Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3‐kinase in rat hepatocyte primary culture

Abstract

Short-term incubation of adult rat hepatocytes with epidermal growth factor (EGF) caused tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 when the cells had been submitted to primary culture from 1±18 h. Tyrosine-phosphorylated IRS-1 and IRS-2 bound to the regulatory subunit (p85) of phosphatidylinositol (PtdIns) 3-kinase, thereby activating the enzymic activity. Tyrosine phosphorylation of the IRSs and activation of PtdIns 3-kinase in 3 h cultured hepatocytes both proceeded similarly to the same actions of insulin; the activation was rapid and transient, with peak values at 15±30 s and with similar EC 50 s in the nm range in both cases. A possible involvement of insulin receptors in these insulin-like actions of EGF was excluded by the following three lines of evidence. Insulin caused tyrosine phosphorylation of the insulin receptor b-subunit but EGF did not. In contrast, the EGF receptor was phosphorylated by EGF, but the insulin receptor was not. The actions of EGF, but not those of insulin, were inhibited by AG1478, a selective inhibitor of EGF receptor tyrosine kinase. Cultured hepatocytes exposed to insulin or insulin-like growth factor-I (IGF-I) for a short period responded to the subsequent addition of EGF, whereas EGF-treated cells responded to insulin. The cells, however, displayed receptor desensitization under the same conditions, that is, no response was observed upon repeated addition of the same agonist, EGF, insulin or IGF-I. Thus, the EGF receptor-initiated signalling was mediated by PtdIns 3-kinase associated with tyrosine-phosphorylated IRSs in short-term cultured rat hepatocytes.Keywords: signal transduction; tyrosine kinase receptor; tyrosine phosphorylation.Membrane receptors having intrinsic protein tyrosine kinase activity are known to be responsible for promoting proliferation of hepatocytes [1] as well as many other types of cells [2]. If kept in a serum-free medium, DNA synthesis in hepatocytes occurs with the addition of growth factors, among which epidermal growth factor (EGF) is effective in vivo and in culture [3,4] as a result of its binding to typical tyrosine kinase related receptors. The EGF receptor (EGFR) is the prototype of the type I ErbB subfamily, which includes three additional members, ErbB-2/Neu, ErbB3 and ErbB4 [5]. All members of the ErbB subfamily have a domain that serves as a docking site for specific SH-2 domains, which are present in signalling proteins such as the p85 regulatory subunit of phosphatidylinositol (PtdIns) 3-kinase. EGF has been shown to activate PtdIns 3-kinase, although it does not bind directly to the EGFR but is instead activated via association with p120 cbl [6,7] or Gab1 [8], which are phosphorylated on tyrosine residues by the growth factor. PtdIns 3-kinase may also interact with heterodimers of other members of the EGFR family, such as ErbB3, in response to EGF [9,10].PtdIns 3-kinase plays important roles in a wide variety of biological responses to hormones, growth factors and cytokines [11]. The typical responses s...

Epidermal Growth Factor and Transforming Growth Factor α Mimic the Effects of Insulin in Human Fat Cells and Augment Downstream Signaling in Insulin Resistance

“…The finding that EGF elicits the phosphorylation of both IRS-1 and IRS-2 is in agreement with the recent work of Fujioka et al in cultured rat hepatocytes as well as A431 cells abundantly expressing EGFR (38,44). These authors reported that EGFR directly phosphorylated IRS-1 in in vitro assays (44) and that this was markedly attenuated if the C-terminal of EGFR was truncated.…”

“…The EGF receptor has three potential PTB-binding sites allowing for an interaction with substrates like IRS-1 (38) and IRS-2. The present data clearly show that EGF increases the tyrosine phosphorylation of the same signaling molecules as seen with insulin and, in addition, Gab1 as well as its cognate receptor.…”

Hepatoprotective Effects of Tribulus terrestris Hydro-Alcholic Extract on Non-Alcoholic Fatty Liver- Induced Rats

“…Steroids are involved in conversion of cholesterol to VLDL and exertion of cholesterol as the bile. Thus, the increased VLDL as long as decreased total serum level of cholesterol in T. terrestris treated rats could be explained by steroids component of the extract (Fujioka & Ui, 2001). Saponin is another component of the extract not only regulates lipid metabolism but also improves hypercholesterolemia and hypertriglyceridemia (Yan et al, 1996).…”

“…Thus, this component of the extract could reduce the absorption of fructose and also interrupt the fructose metabolization to lipids (Gosain et al, 2010). Steroids are involved in conversion of cholesterol to VLDL and exertion of cholesterol as the bile.…”

A Compendium of Signals and Responses Triggered by Prodeath and Prosurvival Cytokines

“…EGF-induced IRS1 modification was not unique to HT-29 cells: EGFR-dependent IRS1-Tyr 896 phosphorylation was also observed in A431 epidermoid carcinoma cells. 4 EGF has previously been reported to promote tyrosine phosphorylation of IRS1, but specific sites of phosphorylation have not been identified (43)(44)(45). To search for candidate IRS1-Tyr 896 kinases, we used the Scansite motif-based searching algorithm (46), which compares Swiss-Prot protein sequences to experimentally determined consensus sites for dozens of protein kinases.…”