scholarly journals Focal adhesion kinase (p125FAK) and paxillin are substrates for sphingomyelinase-induced tyrosine phosphorylation in Swiss 3T3 fibroblasts

1996 ◽  
Vol 315 (3) ◽  
pp. 1035-1040 ◽  
Author(s):  
Takehiko SASAKI ◽  
Kaoru HAZEKI ◽  
Osamu HAZEKI ◽  
Michio UI ◽  
Toshiaki KATADA
Keyword(s):  
Tyrosine Kinase ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Tyrosine Kinase Activity ◽  
3T3 Fibroblasts ◽  
Kinase Cascade ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts

We examined the effect of sphingomyelinase on tyrosine phosphorylation of intracellular proteins in mouse Swiss 3T3 fibroblasts. Incubation of the cells with bacterial sphingomyelinase resulted in the elevation of tyrosine phosphorylation of multiple cellular proteins of 190, 130, 120, 97 and 70 kDa within minutes. The 120 and 70 kDa tyrosine-phosphorylated peptides were identified as p125 focal adhesion kinase (p125FAK) and paxillin respectively by the use of specific antibodies against the proteins. Tyrosine kinase activity associated with anti-p125FAK immunoprecipitate was stimulated by incubation of cells with sphingomyelinase. Cytochalasin D, which selectively disrupts the network of actin filaments, inhibited sphingomyelinase-induced tyrosine phosphorylation of p125FAK and elevation of tyrosine kinase activity in the anti-p125FAK immunoprecipitates. Sphingomyelinase-induced phosphorylation of p125FAK was not inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. This was in sharp contrast with a wortmannin-sensitive phosphorylation of p125FAK observed in platelet-derived growth factor (PGDF)-stimulated cells. Thus hydrolysis of sphingomyelin is considered to regulate the tyrosine kinase cascade including p125FAK and paxillin by a mechanism distinct from PDGF.

scholarly journals Sphingosine 1-phosphate stimulates Rho-mediated tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 fibroblasts

1997 ◽  
Vol 324 (2) ◽  
pp. 481-488 ◽  
Author(s):  
Fang WANG ◽  
Catherine D. NOBES ◽  
Alan HALL ◽  
Sarah SPIEGEL
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Stress Fibre ◽  
3T3 Fibroblasts ◽  
Fibre Formation ◽  
Sphingosine 1 Phosphate ◽  
Swiss 3T3 Fibroblasts ◽  
Stress Fibre Formation ◽  
Stimulation Of

Sphingosine 1-phosphate (SPP), a sphingolipid second messenger implicated in the mitogenic action of platelet-derived growth factor [Olivera, A. and Spiegel, S. (1993) Nature (London) 365, 557–560], induced rapid reorganization of the actin cytoskeleton resulting in stress-fibre formation. SPP also induced transient tyrosine phosphorylation of focal adhesion kinase (p125FAK), a cytosolic tyrosine kinase that localizes in focal adhesions, and of the cytoskeleton-associated protein paxillin. Exoenzyme C3 transferase, which ADP-ribosylates Rho (a Ras-related small GTP binding protein) on asparagine-41 and renders it biologically inactive, inhibited both stress-fibre formation and protein tyrosine phosphorylation induced by SPP. Thus Rho may be an upstream regulator of both stress-fibre formation and tyrosine phosphorylation of p125FAK and paxillin. Pretreatment with PMA, an activator of protein kinase C (PKC), inhibited the stimulation of stress-fibre formation induced by 1-oleoyl-lysophosphatidic acid (LPA) but not that by SPP. Similarly, PMA also decreased LPA-induced tyrosine phosphorylation of p125FAK and paxillin without abrogating the response to SPP. Thus PKC is involved in LPA- but not SPP-dependent signalling. The polyanionic drug suramin, a broad-specificity inhibitor of ligand–receptor interactions, did not inhibit either the mitogenic effect of SPP or its stimulation of tyrosine phosphorylation of p125FAK. However, suramin markedly inhibited these responses induced by LPA. These results suggest that in contrast with LPA, SPP may be acting intracellularly in Swiss 3T3 fibroblasts to stimulate tyrosine phosphorylation of p125FAK and paxillin and cell growth.

scholarly journals Inhibition of mitogen-induced DNA synthesis by bafilomycin A1 in Swiss 3T3 fibroblasts

1996 ◽  
Vol 313 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Andrew J. SAURIN ◽  
Jane HAMLETT ◽  
Michael J. CLAGUE ◽  
Stephen R. PENNINGTON
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Kinase Activity ◽  
Bafilomycin A1 ◽  
Tyrosine Kinase Activity ◽  
3T3 Fibroblasts ◽  
Mitogenic Signalling ◽  
Epidermal Growth ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts

Quiescent cells (in G0) can be stimulated to enter the cell cycle and proceed to DNA synthesis in S-phase by a wide range of growth factors and mitogens. Activation of cell-surface growth factor receptors with intrinsic protein tyrosine kinase activity initiates autophosphorylation of the receptors and subsequent activation of signal transduction cascades. After activation the receptors undergo ligand-induced internalization to endosomes, which become acidified by the action of a vacuolar H+-ATPase (V-ATPase). The extent to which vesicular acidification plays a role in mitogenic signalling by receptors with intrinsic tyrosine kinase activity remains unknown. Here we have shown that bafilomycin A1, a specific inhibitor of V-ATPase, inhibits endosome acidification and mitogen-induced DNA synthesis in Swiss 3T3 fibroblasts. Addition of bafilomycin A1 at successively later times during G1 progressively decreased the inhibition of DNA synthesis such that no inhibition was observed when bafilomycin A1 was added at the onset of S-phase. Bafilomycin A1 also induced a dramatic but reversible change in the morphology of Swiss 3T3 cells. However, the rapid activation of c-fos mRNA accumulation by epidermal growth factor and insulin was unaffected by bafilomycin A1. Together, the results suggest that activation of the V-ATPase plays an important role in the mitogenic signalling pathways that occur during the G1 phase of the cell cycle but is not required for the initial epidermal growth factor and insulin-evoked signalling events that lead to c-fos mRNA expression.

scholarly journals The CD3 chains of the T cell antigen receptor associate with the ZAP-70 tyrosine kinase and are tyrosine phosphorylated after receptor stimulation.

1993 ◽  
Vol 178 (5) ◽  
pp. 1523-1530 ◽  
Author(s):  
D B Straus ◽  
A Weiss
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Antigen Receptor ◽  
Tyrosine Kinase Activity ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Signaling Function ◽  
Signaling Events

Recent work indicates that signaling events resulting from stimulation of the T cell antigen receptor (TCR) can be initiated by the CD3 complex (gamma, delta, epsilon) as well as the zeta chains of the receptor. To help characterize the signaling function of CD3 we examined its associated tyrosine kinase activity since induction of tyrosine phosphorylation is one of the earliest signaling events. Our results indicate that at least two kinases, lck and ZAP-70, contribute to the CD3-associated kinase activity. A likely target of this activity is the CD3 complex itself since we observed that TCR stimulation resulted in rapid tyrosine phosphorylation of the CD3 epsilon and delta chains. To examine the function of the CD3 epsilon chain in particular, we constructed a chimera that fused the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of CD3 epsilon. This chimera demonstrated that CD3 epsilon was independently capable of associating with proteins having tyrosine kinase activity, including ZAP-70. Our results show that the kinase activity that associates with the CD3 complex has characteristics that are quite similar to the previously characterized zeta-associated kinase activity. This finding suggests that both these components of the TCR initiate signaling events using a common mechanism. However, differences in their signaling function could result from recognition of distinct substrates.

Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF

Nature ◽  
1991 ◽  
Vol 350 (6314) ◽  
pp. 158-160 ◽  
Author(s):  
David R. Kaplan ◽  
Dionisio Martin-Zanca ◽  
Luis F. Parada
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Tyrosine Kinase Activity

scholarly journals Functional Proteomic Analysis of Long-term Growth Factor Stimulation and Receptor Tyrosine Kinase Coactivation in Swiss 3T3 Fibroblasts

2012 ◽  
Vol 11 (12) ◽  
pp. 1690-1708 ◽  
Author(s):  
Kohji Nagano ◽  
Akunna Akpan ◽  
Gayathri Warnasuriya ◽  
Steven Corless ◽  
Nick Totty ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Proteomic Analysis ◽  
3T3 Fibroblasts ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts

scholarly journals Signal transduction of the human granulocyte-macrophage colony- stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 706-715 ◽  
Author(s):  
Y Kanakura ◽  
B Druker ◽  
SA Cannistra ◽  
Y Furukawa ◽  
Y Torimoto ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells through their interaction with specific cell-surface receptors. There is a considerable degree of overlap in the biological effects of these two growth factors, but little is known about the mechanisms of postreceptor signal transduction. We have investigated the effects of GM-CSF and IL-3 on protein tyrosine-kinase activity in a human cell line, MO7E, which proliferates in response to either factor. Tyrosine- kinase activity was detected using immunoblotting with a monoclonal antibody (MoAb) specific for phosphotyrosine. GM-CSF and IL-3 were found to induce a nearly identical pattern of protein tyrosine phosphorylation using both one- and two-dimensional gel electrophoresis. Tyrosine phosphorylation of two cytosolic proteins in particular was increased more than 10-fold, a 93-Kd protein (pp93) and a 70-Kd protein (pp70). Tyrosine phosphorylation of pp93 and pp70 was observed within 1 minute, reached a maximum at 5 to 15 minutes, and gradually decreased thereafter. Other proteins of 150, 125, 63, 55, 42, and 36 Kd were also phosphorylated on tyrosine in response to both GM- CSF and IL-3, although to a lesser degree. Tyrosine phosphorylation was dependent on the concentration of GM-CSF over the range of 0.1 to 10 ng/mL and on IL-3 over the range of 1 to 30 ng/mL. Stimulation of MO7E cells with 12–0-tetradecanoyl-phorbol-13-acetate (TPA) or cytokines such as G-CSF, M-CSF, interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon gamma, tumor necrosis factor (TNF), or transforming growth factor-beta (TGF-beta) did not induce tyrosine phosphorylation of pp93 or pp70, suggesting that these two phosphoproteins are specific for GM-CSF-or IL-3-induced activation. The extent and duration of phosphorylation of all the substrates were increased by pretreatment of cells with vanadate, an inhibitor of protein-tyrosine phosphatases. Importantly, culture of MO7E cells with vanadate (up to 10 mumol/L) resulted in a dose-dependent increase in GM- CSF-or IL-3-induced proliferation of up to 1.8-fold. These results suggest that tyrosine phosphorylation may be important for GM-CSF and IL-3 receptor-mediated signal transduction and that cell proliferation may be, at least partially, regulated by a balance between CSF-induced protein-tyrosine kinase activity and protein-tyrosine phosphatase activity.

scholarly journals Activation of the erythropoietin receptor by the Friend spleen focus- forming virus gp55 glycoprotein induces constitutive protein tyrosine phosphorylation

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3070-3078 ◽  
Author(s):  
MO Showers ◽  
JF Moreau ◽  
D Linnekin ◽  
B Druker ◽  
AD D'Andrea
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Kinase Activity ◽  
Parental Cell ◽  
Erythropoietin Receptor ◽  
Parental Cell Line ◽  
Tyrosine Kinase Activity ◽  
Murine Cell

The erythropoietin receptor (EPO-R) can be activated to signal cell growth by binding either EPO or gp55, the Friend spleen focus-forming virus (SFFV) glycoprotein. EPO binding induces tyrosine kinase activity and rapid tyrosine phosphorylation of several cellular substrates. To test for gp55-induced tyrosine kinase activity, we performed immunoblots on two murine cell lines that stably express EPO-R and gp55. Stimulation of the parental cell line, Ba/F3, with murine interleukin-3 (IL-3) resulted in rapid, dose-dependent tyrosine phosphorylation of a 97-Kd substrate. Stimulation with IL-3 or EPO of the Ba/F3 cells expressing the recombinant EPO-R (Ba/F3-EPO-R) resulted in tyrosine phosphorylation of the same p97 substrate. These latter cells, when transformed to growth factor-independence by the Friend gp55 glycoprotein, exhibited constitutive tyrosine phosphorylation of the 97-Kd substrate. Other growth factor-independent Ba/F3 subclones, transformed with either the oncoprotein, v-abl, or with a constitutively activated EPO-R, also had constitutive phosphorylation of a 97-Kd substrate. In CTLL-2-EPO-R cells, a T-lymphocyte line stably transfected with the EPO-R, the 97-Kd substrate was tyrosine- phosphorylated in response to IL-2 or EPO. The 97-Kd protein was constitutively phosphorylated in CTLL-2-EPO-R-gp55 cells. In conclusion, a 97-Kd protein found in two murine cell lines is tyrosine-phosphorylated in response to multiple growth factors and viral oncoproteins, and appears to be a central phosphoprotein in signal transduction.

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