complex containing SHIP2 and a faster-migrating tyrosine-phosphorylated form of p130Cas. The faster-migrating form of p130Cas was no longer recognized by antibodies to the amino terminus of p130Cas and appeared to be generated through proteolysis. Interaction of the SHIP2 protein with the various forms of p130Cas was mediated primarily through the SH2 domain of SHIP2. Immunofluorescence studies indicated that SHIP2 localized to focal contacts and to lamellipodia. Increased adhesion was observed in HeLa cells transiently expressing exogenous WT-SHIP2. These effects were not seen with SHIP2 possessing a mutation in the SH2 domain (R47G). Transfection of a catalytic domain deletion mutant of SHIP2 (ΔRV) inhibited cell spreading. Taken together, our studies suggest an important role for SHIP2 in adhesion and spreading.

scholarly journals Structural Requirements for Function of the Crkl Adapter Protein in Fibroblasts and Hematopoietic Cells

1998 ◽  
Vol 18 (9) ◽  
pp. 5082-5090 ◽  
Author(s):  
Kristen Senechal ◽  
Conor Heaney ◽  
Brian Druker ◽  
Charles L. Sawyers
Keyword(s):  
Hematopoietic Cells ◽  
Sh3 Domain ◽  
Cellular Adhesion ◽  
Sh2 Domain ◽  
Adapter Protein ◽  
Jun Kinase ◽  
Phosphopeptide Mapping ◽  
Fibroblast Transformation

ABSTRACT Crkl is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl oncogene and in signaling by cytokines. When phosphorylated, Crkl binds through its Src homology 2 (SH2) domain to other tyrosine phosphoproteins such as paxillin and Cbl. Overexpression of Crkl in fibroblasts induces transformation. Here we examine the role of Crkl in hematopoietic cells and find that overexpression of Crkl confers a signal leading to increased adhesion to fibronectin. In both fibroblasts and hematopoietic cells, individual mutations or deletions of each SH2 and SH3 domain abrogated transformation and adhesion, respectively, indicating that interactions with other proteins such as Cbl and paxillin (SH2 domain) and Abl, Sos, and C3G (N-terminal SH3 domain) are essential for biological activity. In vivo and in vitro tryptic phosphopeptide mapping studies show that Crkl is phosphorylated on multiple tyrosine residues when overexpressed or when activated by Bcr-Abl. Mutation at tyrosine 207, a residue conserved in c-Crk, abrogates all in vivo tyrosine phosphorylation of Crkl. Despite this loss of phosphotyrosine, mutation at this site enhanced Crkl function as measured by complex formation with SH2 binding proteins, signal transduction to Jun Kinase, and fibroblast transformation. These observations implicate Crkl in cellular adhesion and demonstrate that Y207 functions as a negative regulatory site.

scholarly journals Requisite Role for Nck Adaptors in Cardiovascular Development, Endothelial-to-Mesenchymal Transition, and Directed Cell Migration

2015 ◽  
Vol 35 (9) ◽  
pp. 1573-1587 ◽  
Author(s):  
Derek L. Clouthier ◽  
Cameron N. Harris ◽  
Richard A. Harris ◽  
Claire E. Martin ◽  
Mira C. Puri ◽  
...  
Keyword(s):  
Cell Types ◽  
Actin Dynamics ◽  
Cardiovascular Development ◽  
Vascular Networks ◽  
Mesenchymal Transition ◽  
Perivascular Cell ◽  
Cytoskeleton Organization ◽  
Directed Cell Migration ◽  
Actin Cytoskeleton Organization ◽  
Endothelial To Mesenchymal Transition

Development of the cardiovascular system is critically dependent on the ability of endothelial cells (ECs) to reorganize their intracellular actin architecture to facilitate migration, adhesion, and morphogenesis. Nck family cytoskeletal adaptors function as key mediators of actin dynamics in numerous cell types, though their role in EC biology remains largely unexplored. Here, we demonstrate an essential requirement for Nck within ECs. Mouse embryos lacking endothelial Nck1/2 expression develop extensive angiogenic defects that result in lethality at about embryonic day 10. Mutant embryos show immature vascular networks, with decreased vessel branching, aberrant perivascular cell recruitment, and reduced cardiac trabeculation. Strikingly, embryos deficient in endothelial Nck also fail to undergo the endothelial-to-mesenchymal transition (EnMT) required for cardiac valve morphogenesis, with loss of Nck disrupting expression of major EnMT markers, as well as suppressing mesenchymal outgrowth. Furthermore, we show that Nck-null ECs are unable to migrate downstream of vascular endothelial growth factor and angiopoietin-1, and they exhibit profound perturbations in cytoskeletal patterning, with disorganized cellular projections, impaired focal adhesion turnover, and disrupted actin-based signaling. Our collective findings thereby reveal a crucial role for Nck as a master regulator within the endothelium to control actin cytoskeleton organization, vascular network remodeling, and EnMT during cardiovascular development.

Requirement of phosphatidylinositol-3 kinase modification for its association with p60src

1991 ◽  
Vol 11 (4) ◽  
pp. 1972-1979
Author(s):  
Y Fukui ◽  
H Hanafusa
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Sh2 Domain ◽  
Protein Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Tyrosine Residues ◽  
Phosphorylated Form ◽  
Chicken Embryo Fibroblasts

When purified p60v-src was mixed with lysates of chicken embryo fibroblasts and immunoprecipitated with anti-Src antibody, phosphatidylinositol (PI)-3 kinase activity was found to be present in the Src protein immunoprecipitates. The level of bound PI-3 kinase activity was 5 to 10 times higher in lysates obtained from cells transformed by the src, fps, or yes oncogene than in lysates of uninfected cells. This increase in associated PI-3 kinase activity appears to be due to increased binding of this enzyme to p60v-src. This change most likely resulted from tyrosine phosphorylation of PI-3 kinase or an associated protein, since the PI-3 kinase activity that can bind to p60v-src was depleted by antiphosphotyrosine antibody. Binding of PI-3 kinase did not require either p60src protein kinase activity or autophosphorylation of p60v-src tyrosine residues. Furthermore, binding was markedly decreased by deletions in the N-terminal SH2 region but unchanged by deletion of the C-terminal half of p60v-src containing the catalytic domain. Taking these data together, it appears that PI-3 kinase or its associated protein is phosphorylated on tyrosine and that the phosphorylated form can bind to the N-terminal half of p60v-src, which contains the SH2 domain.

scholarly journals Requirement of phosphatidylinositol-3 kinase modification for its association with p60src.

1991 ◽  
Vol 11 (4) ◽  
pp. 1972-1979 ◽  
Author(s):  
Y Fukui ◽  
H Hanafusa
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Sh2 Domain ◽  
Protein Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Tyrosine Residues ◽  
Phosphorylated Form ◽  
Chicken Embryo Fibroblasts

When purified p60v-src was mixed with lysates of chicken embryo fibroblasts and immunoprecipitated with anti-Src antibody, phosphatidylinositol (PI)-3 kinase activity was found to be present in the Src protein immunoprecipitates. The level of bound PI-3 kinase activity was 5 to 10 times higher in lysates obtained from cells transformed by the src, fps, or yes oncogene than in lysates of uninfected cells. This increase in associated PI-3 kinase activity appears to be due to increased binding of this enzyme to p60v-src. This change most likely resulted from tyrosine phosphorylation of PI-3 kinase or an associated protein, since the PI-3 kinase activity that can bind to p60v-src was depleted by antiphosphotyrosine antibody. Binding of PI-3 kinase did not require either p60src protein kinase activity or autophosphorylation of p60v-src tyrosine residues. Furthermore, binding was markedly decreased by deletions in the N-terminal SH2 region but unchanged by deletion of the C-terminal half of p60v-src containing the catalytic domain. Taking these data together, it appears that PI-3 kinase or its associated protein is phosphorylated on tyrosine and that the phosphorylated form can bind to the N-terminal half of p60v-src, which contains the SH2 domain.

scholarly journals Phg2, a Kinase Involved in Adhesion and Focal Site Modeling in Dictyostelium

2004 ◽  
Vol 15 (8) ◽  
pp. 3915-3925 ◽  
Author(s):  
Leigh Gebbie ◽  
Mohammed Benghezal ◽  
Sophie Cornillon ◽  
Romain Froquet ◽  
Nathalie Cherix ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Genetic System ◽  
Cellular Adhesion ◽  
Specific Role ◽  
Cytoskeleton Organization ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Defective Mutant ◽  
Actin Cytoskeleton Organization

The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility.

Role of PKC isozymes in water transport in toad urinary bladder

1991 ◽  
Vol 49 ◽  
pp. 302-303
Author(s):  
A.J. Mia ◽  
L.X. Oakford ◽  
T. Yorio
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Membrane Surface ◽  
Protein A ◽  
Cell Types ◽  
Leukemic Cells ◽  
Toad Urinary Bladder ◽  
Pkc Isozymes ◽  
Antibody Labeling

Protein kinase C (PKC) isozymes, when activated, are translocated to particulate membrane fractions for transport to the apical membrane surface in a variety of cell types. Evidence of PKC translocation was demonstrated in human megakaryoblastic leukemic cells, and in cardiac myocytes and fibroblasts, using FTTC immunofluorescent antibody labeling techniques. Recently, we reported immunogold localizations of PKC subtypes I and II in toad urinary bladder epithelia, following 60 min stimulation with Mezerein (MZ), a PKC activator, or antidiuretic hormone (ADH). Localization of isozyme subtypes I and n was carried out in separate grids using specific monoclonal antibodies with subsequent labeling with 20nm protein A-gold probes. Each PKC subtype was found to be distributed singularly and in discrete isolated patches in the cytosol as well as in the apical membrane domains. To determine if the PKC isozymes co-localized within the cell, a double immunogold labeling technique using single grids was utilized.

scholarly journals Pan1p, End3p, and Sla1p, Three Yeast Proteins Required for Normal Cortical Actin Cytoskeleton Organization, Associate with Each Other and Play Essential Roles in Cell Wall Morphogenesis

2000 ◽  
Vol 20 (1) ◽  
pp. 12-25 ◽  
Author(s):  
Hsin-Yao Tang ◽  
Jing Xu ◽  
Mingjie Cai
Keyword(s):  
Cell Wall ◽  
Actin Cytoskeleton ◽  
Normal Cell ◽  
Cell Cortex ◽  
Repeat Region ◽  
Cortical Actin ◽  
Cytoskeleton Organization ◽  
Eh Domain ◽  
Actin Cytoskeleton Organization ◽  
Cell Wall Morphogenesis

ABSTRACT The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Δ, andend3Δ mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast.

scholarly journals Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain.

1990 ◽  
Vol 87 (12) ◽  
pp. 4849-4853 ◽  
Author(s):  
A. T. Brunger ◽  
M. V. Milburn ◽  
L. Tong ◽  
A. M. deVos ◽  
J. Jancarik ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Active Form ◽  
Protein A ◽  
Ras Protein
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