Simultaneous cytoplasmic redistribution of ribosomal protein L32 mRNA and phosphorylation of eukaryotic initiation factor 4E after mitogenic stimulation of Swiss 3T3 cells.

ABSTRACT The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKα, and p38MAPKβ. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.

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Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells

Biochemical Journal ◽

10.1042/bj3600087 ◽

2001 ◽

Vol 360 (1) ◽

pp. 87-95 ◽

Cited By ~ 14

Author(s):

Duraisamy SENTHIL ◽

Jennifer L. FAULKNER ◽

Goutam GHOSH CHOUDHURY ◽

Hanna E. ABBOUD ◽

Balakuntalam S. KASINATH

Keyword(s):

Protein Synthesis ◽

Angiotensin Ii ◽

Insulin Signalling ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Tubular Epithelial Cells ◽

Eukaryotic Initiation Factor 4E ◽

Proximal Tubular Epithelial Cells ◽

Proximal Tubular ◽

Stimulation Of

Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1nM) or insulin (1nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/−2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor β-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/−2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II.

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Elevated levels of cyclin D1 protein in response to increased expression of eukaryotic initiation factor 4E

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7358-7363.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7358-7363

Author(s):

I B Rosenwald ◽

A Lazaris-Karatzas ◽

N Sonenberg ◽

E V Schmidt

Keyword(s):

Cyclin D1 ◽

D1 Protein ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

3T3 Cells ◽

Protein Levels ◽

Eukaryotic Initiation Factor 4E ◽

Cyclin D1 Protein ◽

Nih 3T3 ◽

Nih 3T3 Cells

Cyclin D1 is a G1-specific cyclin that has been linked to lymphoid, parathyroid, and breast tumors. Recent studies suggested that high protein levels of cyclin D1 are not always produced when cyclin D1 mRNA is overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 regulation. The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF-4E]) is a potential regulatory of several posttranscriptional events, and it can itself induce neoplastic transformation. Consequently, we examined eIF-4E as a potential regulator of cyclin D1. Overexpression of cyclin D1 mRNA in NIH 3T3 cells did not increase cyclin D1 protein. In contrast, overexpression of eIF-4E markedly increased the amount of cyclin D1 protein in NIH 3T3 cells. This increase was specific to cyclin D1 in comparison with the retinoblastoma gene product, c-Myc, actin, and eukaryotic initiation factor 2 alpha. We also examined cyclin D1 protein in cells expressing an estrogen receptor-Myc fusion protein because we previously found that eIF-4E increases after induction of c-myc function. In these cells, increased levels of eIF-4E protein were closely followed by increases in levels of cyclin D1 protein, but the level of cyclin D1 mRNA was not increased. We conclude that increases in cyclin D1 levels may result from increased expression of eIF-4E, and this regulation may be one determinant of cyclin D1 levels in the cell.

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Elevated levels of cyclin D1 protein in response to increased expression of eukaryotic initiation factor 4E.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7358 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7358-7363 ◽

Cited By ~ 202

Author(s):

I B Rosenwald ◽

A Lazaris-Karatzas ◽

N Sonenberg ◽

E V Schmidt

Keyword(s):

Cyclin D1 ◽

D1 Protein ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

3T3 Cells ◽

Protein Levels ◽

Eukaryotic Initiation Factor 4E ◽

Cyclin D1 Protein ◽

Nih 3T3 ◽

Nih 3T3 Cells

Cyclin D1 is a G1-specific cyclin that has been linked to lymphoid, parathyroid, and breast tumors. Recent studies suggested that high protein levels of cyclin D1 are not always produced when cyclin D1 mRNA is overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 regulation. The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF-4E]) is a potential regulatory of several posttranscriptional events, and it can itself induce neoplastic transformation. Consequently, we examined eIF-4E as a potential regulator of cyclin D1. Overexpression of cyclin D1 mRNA in NIH 3T3 cells did not increase cyclin D1 protein. In contrast, overexpression of eIF-4E markedly increased the amount of cyclin D1 protein in NIH 3T3 cells. This increase was specific to cyclin D1 in comparison with the retinoblastoma gene product, c-Myc, actin, and eukaryotic initiation factor 2 alpha. We also examined cyclin D1 protein in cells expressing an estrogen receptor-Myc fusion protein because we previously found that eIF-4E increases after induction of c-myc function. In these cells, increased levels of eIF-4E protein were closely followed by increases in levels of cyclin D1 protein, but the level of cyclin D1 mRNA was not increased. We conclude that increases in cyclin D1 levels may result from increased expression of eIF-4E, and this regulation may be one determinant of cyclin D1 levels in the cell.

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External Mg2+-dependent early stimulation of nucleotide synthesis in Swiss 3T3 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.6.c1113 ◽

1989 ◽

Vol 257 (6) ◽

pp. C1113-C1118 ◽

Cited By ~ 7

Author(s):

S. Ishijima ◽

K. Kita ◽

M. Tatibana

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Divalent Cation ◽

Metabolic Flux ◽

Ionophore A23187 ◽

3T3 Cells ◽

Nucleotide Synthesis ◽

Mitogenic Stimulation ◽

Swiss 3T3 ◽

Stimulation Of

In quiescent Swiss 3T3 cells, metabolic flux through phosphoribosyl pyrophosphate into nucleotides increased within 1 h when various growth factors were added (S. Ishijima, K. Kita, N. Kinoshita, T. Ishizuka, N. Suzuki, and M. Tatibana. J. Biochem. 104: 570-575, 1988). The divalent cation ionophore A23187 mimicked the stimulatory effect of epidermal growth factor plus insulin, thereby suggesting involvement of divalent cation mobilization in signaling the stimulation by the growth factors. The stimulation induced by the growth factors was nil in medium devoid of added Mg2+ but was not affected by the omission of Ca2+. The dependency on external Mg2+ was also observed with the stimulations by bombesin plus insulin, fibroblast growth factor, and A23187. In contrast, the mitogenic stimulation of glycolysis was observed irrespective of the presence or absence of Mg2+, indicating that initial events in the signaling process, including mitogen binding to receptors, took place in the absence of exogenous Mg2+. These results suggest that a Mg2(+)-dependent process, subsequent to growth factor binding to receptors, plays an essential role in signaling the early mitogenic stimulation of nucleotide synthesis.

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