scholarly journals Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity.

1994 ◽  
Vol 126 (2) ◽  
pp. 575-588 ◽  
Author(s):  
C L Hall ◽  
C Wang ◽  
L A Lange ◽  
E A Turley
Keyword(s):  
Tyrosine Kinase ◽  
Cell Motility ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Peptide Sequence ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Cell Locomotion ◽  
Focal Adhesion Turnover ◽  
Stimulation Of

The molecular mechanisms whereby hyaluronan (HA) stimulates cell motility was investigated in a C-H-ras transformed 10T 1/2 fibroblast cell line (C3). A significant (p < 0.001) stimulation of C3 cell motility with HA (10 ng/ml) was accompanied by an increase in protein tyrosine phosphorylation as detected by anti-phosphotyrosine antibodies using immunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and transient with phosphorylation occurring within 1 min of HA addition and dissipating below control levels 10-15 min later. These responses were also elicited by an antibody generated against a peptide sequence within the HA receptor RHAMM. Treatment of cells with tyrosine kinase inhibitors (genistein, 10 micrograms/ml or herbimycin A, 0.5 micrograms/ml) or microinjection of anti-phosphotyrosine antibodies inhibited the transient protein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimulated tyrosine phosphorylation and the resulting cell locomotion, cytoskeletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapid assembly and disassembly of focal adhesions as revealed by immunofluorescent localization of vinculin. The time course with which HA and antibody induced focal adhesion turnover exactly paralleled the induction of transient protein tyrosine phosphorylation. In addition, phosphotyrosine staining colocalized with vinculin within structures in the lamellapodia of these cells. Notably, the focal adhesion kinase, pp125FAK, was rapidly phosphorylated and dephosphorylated after HA stimulation. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAMM. They also provide a possible molecular basis for focal adhesion turnover, a process that is critical for cell locomotion.

scholarly journals Stimulation of Fc gamma RIIIA results in phospholipase C-gamma 1 tyrosine phosphorylation and p56lck activation.

1992 ◽  
Vol 176 (6) ◽  
pp. 1745-1750 ◽  
Author(s):  
L Azzoni ◽  
M Kamoun ◽  
T W Salcedo ◽  
P Kanakaraj ◽  
B Perussia
Keyword(s):  
T Cells ◽  
Tyrosine Kinase ◽  
Nk Cells ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Tyrosine Residues ◽  
Stimulation Of

Binding of ligand to the alpha subunit of Fc gamma RIIIA(CD16), expressed at the natural killer (NK) cell membrane in association with homo or heterodimers of proteins of the zeta family, results in phosphorylation of several proteins on tyrosine residues. We have analyzed the role of protein tyrosine phosphorylation in the regulation of molecular events induced upon stimulation of Fc gamma RIIIA in NK cells and in T cells expressing the Fc gamma RIII alpha chain in association with endogenous zeta 2 homodimers and devoid of other (CD3, CD2) transducing molecules. Our data indicate that treatment of these cells with protein tyrosine kinase inhibitors prevents not only Fc gamma RIIIA-induced protein tyrosine phosphorylation but also phosphatidylinositol 4,5 diphosphate hydrolysis and increased intracellular Ca2+ concentration, indicating a primary role of tyrosine kinase(s) in the induction of these early activation events. Occupancy of Fc gamma RIIIA by ligand results in phospholipase C (PLC)-gamma 1 tyrosine phosphorylation in NK cells and in Fc gamma RIIIA-transfected CD3-/CD2- T cells, and induces functional activation of p56lck in Fc gamma RIIIA alpha/zeta 2-transfected T cells, suggesting the possibility that the receptor-induced PLC-gamma 1 activation occurs upon phosphorylation of its tyrosine residues mediated by this kinase and is, at least in part, responsible for the signal transduction mediated via CD16 upon ligand binding.

scholarly journals Haemin enhancement of glucose transport in human lymphocytes: stimulation of protein tyrosine phosphatase and activation of p56lck tyrosine kinase

1993 ◽  
Vol 291 (1) ◽  
pp. 281-287 ◽  
Author(s):  
H M Lander ◽  
D M Levine ◽  
A Novogrodsky
Keyword(s):  
Tyrosine Kinase ◽  
Glucose Uptake ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Human Lymphocytes ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Ptpase Activity ◽  
Stimulation Of

Following our previous observation that haemin is mitogenic for human lymphocytes, we investigated the ability of haemin to enhance glucose uptake in these cells. We found that preincubation of human peripheral-blood mononuclear cells (PBMC) with haemin for 60 min increased up to 5-fold the rate of 2-deoxy-D-[1-3H]glucose uptake by the cells. Actinomycin D and cycloheximide did not inhibit the effect, and cytochalasin B completely blocked it. Among the metalloporphyrins tested (Fe-, Ni-, Co-, Zn- and Sn-protoporphyrin), only haemin (Fe-protoporphyrin) induced a marked increase in glucose uptake. Thiourea, a scavenger of oxygen free radicals, and 3-amino-1,2,4-triazole inhibited haemin-induced glucose uptake. Oxidants such as H2O2 and phenylarsine oxide were previously reported to stimulate protein tyrosine phosphorylation and to enhance glucose uptake. We found that incubation of PBMC with haemin resulted in an increase in protein tyrosine phosphatase (PTPase) activity, probably that identified as CD45. Similarly to haemin, we found that phytohaemagglutinin also enhanced PTPase activity. Haemin also activated the tyrosine kinase p56lck, which is negatively controlled by phosphorylation of Tyr-505 at the C-terminus, and increased protein tyrosine phosphorylation in these cells. Tyrphostins, specific inhibitors of tyrosine kinases, at low concentrations markedly enhanced glucose uptake and synergized with haemin in enhancing glucose uptake. At high doses, tyrphostins inhibited the effect of haemin. Taken together, we postulate that haemin enhancement of glucose uptake in human lymphocytes results from its stimulation of PTPase, followed by activation of tyrosine kinase p56lck, leading to an increase in protein tyrosine phosphorylation.

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.

scholarly journals Internalization of the granulocyte-macrophage colony-stimulating factor receptor is not required for induction of protein tyrosine phosphorylation in human myeloid cells

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 1928-1935 ◽  
Author(s):  
K Okuda ◽  
B Druker ◽  
Y Kanakura ◽  
M Koenigsmann ◽  
JD Griffin
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Receptor Internalization ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts its biologic activities through binding to specific high-affinity cell surface receptors. After binding, the ligand/receptor complex is rapidly internalized in most hematopoietic cells. Using a human factor- dependent cell line, MO7, and normal human neutrophils, we found that the internalization is exquisitely temperature-dependent, such that ligand/receptor internalization does not detectably occur at 4 degrees C. Activation of the GM-CSF receptor has previously been shown to stimulate a number of postreceptor signal transduction pathways, including activation of a tyrosine kinase and activation of the serine/threonine kinase, Raf-1. The GM-CSF-stimulated increase in tyrosine kinase activity occurs rapidly at both 4 degrees C and 37 degrees C, and therefore is likely to be independent of receptor internalization. At 37 degrees C, the protein tyrosine phosphorylation was transient in MO7 cells, with maximum phosphorylation observed after 5 to 15 minutes, followed by a rapid decline. At 4 degrees C, the protein tyrosine phosphorylation of the same substrates was greater than at 37 degrees C, and no decline in substrate phosphorylation was observed for at least 90 minutes. In contrast to tyrosine phosphorylation, the activation and hyper-phosphorylation of Raf-1 observed at 37 degrees C in both MO7 cells and neutrophils was markedly diminished at 4 degrees C. These results indicate that at least one postreceptor signal transduction mechanism, activation of a tyrosine kinase, does not require ligand/receptor internalization, and indicate that receptor internalization may be a consequence, rather than the initiator, of signal transduction.

Protein tyrosine phosphorylation mediates TNF-induced endothelial-neutrophil adhesion in vitro

1998 ◽  
Vol 274 (2) ◽  
pp. H513-H519 ◽  
Author(s):  
Susan A. Kelly ◽  
Pascal J. Goldschmidt-Clermont ◽  
Emily E. Milliken ◽  
Toshiyuki Arai ◽  
Elise H. Smith ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Adhesion Molecules ◽  
Neutrophil Adhesion ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Tnf Α ◽  
Dose Dependent

Proinflammatory cytokines initiate the vascular inflammatory response via the upregulation of adhesion molecules on the luminal endothelial surface. We investigated directly the role of protein tyrosine phosphorylation in the upregulation of the endothelial adhesion molecules, intercellular adhesion molecule 1 (ICAM-1) and E-selectin, and the consequent adhesion of neutrophils, after tumor necrosis factor (TNF)-α-stimulation of human aortic endothelial cells in vitro. Time- and dose-dependent TNF-α-stimulated ICAM-1 and E-selectin upregulation and neutrophil adhesion each were suppressed by tyrosine kinase inhibitors, including genistein (200 μM), but not genistin, its isoflavone analog without tyrosine kinase inhibitory activity. Tyrphostin AG 126, a synthetic selective tyrosine kinase inhibitor, also suppressed ICAM-1 and E-selectin upregulation and neutrophil adhesion, each in a dose-dependent manner, whereas tyrphostin AG 1288 had no effect. Tyrosine phosphorylation of two proteins (85 and 145 kDa in the cytoskeleton fraction) found minutes after TNF-α-stimulation was also inhibited by genistein. These findings suggest that, in endothelial cells, TNF-α upregulates ICAM-1 and E-selectin expression and consequent neutrophil adhesion via protein tyrosine phosphorylation.

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