Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts

1999 ◽  
Vol 112 (9) ◽  
pp. 1365-1373 ◽  
Author(s):  
X. Sai ◽  
K. Naruse ◽  
M. Sokabe
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Orienting Response ◽  
Cyclic Stretch ◽  
Wild Type ◽  
Herbimycin A ◽  
Molecular Masses

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120–130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120–130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

Role for tyrosine kinases in carbachol-regulated Ca entry into colonic epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C154-C161 ◽  
Author(s):  
G. Bischof ◽  
B. Illek ◽  
W. W. Reenstra ◽  
T. E. Machen
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Tyrosine Phosphatase ◽  
Inhibitory Effect ◽  
Colonic Epithelial Cells

We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image processing of fura 2 fluorescence. Both carbachol and thapsigargin increased Ca entry to a similar extent and Ca influx was reduced by the tyrosine kinase inhibitor genistein (50 microM). Further experiments were performed in solutions containing 95 mM K to depolarize the membrane potential, and the effects of different inhibitors on influx of Ca, Mn, and Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellular Ca to increase to a plateau of 223 +/- 19 nM. This plateau was reduced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lavendustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, prevented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblots with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa proteins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosphorylation of some key proteins may be important for regulating this pathway.

Induction of autophagy across multiple tumor types through irreversible HER tyrosine kinase blockade.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13517-e13517
Author(s):  
William Rayford Gwin ◽  
Leihua Liu ◽  
Sumin Zhao ◽  
Wenle Xia ◽  
Neil Spector
Keyword(s):  
Prostate Cancer ◽  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Cellular Proliferation ◽  
Kinase Inhibitor ◽  
Significant Inhibition ◽  
Wild Type ◽  
Multiple Tumor ◽  
Mtorc1 Signaling

e13517 Background: Human epidermal growth factor receptor (HER) receptor tyrosine kinases play a key role in solid tumor oncogenesis. Despite broad expression of HER receptors in solid tumors, HER targeted therapies have not shown significant improvement in survival, calling into question the value of wild-type HER receptors as therapeutic targets. Here we found that an irreversible pan-HER tyrosine kinase inhibitor (TKI), neratinib, but not similar HER TKIs, induced morphologic changes in ovarian, TNBC, and prostate cancer cell lines consistent with induction of autophagy. Methods: SKOV3 (ovarian), OVCAR8 (ovarian), HBL-100 (TNBC), and LAPC4 (prostate) cancer cells were treated with lapatinib, gefitinib, CI-1033, afatinib, and neratinib (0.5mM-2.5mM). The activation state of HER2, EGFR, HER3, Akt, Erk, p70S6, 4EBP1, and Ulk1 was determined by Western blot analysis (WB) at various time points of neratinib treatment. LC3 was analyzed by immunofluorescence (IF) microscopy and WB. Analysis of proliferation, apoptosis, and cell cycle were performed using WST-1, annexin V, and PI staining, respectively. Results: Neratinib, but not similar HER TKIs, induced marked cytoplasmic vacuolization in tumors. The conversion of LC3-I to LC3-II in neratinib-treated cells was consistent with induction of autophagy. Moreover, PI3K/Akt, MAPK/Erk1/2 and mTORC1 signaling cascades were inhibited in neratinib-treated cells, and were associated with the inhibition of phospho-Ulk1, a key step in autophagy initiation. Treatment with neratinib alone resulted in G1 cell cycle arrest. Importantly, the combination of neratinib and chloroquine, an autophagy inhibitor, induced a statistically significant inhibition of cellular proliferation (p <0.01) and increased apoptosis compared to treatment with either drug alone. Conclusions: Our data suggest that more effective inhibition of wild-type HER receptors, can lead to mTORC1 inhibition, which in turn triggers autophagy. Here, autophagy appears to protect cells rather than inducing apoptosis. Consequently, targeting both HER receptors and autophagy represents an attractive therapeutic strategy to treat tumors expressing wild-type HER receptors.

scholarly journals Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells.

1993 ◽  
Vol 120 (1) ◽  
pp. 185-195 ◽  
Author(s):  
L P Baker ◽  
H B Peng
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Acetylcholine Receptor ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Cluster Formation ◽  
Protein S ◽  
Muscle Cells ◽  
Nerve Muscle ◽  
Achr Clustering

Aggregation of the nicotinic acetylcholine receptor (AChR) at sites of nerve-muscle contact is one of the earliest events to occur during the development of the neuromuscular junction. The stimulus presented to the muscle by nerve and the mechanisms underlying postsynaptic differentiation are not known. The purpose of this study was to examine the distribution of phosphotyrosine (PY)-containing proteins in cultured Xenopus muscle cells in response to AChR clustering stimuli. Results demonstrated a distinct accumulation of PY at AChR clusters induced by several stimuli, including nerve, the culture substratum, and polystyrene microbeads. AChR microclusters formed by external cross-linking did not show PY colocalization, implying that the accumulation of PY in response to clustering stimuli was not due to the aggregation of basally phosphorylated AChRs. A semi-quantitative determination of the time course for development of PY labeling at bead contacts revealed early PY accumulation within 15 min of contact before significant AChR aggregation. At later stages (within 15 h), the AChR signal came to approximate the PY signal. We have reported the inhibition of bead-induced AChR clustering in response to beads by a tyrphostin tyrosine kinase inhibitor (RG50864) (Peng, H. B., L. P. Baker, and Q. Chen. 1991. Neuron. 6:237-246). RG50864 also inhibited PY accumulation at bead contacts, providing evidence for tyrosine kinase activation in response to the bead stimulus. These results suggest that tyrosine phosphorylation may play an important role in the generative stages of cluster formation, and may involve protein(s) other than or in addition to AChRs.

scholarly journals Modulation of the Kv1.3 Potassium Channel by Receptor Tyrosine Kinases

1997 ◽  
Vol 110 (5) ◽  
pp. 601-610 ◽  
Author(s):  
Mark R. Bowlby ◽  
Debra A. Fadool ◽  
Todd C. Holmes ◽  
Irwin B. Levitan
Keyword(s):  
Tyrosine Kinase ◽  
Potassium Channel ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Mutational Analysis ◽  
Inactivation Kinetics ◽  
Hek 293 ◽  
Voltage Dependent

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (α-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the α-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.

ARG tyrosine kinase activity is inhibited by STI571

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2440-2448 ◽  
Author(s):  
Keiko Okuda ◽  
Ellen Weisberg ◽  
D. Gary Gilliland ◽  
James D. Griffin
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Fusion Partner ◽  
Induced Apoptosis

Abstract The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces hematologic remission in most patients with chronic myeloid leukemia. In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF)β receptor, and c-KIT, but it does not inhibit SRC family kinases, c-FMS, FLT3, the epidermal growth factor receptor, or multiple other tyrosine kinases. ARG is a widely expressed tyrosine kinase that shares substantial sequence identity with c-ABL in the kinase domain and cooperates with ABL to regulate neurulation in the developing mouse embryo. As described here, ARG has recently been implicated in the pathogenesis of leukemia as a fusion partner of TEL. A TEL/ARG fusion was constructed to determine whether ARG can be inhibited by STI571. When expressed in the factor-dependent murine hematopoietic cell line Ba/F3, the TEL/ARG protein was heavily phosphorylated on tyrosine, increased tyrosine phosphorylation of multiple cellular proteins, and induced factor-independent proliferation. The effects of STI571 on Ba/F3 cells transformed with BCR/ABL, TEL/ABL, TEL/PDGFβR, or TEL/ARG were then compared. STI571 inhibited tyrosine phosphorylation and cell growth of Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/PDGFβR, and TEL/ARG with an IC50 of approximately 0.5 μM in each case, but it had no effect on untransformed Ba/F3 cells growing in IL-3 or on Ba/F3 cells transformed by TEL/JAK2. Culture of TEL/ARG-transfected Ba/F3 cells with IL-3 completely prevented STI571-induced apoptosis in these cells, similar to what has been observed with BCR/ABL- or TEL/ABL-transformed cells. These results indicate that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571.

scholarly journals CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae

2002 ◽  
Vol 156 (6) ◽  
pp. 951-957 ◽  
Author(s):  
Shaun W. Lee ◽  
Robert A. Bonnah ◽  
Dustin L. Higashi ◽  
John P. Atkinson ◽  
Sharon L. Milgram ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neisseria Gonorrhoeae ◽  
Tyrosine Phosphorylation ◽  
Bacterial Adhesion ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Host Cells ◽  
Cell Cortex ◽  
Wild Type

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.

scholarly journals Activation of Lyn is a common element of the stimulation of human neutrophils by soluble and particulate agonists

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3567-3574 ◽  
Author(s):  
M Gaudry ◽  
C Gilbert ◽  
F Barabe ◽  
PE Poubelle ◽  
PH Naccache
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Time Course ◽  
Kinase Assay ◽  
Common Element ◽  
Human Neutrophils ◽  
Functional Responsiveness ◽  
Stimulation Of

The functional responsiveness of human neutrophils is known to be initiated and modulated by protein tyrosine phosphorylation. The regulation of the levels of tyrosine phosphorylation is most likely the result of the coordinated actions of tyrosine kinases and phosphatases, which have so far been only very partially characterized. In the present study, we present evidence demonstrating that the stimulation of neutrophils by a variety of agonists (soluble as well as particulate) leads to the activation of the src-related tyrosine kinase lyn. The stimulation of tyrosine kinase activity of lyn was detected using an immune kinase assay as well as an in situ labeling technique. Phosphoaminoacid analysis of lyn indicated that the autophosphorylation of the kinase was exclusively on tyrosine residues. The time course of the activation of lyn is consistent with its playing a role in the early tyrosine phosphorylation responses of neutrophils. The ability of agonists with widely varying functional end responses to stimulate the activity of lyn indicates that this event plays a key and central role in the control of the activation of human neutrophils.

scholarly journals Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.

1992 ◽  
Vol 119 (4) ◽  
pp. 893-903 ◽  
Author(s):  
K Burridge ◽  
C E Turner ◽  
L H Romer
Keyword(s):  
Extracellular Matrix ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Focal Adhesions ◽  
Rat Embryo ◽  
Herbimycin A ◽  
Embryo Fibroblasts ◽  
Coated Surfaces ◽  
In Cells

Cells in culture reveal high levels of protein tyrosine phosphorylation in their focal adhesions, the regions where cells adhere to the underlying substratum. We have examined the tyrosine phosphorylation of proteins in response to plating cells on extracellular matrix substrata. Rat embryo fibroblasts, mouse Balb/c 3T3, and NIH 3T3 cells plated on fibronectin-coated surfaces revealed elevated phosphotyrosine levels in a cluster of proteins between 115 and 130 kD. This increase in tyrosine phosphorylation was also seen when rat embryo fibroblasts were plated on laminin or vitronectin, but not on polylysine or on uncoated plastic. Integrin mediation of this effect was suggested by finding the same pattern of elevated tyrosine phosphorylation in cells plated on the cell-binding fragment of fibronectin and in cells plated on a synthetic polymer containing multiple RGD sequences. We have identified one of the proteins of the 115-130-kD cluster as pp125FAK, a tyrosine kinase recently localized in focal adhesions (Schaller, M. D., C. A. Borgman, B. S. Cobb, R. R. Vines, A. B. Reynolds, and J. T. Parsons. 1992. Proc. Natl. Acad. Sci. USA. 89:5192). A second protein that becomes tyrosine phosphorylated in response to extracellular matrix adhesion is identified as paxillin, a 70-kD protein previously localized to focal adhesions. Treatment of cells with the tyrosine kinase inhibitor herbimycin A diminished the adhesion-induced tyrosine phosphorylation of these proteins and inhibited the formation of focal adhesions and stress fibers. These results suggest a role for integrin-mediated tyrosine phosphorylation in the organization of the cytoskeleton as cells adhere to the extracellular matrix.

scholarly journals A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells.

1993 ◽  
Vol 120 (1) ◽  
pp. 197-204 ◽  
Author(s):  
H B Peng ◽  
L P Baker ◽  
Z Dai
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Electric Fields ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Muscle Cells ◽  
Postsynaptic Membrane ◽  
Achr Clustering

During the development of the neuromuscular junction, acetylcholine receptors (AChRs) become clustered in the postsynaptic membrane in response to innervation. In vitro, several non-neuronal stimuli can also induce the formation of AChR clusters. DC electric field (E field) is one of them. When cultured Xenopus muscle cells are exposed to an E field of 5-10 V/cm, AChRs become clustered along the cathode-facing edge of the cells within 2 h. Recent studies have suggested the involvement of tyrosine kinase activation in the action of several AChR clustering stimuli, including nerve, polymer beads, and agrin. We thus examined the role of tyrosine phosphorylation in E field-induced AChR clustering. An antibody against phosphotyrosine (PY) was used to examine the localization of PY-containing proteins in E field-treated muscle cells. We found that anti-PY staining was colocalized with AChR clusters along the cathodal edge of the cells. In fact, cathodal PY staining could be detected before the first appearance of AChR clusters. When cultures were subjected to E fields in the presence of a tyrosine kinase inhibitor, tyrphostin RG-50864, cathodal AChR clustering was abolished with a half maximal inhibitory dosage of 50 microM. An inactive form of tyrphostin (RG-50862) had no effect on the field-induced clustering. These data suggest that the activation of tyrosine kinases is an essential step in E field-induced AChR clustering. Thus, the actions of several disparate stimuli for AChR clustering seem to converge to a common signal transduction mechanism based on tyrosine phosphorylation at the molecular level.

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