A Novel 160-kDa Phosphotyrosine Protein in Insulin-treated Embryonic Kidney Cells Is a New Member of the Insulin Receptor Substrate Family

Abstract

We have previously identified a 160-kDa protein in human embryonic kidney (HEK) 293 cells that undergoes rapid tyrosine phosphorylation in response to insulin (PY160) (Kuhné , M. R., Zhao, Z., and Lienhard, G. E. (1995) Biochem. Biophys. Res. Commun. 211,[190][191][192][193][194][195][196][197]. The phosphotyrosine form of PY160 was purified from insulin-treated HEK 293 cells by anti-phosphotyrosine immunoaffinity chromatography, the sequences of peptides determined, and its cDNA cloned. The PY160 cDNA encodes a 1257-amino acid protein that contains, in order from its N terminus, a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain, and, spread over the C-terminal portion, 12 potential tyrosine phosphorylation sites. Several of these sites are in motifs expected to bind specific SH2 domain-containing proteins: YXXM (7 sites), phosphatidylinositol 3-kinase; YVNM (1 site), Grb-2; and YIEV (1 site), either the protein-tyrosine phosphatase SHP-2 or phospholipase C␥. Furthermore, the PH and PTB domains are highly homologous (at least 40% identical) to those found in insulin receptor substrates 1, 2, and 3 (IRS-1, IRS-2, and IRS-3). Thus, PY160 is a new member of the IRS family, which we have designated IRS-4.The insulin receptor is a tyrosine kinase, which when activated by insulin binding phosphorylates cellular substrates. The most well characterized of these are two members of the IRS 1 family, IRS-1 and IRS-2, and the protein Shc. Tyrosine phosphorylation of the IRS proteins creates binding sites for SH2 domain-containing signaling molecules, including PI 3-kinase, the adapter molecule Grb-2, and the protein-tyrosine phosphatase SHP-2. Docking of these proteins in turn activates specific signal transduction pathways (reviewed in Refs. 1 and 2). Recently, we have identified, by purification and cloning, a third member of the IRS family, called IRS-3, which in insulintreated adipocytes is tyrosine-phosphorylated and associated with PI 3-kinase (3, 4). All three IRS family members possess a common domain structure that includes PH and PTB domains at the N terminus and, C-terminal to these, a number of potential tyrosine phosphorylation sites (1,2,4,5). The presence of these features can therefore be viewed as defining an IRS. Previously, we have identified a 160-kDa protein in HEK 293 cells, termed PY160, which is rapidly tyrosine-phosphorylated in response to insulin but which is immunologically unrelated to IRS-1 (6). In the present study we have isolated PY160 from insulin-treated HEK 293 cells and cloned its cDNA. The predicted amino acid sequence shows that PY160 is a new member of the IRS family. EXPERIMENTAL PROCEDURESCell Culture and Preparation of Lysates-HEK 293 cells were grown on 10-cm plates as described previously (6). Before use, confluent plates of cells were incubated in serum-free medium for 2 h and then incubated for 5 min further with either no addition or the addition of 1 M insulin to activate fully the insulin and IGF-1 receptors present on these cells (7). Each plate was ...

Adenovirus 5 E1A is responsible for increased expression of insulin receptor substrate 4 in established adenovirus 5-transformed cell lines and interacts with IRS components activating the PI3 kinase/Akt signalling pathway

“…IRS-4 expression increases cellular proliferation (White, 1998;Fantin et al, 1999), and may be implicated in cellular differentiation (Giovannone et al, 2000;Tseng et al, 2004). Notably for our study, IRS-4 was first discovered and characterized in Ad5E1HEK293 cells, where it is the predominantly expressed IRS protein (Lavan et al, 1997;Fantin et al, 1998).…”

An arginine to cysteine252 mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events

“…Insulin binding to the α-subunit elicits a conformational change of the receptor resulting in activation of the β-subunit tyrosine kinase which, in turn, phosphorylates multiple endogenous proteins including the IR substrates (IRS 1 to 4), Shc and other cellular proteins [1,2,3,4,5,6,7,8]. IRS and Shc serve as docking proteins for several specific signalling molecules [9].…”

Discordant Effects of Glucosamine on Insulin-stimulated Glucose Metabolism and Phosphatidylinositol 3-Kinase Activity

“…The metabolic actions of insulin mediated by PI3K include glucose uptake (14), GLUT4 translocation (15), and glycogen synthase activation (16). Additionally, PI3K binds to all four insulin receptor substrates (IRS-1 to 4) as yet identified (17), highlighting its vital role in insulin signaling. Decreased PI3K activity in skeletal muscle has been observed in in vivo models of insulin resistance (18 -20).…”