scholarly journals Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption

2003 ◽  
Vol 116 (6) ◽  
pp. 975-986 ◽  
Author(s):  
J. Kirchner
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesions ◽  
Live Cell ◽  
Microtubule Disruption ◽  
Cell Monitoring

Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK

1993 ◽  
Vol 13 (2) ◽  
pp. 785-791
Author(s):  
M D Schaller ◽  
C A Borgman ◽  
J T Parsons
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Signal Transduction Pathway ◽  
Focal Adhesions ◽  
Cellular Adhesion ◽  
Protein Tyrosine ◽  
Intracellular Signals ◽  
Embryo Cells

Integrins play a central role in cellular adhesion and anchorage of the cytoskeleton and participate in the generation of intracellular signals, including tyrosine phosphorylation. We have recently isolated a cDNA encoding a unique, focal adhesion-associated protein tyrosine kinase (FAK) that is a component of an integrin-mediated signal transduction pathway. Here we report the isolation of cDNAs encoding the C-terminal, noncatalytic domain of the FAK kinase, termed FRNK (FAK-related nonkinase). Both the FAK- and FRNK-encoded polypeptides, pp125FAK and p41/p43FRNK, are expressed in normal chicken embryo cells. pp125FAK and p41/p43FRNK were localized to focal adhesions, suggesting that pp125FAK is directed to the focal adhesions by sequences within its C-terminal domain. We also show that the fibronectin-dependent increase in tyrosine phosphorylation of pp125FAK is accompanied by a concomitant posttranslational modification of p41FRNK.

Ca2+ channels mediate protein tyrosine kinase activation by endothelin-1

1996 ◽  
Vol 270 (5) ◽  
pp. F790-F797 ◽  
Author(s):  
M. S. Simonson ◽  
Y. Wang ◽  
W. H. Herman
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Mesangial Cells ◽  
Focal Adhesions ◽  
Cellular Protein ◽  
Early Gene ◽  
Glomerular Mesangial Cells ◽  
Protein Tyrosine ◽  
Endothelin 1 ◽  
Ca2 Channels

To investigate the novel interaction between endothelin-1 (ET-1) and cellular protein tyrosine kinases (PTK), we asked whether Ca2+ influx links ET-1 receptors to PTK activation. In glomerular mesangial cells, ET-1 stimulated a biphasic increase in PTK activity in anti-phosphotyrosine immunoprecipitates that temporally correlated with increased tyrosine phosphorylation of cellular proteins. ET-1 increased tyrosine phosphorylation of proteins in the cytosol and in a puncture distribution consistent with focal adhesions. Addition of ionomycin to increase Ca2+ influx stimulated PTK activity, and inhibition of extracellular Ca2+ influx blocked PTK activation by ET-1. ET-1 increased autophosphorylation of pp60c-src, which was mimicked by addition of ionomycin and inhibited by chelation of extracellular Ca2+. In addition, a selective PTK inhibitor blocked induction of c-fos mRNA by ionomycin, suggesting that Ca(2+)-stimulated PTKs contribute to a signaling pathway regulating immediate early gene expression. Taken together, these results demonstrate that ET-1 stimulates nonreceptor PTK activity, including pp60c-src, by activating Ca2+ channels and subsequent influx of extracellular Ca2+.

Role of tyrosine kinase pathways in ETB receptor activation of NHE3

1996 ◽  
Vol 271 (3) ◽  
pp. C763-C771 ◽  
Author(s):  
T. S. Chu ◽  
H. Tsuganezawa ◽  
Y. Peng ◽  
A. Cano ◽  
M. Yanagisawa ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Integral Membrane Protein ◽  
Receptor Activation ◽  
Kinase C ◽  
Focal Adhesion Proteins ◽  
Etb Receptor

Endothelin-1 (ET-1) binding to ETB receptors increases the activity of the apical membrane Na+/H+ antiporter (NHE3) of renal proximal tubule and cultured OKP cells. In OKPETB6 cells, a clonal cell line of OKP cells that overexpresses ETB receptors, ET-1-induced increases in Na+/H+ antiporter activity are mediated 50% by Ca2(+)-dependent pathways and 50% by tyrosine kinase pathways. ET-1 induces tyrosine phosphorylation of proteins of 68, 110, 125, 130, and 210 kDa. ET-1-induced tyrosine phosphorylation is mediated by the ETB receptor and is not dependent on increases in cell Ca2+ or protein kinase C. The 68-, 110-, 125-, and 130-kDa phosphoproteins are cytosolic, whereas the 210-kDa phosphoprotein is an integral membrane protein. Immunoprecipitation studies showed that the 68-kDa protein is paxillin and the 125-kDa protein is p125FAK (focal adhesion kinase). Cytochalasin D, which disrupts focal adhesions, prevented ET-1-induced tyrosine phosphorylation of paxillin, p110, p125FAK, and p130 but did not prevent tyrosine phosphorylation of p210 and did not prevent ET-1-induced increases in Na+/H+ antiporter activity. Thus 50% of ETB receptor-induced Na+/H+ antiporter activation is mediated by tyrosine kinase pathways, possibly involving p210. ETB receptor activation also induces tyrosine phosphorylation of focal adhesion proteins, but this is not required for antiporter activation.

scholarly journals Live Cell Monitoring of μ-Opioid Receptor-mediated G-protein Activation Reveals Strong Biological Activity of Close Morphine Biosynthetic Precursors

2007 ◽  
Vol 282 (37) ◽  
pp. 27126-27132 ◽  
Author(s):  
Viacheslav O. Nikolaev ◽  
Chotima Boettcher ◽  
Christian Dees ◽  
Moritz Bünemann ◽  
Martin J. Lohse ◽  
...  
Keyword(s):  
Biological Activity ◽  
G Protein ◽  
Opioid Receptor ◽  
Live Cell ◽  
Protein Activation ◽  
G Protein Activation ◽  
Cell Monitoring ◽  
Μ Opioid Receptor

scholarly journals pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk.

1995 ◽  
Vol 15 (5) ◽  
pp. 2635-2645 ◽  
Author(s):  
M D Schaller ◽  
J T Parsons
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Focal Adhesions ◽  
Sh2 Domain ◽  
Binding Motif ◽  
Adapter Protein ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
High Affinity Binding Site

Paxillin, a focal-adhesion-associated protein, becomes phosphorylated in response to a number of stimuli which also induce the tyrosine phosphorylation of the focal-adhesion-associated protein tyrosine kinase pp125FAK. On the basis of their colocalization and coordinate phosphorylation, paxillin is a candidate for a substrate of pp125FAK. We describe here conditions under which the phosphorylation of paxillin on tyrosine is pp125FAK dependent, supporting the hypothesis that paxillin phosphorylation is regulated by pp125FAK. pp125FAK must localize to focal adhesions and become autophosphorylated to induce paxillin phosphorylation. Phosphorylation of paxillin on tyrosine creates binding sites for the SH2 domains of Crk, Csk, and Src. We identify two sites of phosphorylation as tyrosine residues 31 and 118, each of which conforms to the Crk SH2 domain binding motif, (P)YXXP. These observations suggest that paxillin serves as an adapter protein, similar to insulin receptor substrate 1, and that pp125FAK may regulate the formation of signaling complexes by directing the phosphorylation of paxillin on tyrosine.

scholarly journals Hormonal regulation of focal adhesions in bovine adrenocortical cells: induction of paxillin dephosphorylation by adrenocorticotropic hormone

1998 ◽  
Vol 332 (2) ◽  
pp. 533-540 ◽  
Author(s):  
Isabelle VILGRAIN ◽  
Anna CHINN ◽  
Isabelle GAILLARD ◽  
Edmond M. CHAMBAZ ◽  
Jean-Jacques FEIGE
Keyword(s):  
Tyrosine Phosphorylation ◽  
Hormonal Regulation ◽  
Adrenocorticotropic Hormone ◽  
Focal Adhesions ◽  
Insoluble Fraction ◽  
Dependent Manner ◽  
Sodium Orthovanadate ◽  
Adrenocortical Cells ◽  
Acth Challenge ◽  
Phosphatase Inhibitor

A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes.

scholarly journals Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs

PLoS ONE ◽  
2010 ◽  
Vol 5 (7) ◽  
pp. e11834 ◽  
Author(s):  
Masakazu Kamata ◽  
Min Liang ◽  
Shirley Liu ◽  
Yoshiko Nagaoka ◽  
Irvin S. Y. Chen
Keyword(s):  
Differential Expression ◽  
Live Cell ◽  
Cell Monitoring

scholarly journals Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells.

1996 ◽  
Vol 7 (3) ◽  
pp. 415-423
Author(s):  
D A Troyer ◽  
A Bouton ◽  
R Bedolla ◽  
R Padilla
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Actin Filaments ◽  
Mesangial Cells ◽  
Close Contact ◽  
Focal Adhesions ◽  
Cytochalasin D ◽  
Fiber Formation ◽  
Stress Fibers

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.

scholarly journals Beta 3 integrin-mediated fibrin clot retraction by nucleated cells: differing behavior of alpha IIb beta 3 and alpha v beta 3

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2606-2615 ◽  
Author(s):  
YP Chen ◽  
TE O'Toole ◽  
L Leong ◽  
BQ Liu ◽  
F Diaz-Gonzalez ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Tyrosine Phosphorylation ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Fibrin Clot ◽  
Clot Retraction ◽  
Functional Roles ◽  
Binding Function ◽  
Alpha V Beta 3

Fibrin clot retraction may be important in resolution of thrombi and, in platelets, is mediated by integrin alpha IIb beta 3 (GPIIb-IIIa). Nucleated cells that lack alpha IIb beta 3 can retract fibrin clots, and we now report that integrin alpha v beta 3 can support this process. In addition, we compared the capacities of recombinant beta 3 integrins to mediate clot retraction in Chinese hamster ovary and M21 melanoma cells. We found that alpha v beta 3, but not alpha IIb beta 3, could spontaneously support retraction. Transferring the cytoplasmic domain of alpha v to alpha IIb enabled the resulting chimeric alpha IIb beta 3 to support clot retraction. The capacity of the alpha v cytoplasmic domain to support clot retraction was not caused by activation of the ligand binding function of alpha IIb beta 3 or by enhancement of alpha IIb beta 32s capacity to stimulate the formation of focal adhesions or the tyrosine phosphorylation of pp125FAK. These experiments define requirements for alpha IIb beta 3-mediating clot retraction, establish the capacity of alpha v beta 3 to mediate this process, and suggest differing functional roles of the alpha v and alpha IIb cytoplasmic domains.

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