scholarly journals Correlation of α-Fetoprotein Expression in Normal Hepatocytes during Development with Tyrosine Phosphorylation and Insulin Receptor Expression

1998 ◽  
Vol 9 (5) ◽  
pp. 1093-1105 ◽  
Author(s):  
Leila Khamzina ◽  
Pierre Borgeat
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Receptor Expression ◽  
Regulatory Subunit ◽  
Β Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Fetal Hepatocytes ◽  
In Cells ◽  
Phosphatidylinositol 3

The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the α-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP−) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+clones and likewise for adult hepatocytes and AFP− clones. The tyrosine phosphorylation of several proteins, including the β-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, andras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP− clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor β-subunit compared with adult hepatocytes and AFP− clones and, accordingly, that these AFP+clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP− clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.

Changes in tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) and association of p85 subunit of phosphatidylinositol 3-kinase with IRS-1 after feeding in rat liver in vivo

1997 ◽  
Vol 154 (2) ◽  
pp. 267-273 ◽  
Author(s):  
Y Ito ◽  
M Ariga ◽  
S-I Takahashi ◽  
A Takenaka ◽  
T Hidaka ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Rat Liver ◽  
Insulin Receptor Substrate ◽  
Β Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Substrate 1 ◽  
Insulin Receptor Tyrosine Kinase ◽  
Phosphatidylinositol 3

Abstract The binding of insulin to its receptor rapidly induces intrinsic insulin receptor tyrosine kinase activity, resulting in tyrosine phosphorylation of various cytosolic substrates, such as insulin receptor substrate-1 (IRS-1) which, in turn, associates with a p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) followed by activation of this enzyme. In the present study, we have examined these early steps of insulin signalling in rat liver in vivo after food ingestion. After fasting for 22 h, a 12% casein diet was available ad libitum throughout the 8-h experimental period. Plasma insulin concentrations increased within 45 min after feeding, reached a maximum at 1·5 h and gradually decreased until 8 h. Autophosphorylation of the insulin receptor β-subunit in liver was detected even during fasting and increased about 1·5-fold at 1·5 h after feeding. Basal tyrosine phosphorylation of IRS-1 was detectable during starvation, increased about twofold at 3 h after feeding and levels were maintained until 8 h. The content of the p85 subunit of PI 3-kinase associated with IRS-1 also increased after feeding in parallel with the changes in tyrosine phosphorylation of IRS-1. Because tyrosine phosphorylation of the insulin receptor β-subunit and IRS-1 and the association of the p85 subunit of PI 3-kinase with IRS-1 in liver were closely correlated with the changes in the plasma concentration of insulin, we concluded that endogenous insulin secreted in response to eating caused these insulin-dependent intracellular changes in the liver. Journal of Endocrinology (1997) 154, 267–273

scholarly journals Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

1995 ◽  
Vol 15 (10) ◽  
pp. 5403-5411 ◽  
Author(s):  
M J Quon ◽  
H Chen ◽  
B L Ing ◽  
M L Liu ◽  
M J Zarnowski ◽  
...  
Keyword(s):  
Cell Surface ◽  
Insulin Receptor ◽  
Glucose Transport ◽  
Expression Vectors ◽  
Regulatory Subunit ◽  
Dominant Negative Effect ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Adipose Cells ◽  
Phosphatidylinositol 3

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.

Prolactin induced tyrosine phosphorylation of p59fyn may mediate phosphatidylinositol 3-kinase activation in Nb2 cells

1997 ◽  
Vol 19 (3) ◽  
pp. 347-350 ◽  
Author(s):  
KA al-Sakkaf ◽  
PR Dobson ◽  
BL Brown
Keyword(s):  
Tyrosine Phosphorylation ◽  
Pi3 Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Src Tyrosine Kinase ◽  
Kinase Activation ◽  
Nb2 Cells ◽  
Co Precipitation ◽  
Dose Dependent ◽  
Phosphatidylinositol 3

Our previous studies indicated that PI3-kinase is involved in prolactin (PRL) signalling. We have now examined the involvement of the src tyrosine kinase, fyn, in PRL-induced the activation of PI3-kinase in the rat lymphoma cell line, Nb2. Cells were stimulated with increasing doses of PRL, lysed and immunoprecipitated with anti-fyn specific antibody. Then PI3-kinase activity was measured as the increase in the phosphorylation of phosphatidylinositol to phosphatidylinositol 3-phosphate separated by TLC. Our data indicated that, in PRL treated cells, co-precipitation of PI3-kinase with anti-fyn antiserum led to time and dose-dependent activation of PI3-kinase in vitro and that this activation was blocked by the addition of LY294002. However, LY294002 appeared to have no effect on fyn autophosphorylation. Furthermore, the physical association of PI3-kinase with fyn was confirmed by Western blot analysis employing the same specific antisera. These data provide evidence that PRL-induced activation of PI3-kinase may be mediated by the tyrosine phosphorylation of fyn in Nb2 cells.

scholarly journals Phosphatidylinositol 3-kinase associates, via its Src homology 2 domains, with the activated erythropoietin receptor

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3204-3210 ◽  
Author(s):  
JE Damen ◽  
AL Mui ◽  
L Puil ◽  
T Pawson ◽  
G Krystal
Keyword(s):  
Erythropoietin Receptor ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Protein Substrates ◽  
Sh2 Domains ◽  
Src Homology 2 ◽  
Src Homology ◽  
Specific Association ◽  
Phosphatidylinositol 3

The erythropoietin receptor (EpR) belongs to a family of hematopoietin receptors whose members lack tyrosine kinase activity. Nonetheless, within minutes of binding Ep, a number of cellular proteins become transiently phosphorylated on tyrosine residues. One of these proteins, as we and others have shown previously, is the EpR itself. To identify the remaining protein substrates, we have examined the antiphosphotyrosine immunoprecipitates of lysates from Ba/F3 cells expressing high levels of cell surface EpRs. We now present data showing that, in response to Ep, the 85-Kd regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) becomes immunoprecipitable with antiphosphotyrosine antibodies. This appears to be due, in large part, to the specific association of PI 3-kinase with the tyrosine- phosphorylated EpR, either directly or through a 93- or 70-Kd tyrosine- phosphorylated intermediate. The activity of this EpR associated PI 3- kinase, assessed in anti-EpR immunoprecipitates, is maximal within 2 minutes of incubation with Ep and returns almost to baseline levels by 10 minutes. In vitro studies suggest that the interaction between PI 3- kinase and the activated EpR is mediated by the N- and C-terminal SH2 domains of p85 and tyrosine-phosphorylated motifs on the EpR.

scholarly journals The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase.

1995 ◽  
Vol 15 (8) ◽  
pp. 4453-4465 ◽  
Author(s):  
S Pons ◽  
T Asano ◽  
E Glasheen ◽  
M Miralpeix ◽  
Y Zhang ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Regulatory Subunit ◽  
Stable Complex ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Cellular Processes ◽  
Src Homology 2 ◽  
Src Homology ◽  
And Function ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55PIK, a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identify to those in p85. p55PIK mRNA is expressed early during development, remains abundant in adult mouse brain and testis tissue, and is detectable in adult adipocytes and heart and kidney tissues. p55PIK forms a stable complex with p110, and it associates with IRS-1 during insulin stimulation. Moreover, the activated insulin receptor phosphorylates p55PIK in Sf9 cells, and insulin stimulates p55PIK phosphorylation in CHOIR/p55PIK cells. The unique features of p55PIK suggest that it is important in receptor signaling.

scholarly journals Detection of a 60 kDa tyrosine-phosphorylated protein in insulin-stimulated hepatoma cells that associates with the SH2 domain of phosphatidylinositol 3-kinase

1995 ◽  
Vol 308 (2) ◽  
pp. 579-583 ◽  
Author(s):  
K L Milarski ◽  
D F Lazar ◽  
R J Wiese ◽  
A R Saltiel
Keyword(s):  
Signal Transduction ◽  
Insulin Receptor ◽  
Hepatoma Cells ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphorylated Protein ◽  
Tyrosine Phosphorylated Protein ◽  
Phosphatidylinositol 3

Activation of the tyrosine kinase activity of the insulin receptor by autophosphorylation leads to phosphorylation of cellular substrates on tyrosine. Thus far, the best characterized is the insulin receptor substrate (IRS) 1, which has been proposed to serve as a docking protein for other molecules involved in signal transduction. A number of other proteins that become phosphorylated in response to insulin have been identified, some of which are reported to be tissue-specific. A 60 kDa phosphoprotein has been detected in adipocytes after insulin stimulation [Lavan and Lienhard (1993) J. Biol. Chem. 268, 5921-5928]. We have identified a protein of similar molecular mass in rat hepatoma cells transfected with the human insulin receptor. The 60 kDa protein in hepatoma cells is tyrosine-phosphorylated in response to insulin in a dose-dependent manner, with maximal phosphorylation occurring at 50 nM insulin. Although the dose-response of p60 phosphorylation mirrors that of IRS-1, the time course is slightly slower, with maximal phosphorylation observed 5 min after addition of insulin. Like the adipocyte protein, the 60 kDa protein detected in liver cells binds to the SH2 domain of the p85 regulatory subunit of phosphatidylinositol 3-kinase, but not to other SH2 domains. Binding of p60 to p85 is similar to the interaction between p85 and IRS-1 in that a tyrosine-phosphorylated peptide containing the YVXM motif can inhibit the association. The presence of this 60 kDa tyrosine-phosphorylated protein in adipocytes and hepatoma cells suggests that it represents another important intermediate in the insulin-receptor signal-transduction pathway.

Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression

1994 ◽  
Vol 14 (11) ◽  
pp. 7466-7475
Author(s):  
B H Jhun ◽  
D W Rose ◽  
B L Seely ◽  
L Rameh ◽  
L Cantley ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Dna Synthesis ◽  
Insulin Receptor ◽  
Signal Transduction Pathway ◽  
Sh2 Domain ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Substrate 1 ◽  
Phosphatidylinositol 3

We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction.

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