scholarly journals Phosphorylation of N-Cadherin-associated Cortactin by Fer Kinase Regulates N-Cadherin Mobility and Intercellular Adhesion Strength

2005 ◽  
Vol 16 (12) ◽  
pp. 5514-5527 ◽  
Author(s):  
Tarek Y. El Sayegh ◽  
Pamela D. Arora ◽  
Lingzhi Fan ◽  
Carol A. Laschinger ◽  
Peter A. Greer ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Adhesion Strength ◽  
Fluorescence Recovery After Photobleaching ◽  
Kinase Activity ◽  
Intercellular Adhesion ◽  
Wild Type ◽  
Nonreceptor Tyrosine Kinase ◽  
Junction Formation ◽  
Intercellular Adhesions ◽  
Dependent Mechanism

Cortactin regulates the strength of nascent N-cadherin-mediated intercellular adhesions through a tyrosine phosphorylation-dependent mechanism. Currently, the functional significance of cortactin phosphorylation and the kinases responsible for the regulation of adhesion strength are not defined. We show that the nonreceptor tyrosine kinase Fer phosphorylates cadherin-associated cortactin and that this process is involved in mediating intercellular adhesion strength. In wild-type fibroblasts N-cadherin ligation-induced transient phosphorylation of Fer, indicating that junction formation activates Fer kinase. Tyrosine phosphorylation of cortactin after N-cadherin ligation was strongly reduced in fibroblasts expressing only catalytically inactive Fer (D743R), compared with wild-type cells. In wild-type cells, N-cadherin-coated bead pull-off assays induced fourfold greater endogenous N-cadherin association than in D743R cells. Fluorescence recovery after photobleaching showed that GFP-N-cadherin mobility at nascent contacts was 50% faster in wild-type than D743R cells. In shear wash-off assays, nascent intercellular adhesion strength was twofold higher in wild-type than D743R cells. Cortactin recruitment to adhesions was independent of Fer kinase activity, but was impacted by N-cadherin ligation-provoked Rac activation. We conclude that N-cadherin ligation induces Rac-dependent cortactin recruitment and Fer-dependent cortactin phosphorylation, which in turn promotes enhanced mobilization and interaction of surface expressed N-cadherin in contacting cells.

scholarly journals Phosphatidylinositol-4,5 Bisphosphate Produced by PIP5KIγ Regulates Gelsolin, Actin Assembly, and Adhesion Strength of N-Cadherin Junctions

2007 ◽  
Vol 18 (8) ◽  
pp. 3026-3038 ◽  
Author(s):  
T. Y. El Sayegh ◽  
P. D. Arora ◽  
K. Ling ◽  
C. Laschinger ◽  
P. A. Janmey ◽  
...  
Keyword(s):  
Adhesion Strength ◽  
Intercellular Adhesion ◽  
Actin Binding ◽  
Type I ◽  
Actin Assembly ◽  
Wild Type ◽  
Binding Region ◽  
A Cell ◽  
Intercellular Adhesions ◽  
Phosphatidylinositol 4

Phosphoinositides regulate several actin-binding proteins but their role at intercellular adhesions has not been defined. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was generated at sites of N-cadherin–mediated intercellular adhesion and was a critical regulator of intercellular adhesion strength. Immunostaining for PI(4,5)P2 or transfection with GFP-PH-PLCδ showed that PI(4,5)P2 was enriched at sites of N-cadherin adhesions and this enrichment required activated Rac1. Isoform-specific immunostaining for type I phosphatidylinositol 4-phosphate 5 kinase (PIP5KI) showed that PIP5KIγ was spatially associated with N-cadherin–Fc beads. Association of PIP5KIγ with N-cadherin adhesions was in part dependent on the activation of RhoA. Transfection with catalytically inactive PIP5KIγ blocked the enrichment of PI(4,5)P2 around beads. Catalytically inactive PIP5KIγ or a cell-permeant peptide that mimics and competes for the PI(4,5)P2-binding region of the actin-binding protein gelsolin inhibited incorporation of actin monomers in response to N-cadherin ligation and reduced intercellular adhesion strength by more than twofold. Gelsolin null fibroblasts transfected with a gelsolin severing mutant containing an intact PI(4,5)P2 binding region, demonstrated intercellular adhesion strength similar to wild-type transfected controls. We conclude that PIP5KIγ-mediated generation of PI(4,5)P2 at sites of N-cadherin contacts regulates intercellular adhesion strength, an effect due in part to PI(4,5)P2-mediated regulation of gelsolin.

scholarly journals Regulation of the Rho Family Small GTPase Wrch-1/RhoU by C-Terminal Tyrosine Phosphorylation Requires Src

2010 ◽  
Vol 30 (17) ◽  
pp. 4324-4338 ◽  
Author(s):  
Jamie K. Alan ◽  
Anastacia C. Berzat ◽  
Brian J. Dewar ◽  
Lee M. Graves ◽  
Adrienne D. Cox
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Three Dimensional ◽  
Small Gtpase ◽  
Cell Morphogenesis ◽  
New Paradigm ◽  
Nonreceptor Tyrosine Kinase ◽  
Serum Stimulation ◽  
Rho Family

ABSTRACT Wrch-1 is an atypical Rho family small GTPase with roles in migration, epithelial cell morphogenesis, osteoclastogenesis, and oncogenic transformation. Here, we observed rapid relocalization of Wrch-1 from the plasma membrane upon serum stimulation. Studies revealed a requirement for serum-stimulated tyrosine phosphorylation of Wrch-1 at residue Y254 within its C-terminal membrane targeting domain, mediated by the nonreceptor tyrosine kinase Src. Genetic or pharmacological loss of Src kinase activity blocked both phosphorylation and relocalization of Wrch-1. Functionally, Y254 was required for proper Wrch-1 modulation of cystogenesis in three-dimensional culture, and the phospho-deficient mutant, Y254F, was enhanced in Wrch-1-mediated anchorage-independent growth. Mechanistically, C-terminal tyrosine phosphorylation and subsequent relocalization of Wrch-1 downregulated its ability to interact with and activate its effectors by decreasing active Wrch-1-GTP, perhaps by altering proximity to a GEF or GAP. Phospho-deficient Wrch-1(Y254F) remained at the plasma membrane and GTP bound and continued to recruit and activate its effector PAK, even upon serum stimulation. In contrast, a phospho-mimetic mutant, Y254E, was constitutively endosomally localized and GDP bound and failed to recruit PAK unless mutated to be constitutively active/GAP insensitive. C-terminal tyrosine phosphorylation thus represents a new paradigm in posttranslational control of small GTPase localization, activation, and biological function.

scholarly journals Inhibition of Bcr serine kinase by tyrosine phosphorylation.

1996 ◽  
Vol 16 (3) ◽  
pp. 998-1005 ◽  
Author(s):  
J Liu ◽  
Y Wu ◽  
G Z Ma ◽  
D Lu ◽  
L Haataja ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Philadelphia Chromosome ◽  
Wild Type ◽  
Serine Kinase ◽  
Serine Threonine Kinase ◽  
Threonine Kinase

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

scholarly journals Functional analysis of Csk in signal transduction through the B-cell antigen receptor

1994 ◽  
Vol 14 (11) ◽  
pp. 7306-7313
Author(s):  
A Hata ◽  
H Sabe ◽  
T Kurosaki ◽  
M Takata ◽  
H Hanafusa
Keyword(s):  
Signal Transduction ◽  
B Cells ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
B Cell Antigen Receptor ◽  
Wild Type ◽  
Inactive State ◽  
Cell Clones ◽  
Bcr Signaling

In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.

scholarly journals Erythropoietin induces tyrosine phosphorylation and kinase activity of the c-fps/fes proto-oncogene product in human erythropoietin-responsive cells

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3193-3196 ◽  
Author(s):  
Y Hanazono ◽  
S Chiba ◽  
K Sasaki ◽  
H Mano ◽  
Y Yazaki ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Protein S ◽  
Receptor Activation ◽  
Kinase Domain ◽  
Cellular Protein ◽  
Erythroid Progenitor ◽  
Nonreceptor Tyrosine Kinase ◽  
Human Erythropoietin

Erythropoietin (EPO) is a hematopoietic growth factor that stimulates the proliferation and differentiation of erythroid progenitor cells. Although the EPO receptor has no kinase domain, EPO rapidly induces tyrosine phosphorylation of several proteins in EPO-responsive cells. Therefore, the receptor activation by the ligand could induce tyrosine- kinase activity of unidentified cellular protein(s). Here we show that c-fps/fes proto-oncogene product (p92c-fes), nonreceptor tyrosine kinase, is tyrosine-phosphorylated on treatment with EPO in a human erythroleukemia cell line TF-1 that is responsive to granulocyte- macrophage colony-stimulating factor, interleukin-3, and EPO. In addition, the kinase activity of p92c-fes was shown to be enhanced by treatment with EPO. Therefore, p92c-fes could be implicated in a signaling pathway triggered by EPO in human EPO-responsive cells.

ZAP-70 Enhances IgM Signaling in Chronic Lymphocytic Leukemia Cells Independent of Its Tyrosine Kinase Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 585-585 ◽  
Author(s):  
Liguang Chen ◽  
Lang Huynh ◽  
Arthur Weiss ◽  
Thomas J. Kipps
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Protein Tyrosine Phosphorylation ◽  
Wild Type ◽  
Tyrosine Kinase Activity ◽  
Bcr Signaling

Abstract We found that expression of the zeta-associated protein of 70 kD (ZAP-70) by chronic lymphocytic leukemia (CLL) B cells enhanced IgM-receptor signaling, even though such CLL cells also expressed a highly-related and more active tyrosine kinase, p72Syk. To investigate whether the kinase activity of ZAP-70 is necessary for this effect, we transfected ZAP-70-negative primary leukemia cells with expression vectors encoding either wild-type ZAP-70 or ZAP-70-KI, a mutant ZAP-70 that has an inactivating point mutation in the ATP-binding site (Lys369-Ala) and that lacks tyrosine kinase activity. We achieved high-level expression of ZAP-70 or ZAP-70-KI in transfected CLL cell samples and compared these cells with each other and with the same CLL cell samples that had been mock transfected with a control vector and that remained negative for expression of ZAP-70 (n = 7). To examine the B-cell-receptor (BCR) signaling potential of these cells we assessed the extent of tyrosine phosphorylation of p72Syk, B-cell linker protein (BLNK), and phospholipase C gamma (PLC-γ), and measured intracellular calcium flux ([Ca2+]I) before and 5–10 minutes after surface IgM ligation with F(ab)2 anti- μantibody. Treatment of control mock-transfected ZAP-70-negative CLL cells with anti-μ resulted in negligible-to-minimal increases in phosphorylation of these cytosolic proteins, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of only 46% ± 47% (S.D), 173% ± 112%, and 73% ± 76%, respectively. CLL cells engineered to express non-mutated ZAP-70 experienced significantly higher levels of protein tyrosine phosphorylation following treatment with anti-μ, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of 186% ± 102%, 490% ± 323%, and 722% ± 836%, respectively, as had been noted in earlier studies. Surprisingly, CLL cells made to express the tyrosine-kinase-defective mutant ZAP-70KI also experienced significantly higher levels of protein tyrosine phosphorylation following treatment with anti-μ than did the mock-transfected CLL cells, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of 172% ± 94%, 431% ± 261%, and 759% ± 637%, respectively. These values were similar to those noted for anti-μ treated CLL cells that expressed the wild-type ZAP-70 protein. These results were reflected also in the ([Ca2+]I) induced by anti-μ in each of the three groups of CLL cells. Whereas anti-μ treatment of mock-transfected CLL cells resulted in negligible-to-minimal increases in ([Ca2+]I) of 0.25 units ± 0.19, anti-μ treatment of ZAP-70-transfected or ZAP-70KI-transfected CLL cells resulted in increases in ([Ca2+]I) of 1.05 units ± 0.53 and 0.95 units ± 0.46, respectively. These values each were significantly higher than that noted for mock-transfected CLL cells. We conclude that the tyrosine-kinase activity of ZAP-70 is not required for ZAP-70 to enhance BCR signaling in CLL cells.

Erythropoietin induces tyrosine phosphorylation and kinase activity of the c-fps/fes proto-oncogene product in human erythropoietin-responsive cells

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3193-3196 ◽  
Author(s):  
Y Hanazono ◽  
S Chiba ◽  
K Sasaki ◽  
H Mano ◽  
Y Yazaki ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Protein S ◽  
Receptor Activation ◽  
Kinase Domain ◽  
Cellular Protein ◽  
Erythroid Progenitor ◽  
Nonreceptor Tyrosine Kinase ◽  
Human Erythropoietin

Abstract Erythropoietin (EPO) is a hematopoietic growth factor that stimulates the proliferation and differentiation of erythroid progenitor cells. Although the EPO receptor has no kinase domain, EPO rapidly induces tyrosine phosphorylation of several proteins in EPO-responsive cells. Therefore, the receptor activation by the ligand could induce tyrosine- kinase activity of unidentified cellular protein(s). Here we show that c-fps/fes proto-oncogene product (p92c-fes), nonreceptor tyrosine kinase, is tyrosine-phosphorylated on treatment with EPO in a human erythroleukemia cell line TF-1 that is responsive to granulocyte- macrophage colony-stimulating factor, interleukin-3, and EPO. In addition, the kinase activity of p92c-fes was shown to be enhanced by treatment with EPO. Therefore, p92c-fes could be implicated in a signaling pathway triggered by EPO in human EPO-responsive cells.

scholarly journals Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers

2010 ◽  
Vol 299 (1) ◽  
pp. G186-G195 ◽  
Author(s):  
Shyamali Basuroy ◽  
Mitzi Dunagan ◽  
Parimal Sheth ◽  
Ankur Seth ◽  
R. K. Rao
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Cell Migration ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Src Kinase ◽  
Pi3 Kinase ◽  
Cell Monolayers ◽  
Dependent Mechanism

Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl]pyrazolo[3–4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-SrcY527F enhanced cell migration, whereas the expression of dominant negative c-SrcK296R/Y528F attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130CAS in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism.

scholarly journals Nonreceptor Tyrosine Kinase c-Yes Interacts with Occludin during Tight Junction Formation in Canine Kidney Epithelial Cells

2002 ◽  
Vol 13 (4) ◽  
pp. 1227-1237 ◽  
Author(s):  
Yan-Hua Chen ◽  
Qun Lu ◽  
Daniel A. Goodenough ◽  
Beverly Jeansonne
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tight Junction ◽  
Tyrosine Phosphorylation ◽  
Nonreceptor Tyrosine Kinase ◽  
Tight Junction Formation ◽  
Kinase C ◽  
Junction Formation ◽  
Kidney Epithelial Cells ◽  
Canine Kidney

Occludin is an integral membrane protein that is tyrosine phosphorylated when localized at tight junctions. When Ca2+was depleted from the culture medium, occludin tyrosine phosphorylation was diminished from Madin-Darby canine kidney epithelial cells in 2 min. This dephosphorylation was correlated with a significant reduction in transepithelial electrical resistance (TER), indicating a global loss of the tight junction barrier function. Reconstitution of Ca2+ resulted in a robust tyrosine rephosphorylation of occludin that was temporally associated with an increase in TER. Moreover, we demonstrate in this study that occludin was colocalized with the nonreceptor tyrosine kinase c-Yes at cell junction areas and formed an immunoprecipitable complex with c-Yes in vivo. This complex dissociated when the cells were incubated in medium without Ca2+ or treated with a c-Yes inhibitor, CGP77675. In the presence of CGP77675 after Ca2+ repletion, occludin tyrosine phosphorylation was completely abolished and both tight junction formation and the increase of the TER were inhibited. Our study thus provides strong evidence that occludin tyrosine phosphorylation is tightly linked to tight junction formation in epithelial cells, and that the nonreceptor tyrosine kinase c-Yes is involved in the regulation of this process.

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