scholarly journals Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts.

1983 ◽  
Vol 3 (3) ◽  
pp. 380-390 ◽  
Author(s):  
K D Nakamura ◽  
R Martinez ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Chicken Embryo ◽  
Sodium Dodecyl ◽  
Biological Response ◽  
Egf Receptors ◽  
Phosphorylation Of Proteins ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts

1983 ◽  
Vol 3 (3) ◽  
pp. 380-390
Author(s):  
K D Nakamura ◽  
R Martinez ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Chicken Embryo ◽  
Sodium Dodecyl ◽  
Biological Response ◽  
Egf Receptors ◽  
Phosphorylation Of Proteins ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.

scholarly journals Counter-transport in chick embryo fibroblasts. A significant factor in measurement of glucose entry

1982 ◽  
Vol 206 (2) ◽  
pp. 301-309 ◽  
Author(s):  
R J Gay ◽  
H Amos
Keyword(s):  
Chick Embryo ◽  
Glucose Transport ◽  
Cell Cultures ◽  
Chicken Embryo ◽  
Primary Cell ◽  
Primary Cell Cultures ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

Enhanced rates of carrier-mediated 3-O-methyl-D-glucose (0.1 mM) transport were observed in primary cell cultures of chicken embryo fibroblasts deprived of glucose for 1 day. The addition of 5.5 mM-glucose, glucosamine or 2-deoxy-D-glucose for 15 min (37 degrees C) to glucose-starved cultures followed by washing and immediate measurement of 3-O-methyl-D-glucose transport resulted in an apparent further stimulation of transport. Transport stimulation increased with increasing concentrations of the added preincubation sugar and was observed at test concentrations ranging from 0.1 mM- to 10 mM-3-O-methyl-D-glucose. This enhancement occurred when the preloaded sugar was rapidly effluxing from cells and was eliminated by allowing cultures to incubate in buffer without sugar for 30 min (37 degrees C) after the removal of hexose and before measuring transport. A transient overshoot in the cumulative uptake of 3-O-methyl-D-glucose was observed in glucose-starved cultures that were pre-incubated in the presence of 55 mM-glucose or -glucosamine for 15 min (37 degrees C). These data suggest that counter-transport accounts for the apparent enhancement of glucose-transport capability observed in glucose-starved cells when they are briefly re-exposed to hexose.

Expression of 9E3 mRNA is associated with mitogenicity, phosphorylation, and morphological alteration in chicken embryo fibroblasts

1990 ◽  
Vol 10 (7) ◽  
pp. 3813-3817
Author(s):  
K Barker ◽  
H Hanafusa
Keyword(s):  
Mrna Expression ◽  
Tyrosine Phosphorylation ◽  
Okadaic Acid ◽  
Chicken Embryo ◽  
Cell Shape ◽  
Morphological Alteration ◽  
Rapid Induction ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Transformation of chicken embryo fibroblasts (CEF) with viruses encoding src, ros, yes, and fps as well as ras, mos, middle T, erbA and erbB, myc, and crk stimulated 9E3 mRNA expression. Treatment of CEF with agents that modulate cell shape or attachment to the substratum caused an increase in 9E3 mRNA without an increase in tyrosine phosphorylation. 9E3 mRNA was also increased in CEF in response to several agents which modulate phosphorylation, including phorbol myristic acetate, vanadate, and okadaic acid, which suggests that the rapid induction of 9E3 mRNA expression in CEF by the src protein occurs downstream of morphological or phosphorylation events.

scholarly journals Expression of 9E3 mRNA is associated with mitogenicity, phosphorylation, and morphological alteration in chicken embryo fibroblasts.

1990 ◽  
Vol 10 (7) ◽  
pp. 3813-3817 ◽  
Author(s):  
K Barker ◽  
H Hanafusa
Keyword(s):  
Mrna Expression ◽  
Tyrosine Phosphorylation ◽  
Okadaic Acid ◽  
Chicken Embryo ◽  
Cell Shape ◽  
Morphological Alteration ◽  
Rapid Induction ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Transformation of chicken embryo fibroblasts (CEF) with viruses encoding src, ros, yes, and fps as well as ras, mos, middle T, erbA and erbB, myc, and crk stimulated 9E3 mRNA expression. Treatment of CEF with agents that modulate cell shape or attachment to the substratum caused an increase in 9E3 mRNA without an increase in tyrosine phosphorylation. 9E3 mRNA was also increased in CEF in response to several agents which modulate phosphorylation, including phorbol myristic acetate, vanadate, and okadaic acid, which suggests that the rapid induction of 9E3 mRNA expression in CEF by the src protein occurs downstream of morphological or phosphorylation events.

scholarly journals Rap2B-Dependent Stimulation of Phospholipase C-ε by Epidermal Growth Factor Receptor Mediated by c-Src Phosphorylation of RasGRP3

2004 ◽  
Vol 24 (11) ◽  
pp. 4664-4676 ◽  
Author(s):  
Matthias B. Stope ◽  
Frank vom Dorp ◽  
Daniel Szatkowski ◽  
Anja Böhm ◽  
Melanie Keiper ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Dominant Negative ◽  
Nucleotide Exchange ◽  
Egf Receptors ◽  
Epidermal Growth ◽  
Stimulation Of

ABSTRACT Receptor tyrosine kinase regulation of phospholipase C-ε (PLC-ε), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca2+ signaling by the EGF receptor, which activated both PLC-γ1 and PLC-ε, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-ε, and Rap2B-dependent translocation of PLC-ε to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca2+ and expression of lipase-inactive PLC-γ1 but not of PLC-ε. Expression of RasGRP3, a Ca2+/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca2+ signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-γ1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-ε.

scholarly journals Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

1987 ◽  
Vol 7 (9) ◽  
pp. 3147-3155 ◽  
Author(s):  
E Erikson ◽  
D Stefanovic ◽  
J Blenis ◽  
R L Erikson ◽  
J L Maller
Keyword(s):  
Chicken Embryo ◽  
Kinase Activity ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Cell Types ◽  
Polyclonal Antiserum ◽  
Xenopus Laevis Oocytes ◽  
S6 Kinase ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Ribosomal protein S6 becomes highly phosphorylated during progesterone- or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographically distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20- to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.

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