scholarly journals Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells

2000 ◽  
Vol 348 (2) ◽  
pp. 409-416 ◽  
Author(s):  
Miyako ARIGA ◽  
Taku NEDACHI ◽  
Masakazu AKAHORI ◽  
Hideki SAKAMOTO ◽  
Yoshiaki ITO ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Signalling Pathways ◽  
Insulin Like Growth Factor ◽  
Dibutyryl Camp ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiates the mitogenic response to insulin-like growth factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of known IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or dibutyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In contrast, cAMP pretreatment potentiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2. We found that the IGF-I-dependent tyrosine phosphorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in the levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound protein 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-activated protein kinase) induced by IGF-I was also enhanced by cAMP. The presence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treatment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increased binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to IRS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatment with and without TSH or dibutyryl cAMP. Our results suggest that the interaction between cAMP-dependent and IGF-I-dependent pathways leads to an augmentation of cell proliferation, which is mediated, at least in part, through the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrates, including IRS-2 and Shc.

scholarly journals Increased basal and induced tyrosine phosphorylation of the insulin- like growth factor I receptor beta subunit in circulating mononuclear cells of patients with polycythemia vera

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 877-882 ◽  
Author(s):  
AM Mirza ◽  
PN Correa ◽  
AA Axelrad
Keyword(s):  
Growth Factor ◽  
Polycythemia Vera ◽  
Tyrosine Phosphorylation ◽  
Mononuclear Cells ◽  
Beta Subunit ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I ◽  
Receptor Beta

We have previously shown that circulating progenitor cells in patients with polycythemia vera (PV) are hypersensitive to insulin-like growth factor I (IGF-I) with respect to erythroid burst formation in serum- free medium, and that this effect occurs through the IGF-I receptor. To investigate the molecular basis of this IGF-I hypersensitivity phenomenon, we examined tyrosine phosphorylation of the IGF-I receptor beta subunit in peripheral blood mononuclear cells (PBMNC) from eight PV patients and six normals. Cells were exposed to IGF-I at concentrations of 10(-8) and 10(-10) mol/L for 0, 1, 3, and 10 minutes, and then lysed. The IGF-I receptor beta subunit was immunoprecipitated, and the protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted with antiphosphotyrosine antibody (4G10). We found that, in the absence of exogenous IGF-I, there was a basal level of tyrosine phosphorylation of the IGF-I receptor beta subunit, and it was substantially greater in PV than in normal. At 10(-10) mol/L IGF-I in normals, no evidence of increased tyrosine phosphorylation was detected; however in PV, a pronounced increase in tyrosine phosphorylation was observed at both 10(-10) and 10(-8) mol/L IGF-I, and it occurred earlier and attained a higher level than in normal. In contrast, in PBMNC from three patients with erythrocytosis, no significant increase above normal was seen in either basal or induced tyrosine phosphorylation of the IGF-I receptor beta subunit. Thus, our findings show two distinctive features of the PV phenotype in PBMNC: (1) an increased basal tyrosine phosphorylation of the IGF-I receptor beta subunit, and (2) a hypersensitive and hyperresponsive receptor with respect to tyrosine phosphorylation. These features may influence the ability of the receptor to transmit a proliferative signal; thus, they may play a role in the pathogenesis of PV.

scholarly journals Activation of the phosphatidylinositol-3' kinase pathway and DNA synthesis by a mutant insulin-like growth factor I receptor lacking the NPXY motif

2004 ◽  
Vol 181 (1) ◽  
pp. 139-146 ◽  
Author(s):  
K Kataoka ◽  
D Yu ◽  
M Miura
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Insulin Like Growth Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Factor I ◽  
Igf I ◽  
Npxy Motif ◽  
Growth Factor I ◽  
Phosphatidylinositol 3

We have investigated the role of the NPXY motif in the insulin-like growth factor I receptor (IGF-IR) by focusing on the activation of the phosphatidylinositol-3' kinase (PI3-K) pathway and DNA synthesis following IGF-I stimulation. For this purpose, we established stable R-cell lines, which are deficient in endogenous IGF-IR, and express human IGF-IR lacking the whole NPEY(950) sequence (DeltaNPEY). The DeltaNPEY cells showed an apparent autophosphorylation of IGF-IR, albeit with reduced sensitivity to stimulation compared with cells expressing similar levels of wild-type IGF-IR. Activation of insulin receptor substrate (IRS)-1 and IRS-2 was severely impaired in DeltaNPEY cells even at high concentrations of IGF-I. However, recruitment of p85, a regulatory subunit of PI3-K, to activated IRS-2 was similar between the cell lines, but recruitment of p85 to IRS-1 was reduced in DeltaNPEY cells. Essentially similar levels of p85- or phosphotyrosine-associated PI3-K and Akt activities were observed between the cell lines, although the sensitivity to stimulation was reduced in DeltaNPEY cells. Activation of extracellular signal-regulated kinase and DNA synthesis were virtually unaffected by the mutation, in terms of both sensitivity to stimulation and responsiveness. DNA synthesis was completely inhibited by the PI3-K inhibitor, LY294002. These results indicate that the IGF-IR is able to activate the PI3-K pathway and induce DNA synthesis in a normal fashion without the NPXY motif when the receptor is fully activated.

scholarly journals Zinc Deprivation of Murine 3T3 Cells by Use of Diethylenetrinitrilopentaacetate Impairs DNA Synthesis upon Stimulation with Insulin-Like Growth Factor-I (IGF-I)

1998 ◽  
Vol 128 (10) ◽  
pp. 1600-1605 ◽  
Author(s):  
Ruth S. MacDonald ◽  
Lavonna C. Wollard-Biddle ◽  
Jimmy D. Browning ◽  
William H. Thornton ◽  
Boyd L. O'Dell
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Insulin Like Growth Factor ◽  
3T3 Cells ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I

scholarly journals Increased basal and induced tyrosine phosphorylation of the insulin- like growth factor I receptor beta subunit in circulating mononuclear cells of patients with polycythemia vera

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 877-882 ◽  
Author(s):  
AM Mirza ◽  
PN Correa ◽  
AA Axelrad
Keyword(s):  
Growth Factor ◽  
Polycythemia Vera ◽  
Tyrosine Phosphorylation ◽  
Mononuclear Cells ◽  
Beta Subunit ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I ◽  
Receptor Beta

Abstract We have previously shown that circulating progenitor cells in patients with polycythemia vera (PV) are hypersensitive to insulin-like growth factor I (IGF-I) with respect to erythroid burst formation in serum- free medium, and that this effect occurs through the IGF-I receptor. To investigate the molecular basis of this IGF-I hypersensitivity phenomenon, we examined tyrosine phosphorylation of the IGF-I receptor beta subunit in peripheral blood mononuclear cells (PBMNC) from eight PV patients and six normals. Cells were exposed to IGF-I at concentrations of 10(-8) and 10(-10) mol/L for 0, 1, 3, and 10 minutes, and then lysed. The IGF-I receptor beta subunit was immunoprecipitated, and the protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted with antiphosphotyrosine antibody (4G10). We found that, in the absence of exogenous IGF-I, there was a basal level of tyrosine phosphorylation of the IGF-I receptor beta subunit, and it was substantially greater in PV than in normal. At 10(-10) mol/L IGF-I in normals, no evidence of increased tyrosine phosphorylation was detected; however in PV, a pronounced increase in tyrosine phosphorylation was observed at both 10(-10) and 10(-8) mol/L IGF-I, and it occurred earlier and attained a higher level than in normal. In contrast, in PBMNC from three patients with erythrocytosis, no significant increase above normal was seen in either basal or induced tyrosine phosphorylation of the IGF-I receptor beta subunit. Thus, our findings show two distinctive features of the PV phenotype in PBMNC: (1) an increased basal tyrosine phosphorylation of the IGF-I receptor beta subunit, and (2) a hypersensitive and hyperresponsive receptor with respect to tyrosine phosphorylation. These features may influence the ability of the receptor to transmit a proliferative signal; thus, they may play a role in the pathogenesis of PV.

scholarly journals Insulin-like growth factor-I-induced DNA synthesis in insulin-secreting cell line RINm5F is associated with phosphorylation of the insulin-like growth factor-I receptor and the insulin receptor substrate-2

1998 ◽  
Vol 156 (3) ◽  
pp. 573-581 ◽  
Author(s):  
Q Zhang ◽  
PO Berggren ◽  
A Hansson ◽  
M Tally
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Insulin Receptor ◽  
Cell Line ◽  
Insulin Receptor Substrate ◽  
Insulin Like Growth Factor ◽  
Rinm5f Cells ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I

A proliferative effect of insulin-like growth factor-I (IGF-I) was previously shown in pancreatic islets. However, the mechanism under which IGF-I actions are exerted in insulin-secreting cells is not clear. The rat insulinoma cell line, RINm5F, was shown to have both IGF-I receptors and IGF-Il/mannose-6-phosphate receptors. IGF-I binding to cell surface receptors stimulated phosphorylation of 97 kDa and 93 kDa subunits of the IGF-I receptor and incorporation of [3H]thymidine into RINm5F cells. Both the IGF-I-induced protein phosphorylation and [3H]thymidine incorporation were abolished in the presence of the tyrosine kinase inhibitor, genistein. Under basal conditions, IGF-I did not induce insulin release or changes in cytosolic free Ca2+ concentration. Immunoprecipitation of proteins from RINm5F cells, using phosphotyrosine antibodies, followed by western blotting using antibody against IRS-1 revealed no distinct band of phosphorylated insulin receptor substrate (IRS)-1. Instead, tyrosine-phosphorylated IRS-2 was detected and stimulated by IGF-I when western blotting was performed using antibody against IRS-2. These results indicate that IRS-1 is not likely to be involved in IGF-I signalling in RINm5F cells. Hence, IGF-I stimulated DNA synthesis in RINm5F cells was associated with phosphorylation of IGF-I receptors and IRS-2.

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