Differentiation between serum stimulation of ouabain-resistant and sensitive Rb influx in quiescent NIH 3T3 cells

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.

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Serum stimulation of NIH 3T3 cells induces the production of lipids able to inhibit GTPase-activating protein activity

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6683-6689.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6683-6689

Author(s):

C L Yu ◽

M H Tsai ◽

D W Stacey

Keyword(s):

Arachidonic Acid ◽

Cellular Proliferation ◽

Gel Chromatography ◽

Gtpase Activating Protein ◽

3T3 Cells ◽

Protein Activity ◽

Serum Stimulation ◽

Silica Gel Chromatography ◽

Nih 3T3 ◽

Nih 3T3 Cells

Quiescent NIH 3T3 cells were stimulated with serum prior to the extraction of total cellular lipids. These lipids were fractionated on thin-layer chromatography plates, and individual fractions were tested for the ability to inhibit GTPase-activating protein (GAP) activity. Two separate GAP inhibitory lipids were produced. One behaved similarly to arachidonic acid during silica gel chromatography, whereas the other was related to a phosphoinositide. Further study of the arachidonic acid-related material indicated that it was produced between 1 and 5 min after serum addition but was never observed in high-density, contact-inhibited cultures. The identity of these lipids is under investigation. The possibility raised by these results, that a metabolite of arachidonic acid is involved in mitogenic signaling, was supported by the finding that several lipoxygenase products of arachidonic acid efficiently inhibited GAP activity. These results provide further support for the hypothesis that lipids, GAP, and ras activity function together in the control of cellular proliferation.

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Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3903 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3903-3909 ◽

Cited By ~ 18

Author(s):

C J Molloy ◽

T P Fleming ◽

D P Bottaro ◽

A Cuadrado ◽

S A Aaronson

Keyword(s):

Growth Factor ◽

Tyrosine Phosphorylation ◽

Platelet Derived Growth Factor ◽

Gtpase Activating Protein ◽

3T3 Cells ◽

Pdgf Stimulation ◽

Nih 3T3 ◽

Stimulation Of ◽

Sustained Increase ◽

Nih 3T3 Cells

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.

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Comparison of the mode of action of ras p21 with those of protein kinases A and C in the stimulation of gene expression in NIH/3T3 cells

FEBS Letters ◽

10.1016/0014-5793(90)81141-a ◽

1990 ◽

Vol 269 (1) ◽

pp. 148-152 ◽

Cited By ~ 2

Author(s):

Naohisa Oku ◽

Kozo Kaibuchi ◽

Yasuo Fukumoto ◽

Yuichi Hori ◽

Hiroyuki Fujioka ◽

...

Keyword(s):

Gene Expression ◽

Protein Kinases ◽

Mode Of Action ◽

3T3 Cells ◽

Ras P21 ◽

Nih 3T3 ◽

Stimulation Of ◽

Nih 3T3 Cells

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Ectopic Expression of Protein Kinase CβII, -δ, and -ϵ, but Not -βI or -ζ, Provide for Insulin Stimulation of Glucose Uptake in NIH-3T3 Cells

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.2001.2300 ◽

2001 ◽

Vol 388 (1) ◽

pp. 178

Author(s):

Denise R. Cooper ◽

James E. Watson ◽

Niketa Patel ◽

Philip Illingworth ◽

Mildred Acevedo-Duncan ◽

...

Keyword(s):

Protein Kinase ◽

Glucose Uptake ◽

Ectopic Expression ◽

3T3 Cells ◽

Insulin Stimulation ◽

Nih 3T3 ◽

Stimulation Of ◽

Nih 3T3 Cells

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Serum stimulation of NIH 3T3 cells induces the production of lipids able to inhibit GTPase-activating protein activity.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6683 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6683-6689 ◽

Cited By ~ 39

Author(s):

C L Yu ◽

M H Tsai ◽

D W Stacey

Keyword(s):

Arachidonic Acid ◽

Cellular Proliferation ◽

Gel Chromatography ◽

Gtpase Activating Protein ◽

3T3 Cells ◽

Protein Activity ◽

Serum Stimulation ◽

Silica Gel Chromatography ◽

Nih 3T3 ◽

Nih 3T3 Cells

Quiescent NIH 3T3 cells were stimulated with serum prior to the extraction of total cellular lipids. These lipids were fractionated on thin-layer chromatography plates, and individual fractions were tested for the ability to inhibit GTPase-activating protein (GAP) activity. Two separate GAP inhibitory lipids were produced. One behaved similarly to arachidonic acid during silica gel chromatography, whereas the other was related to a phosphoinositide. Further study of the arachidonic acid-related material indicated that it was produced between 1 and 5 min after serum addition but was never observed in high-density, contact-inhibited cultures. The identity of these lipids is under investigation. The possibility raised by these results, that a metabolite of arachidonic acid is involved in mitogenic signaling, was supported by the finding that several lipoxygenase products of arachidonic acid efficiently inhibited GAP activity. These results provide further support for the hypothesis that lipids, GAP, and ras activity function together in the control of cellular proliferation.

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Activation of inositol phospholipid breakdown by prostaglandin F2α without any stimulation of proliferation in quiescent NIH-3T3 fibroblasts

Biochemical Journal ◽

10.1042/bj2660661 ◽

1990 ◽

Vol 266 (3) ◽

pp. 661-667 ◽

Cited By ~ 15

Author(s):

F M Black ◽

M J O Wakelam

Keyword(s):

Cell Proliferation ◽

Inositol Phosphates ◽

Calf Serum ◽

Prostaglandin F2α ◽

3T3 Cells ◽

Inositol Phospholipid ◽

Inositol Phospholipid Breakdown ◽

Nih 3T3 ◽

Stimulation Of ◽

Nih 3T3 Cells

Stimulation of NIH-3T3 cells with prostaglandin F2 alpha (PGF2 alpha) caused a dose- and time-dependent generation of inositol phosphates. The first detectable changes were in the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Increases in Ins(1,3,4)P3, InsP2 and InsP were detected later, and only minor changes were observed in putative InsP5 or InsP6. The accumulation of inositol phosphates was synergistically increased by the addition of calf serum, whereas PGF2 alpha had no effects on cell proliferation in either the presence or the absence of calf serum. Stimulation of a different clone of NIH-3T3 cells (AmNIH-3T3) or Swiss 3T3 cells with PGF2 alpha resulted in both inositol phospholipid breakdown and cell proliferation. No differences were found in the characteristics of PGF2 alpha-stimulated inositol phosphate generation between the two clones of NIH-3T3 cells, nor was there any difference in receptor number of Kd. These results question the role of inositol phospholipid breakdown in mitogenesis and demonstrate significant differences in the biochemical properties of apparently the ‘same’ cells.

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