Faculty Bibliography

BACKGROUND: In insulin-sensitive cells, such as adipocytes and skeletal muscle, the activation of phosphoinositide 3-kinase (PI 3-kinase) is thought to be critical in allowing insulin to stimulate both the uptake of glucose and the translocation of a specialized glucose transporter, GLUT4, to the plasma membrane. However, the downstream mediators that couple PI 3-kinase to GLUT4 translocation are still not known. Recent studies have shown that the GTP-binding protein Rac mediates some of the biological effects of PI 3-kinase, and these findings have led to the suggestion that Rac may be a common mediator for a variety of responses mediated by PI 3-kinase. To determine whether Rac couples PI 3-kinase to glucose uptake in adipocytes, we produced 3T3-L1 cells expressing either a constitutively active Rac1 (V12 Rac1, containing a valine residue at position 12) or a dominant-inhibitory Rac1 (N17 Rac1, containing an asparagine residue at position 17). RESULTS: The stable expression of both V12 Rac1 and N17 Rac1 led to observable phenotypes in 3T3-L1 cells; expression of V12 Rac1 resulted in constitutive formation of lamellipodia and constitutive activation of the cJun-N-terminal kinase (JNK), whereas expression of N17 Rac1 inhibited the insulin-stimulated formation of lamellipodia. However, neither basal glucose uptake nor insulin-stimulated glucose uptake was affected by the expression of either mutant Rac protein. In addition, expression of V12 Rac1 did not reverse the inhibition of insulin-stimulated glucose uptake caused by the PI 3-kinase inhibitor wortmannin. CONCLUSIONS: These findings provide direct evidence that PI 3-kinase does not use Rac to couple the insulin receptor to glucose uptake in adipocytes. Furthermore, the finding that Rac does not mediate glucose uptake in response to insulin is consistent with the idea that PI 3-kinase couples to a variety of different effector molecules in cells, and suggests that some of the specificity in the biological responses elicited by PI 3-kinase may be mediated by the activation of different effector molecules

Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells

Ras, a small GTP-binding protein, is an important component of the signal transduction pathway used by growth factors to initiate cell growth and differentiation. Cell activation with growth factors such as epidermal growth factor (EGF) induces Ras to move from an inactive GDP-bound state to an active GTP-bound state. Recently, a combination of genetic and biochemical studies has resulted in the elucidation of a signaling pathway that leads from growth factor receptors to Ras. After binding EGF, the EGF receptor tyrosine kinase is activated, leading to receptor autophosphorylation on multiple tyrosine residues. Signaling proteins with Src homology 2 (SH2) domains then bind to these tyrosine-phosphorylated residues, initiating multiple signaling cascades. One of these SH2 domain proteins, Grb2, exists in the cytoplasm in a preformed complex with a second protein, Son of Sevenless (Sos), which can catalyze Ras GTP/GDP exchange. After growth factor stimulation, the tyrosine phosphorylated EGF receptor binds the Grb2/Sos complex, translocating it to the plasma membrane. This translocation is thought to bring Sos into close proximity with Ras, leading to the activation of Ras. In contrast, the insulin receptor does not bind Grb2 directly but rather induces the tyrosine phosphorylation of two proteins, insulin receptor substrate-1 and Shc, that bind the Grb2/Sos complex. Once Ras is activated, it proceeds to stimulate a cascade of protein kinases that are important in a myriad of growth factor responses

We analyzed the binding site(s) for Grb2 on the epidermal growth factor (EGF) receptor (EGFR), using cell lines overexpressing EGFRs containing various point and deletion mutations in the carboxy-terminal tail. Results of co-immunoprecipitation experiments suggest that phosphotyrosines Y-1068 and Y-1173 mediate the binding of Grb2 to the EGFR. Competition experiments with synthetic phosphopeptides corresponding to known autophosphorylation sites on the EGFR demonstrated that phosphopeptides containing Y-1068, and to a lesser extent Y-1086, were able to inhibit the binding of Grb2 to the EGFR, while a Y-1173 peptide did not. These findings were confirmed by using a dephosphorylation protection assay and by measuring the dissociation constants of Grb2's SH2 domain to tyrosine-phosphorylated peptides, using real-time biospecific interaction analysis (BIAcore). From these studies, we concluded that Grb2 binds directly to the EGFR at Y-1068, to a lesser extent at Y-1086, and indirectly at Y-1173. Since Grb2 also binds Shc after EGF stimulation, we investigated whether Y-1173 is a binding site for the SH2 domain of Shc on the EGFR. Both competition experiments with synthetic phosphopeptides and dephosphorylation protection analysis demonstrated that Y-1173 and Y-992 are major and minor binding sites, respectively, for Shc on the EGFR. However, other phosphorylation sites in the carboxy-terminal tail of the EGFR are able to compensate for the loss of the main binding sites for Shc. These analyses reveal a hierarchy of interactions between Grb2 and Shc with the EGFR and indicate that Grb2 can bind the tyrosine-phosphorylated EGFR directly, as well as indirectly via Shc

Nck, an oncogenic protein composed of one SH2 and three SH3 domains, is a common target for various cell surface receptors. Nck is thought to function as an adaptor protein to couple cell surface receptors to downstream effector molecules that regulate cellular responses induced by receptor activation. In this report, we show that Nck forms a stable complex in vivo with IRS-1 in insulin-stimulated cells. The interaction between IRS-1 and Nck is mediated by the binding of the SH2 domain of Nck to tyrosine-phosphorylated IRS-1. Although Nck associates with IRS-1, Nck phosphorylation is not affected by insulin stimulation. Furthermore, in vitro and in vivo studies show that the SH2 domains of Nck, GRB2, and p85 bind distinct phosphotyrosine residues in IRS-1. After insulin stimulation all three signaling molecules can be found complexed to a single IRS-1 molecule. These findings provide further evidence that, in response to insulin stimulation, IRS-1 acts as an SH2 docking protein that coordinates the regulation of various different signaling pathways activated by the insulin receptor

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85

Many of the actions of receptor tyrosine kinases are mediated by the protein Ras, including the activation of various downstream serine/threonine kinases and the stimulation of growth and differentiation. The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its Src-homology (SH) domains SH2 and SH3, respectively, and like its homologue from Caenorhabditis elegans, Sem-5, apparently forms part of a highly conserved pathway by which these receptors can control Ras activity. Here we show that the SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homologue of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by epidermal growth factor receptor and sevenless. Moreover, a synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction. These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signalling

Insulin-induced activation of extracellular signal-regulated kinases [ERKs, also known as mitogen-activated protein (MAP) kinases] is mediated by Ras. Insulin activates Ras primarily by increasing the rate of guanine nucleotide-releasing activity. Here, we show that insulin-induced activation of ERKs was enhanced by stable overexpression of growth factor receptor-bound protein 2 (GRB2) but not by overexpression of GRB2 proteins with point mutations in the Src homology 2 and 3 domains. Moreover, a dominant negative form of Ras (with Ser17 substituted with Asn) blocked insulin-induced activation of ERKs in cells that overexpressed GRB2. GRB2 overexpression led to increased formation of a complex between the guanine nucleotide-releasing factor Sos (the product of the mammalian homolog of son of sevenless gene) and GRB2. In response to insulin stimulation, this complex bound to tyrosine-phosphorylated IRS-1 (insulin receptor substrate-1) and Shc. In contrast to the activated epidermal growth factor receptor that binds the GRB2-Sos complex directly, activation of the insulin receptor results in the interaction of GRB2-Sos with IRS-1 and Shc, thus linking the insulin receptor to Ras signaling pathways

GRB2, a small protein comprising one SH2 domain and two SH3 domains, represents the human homologue of the Caenorhabditis elegans protein, sem-5. Both GRB2 and sem-5 have been implicated in a highly conserved mechanism that regulates p21ras signalling by receptor tyrosine kinases. In this report we show that in response to insulin, GRB2 forms a stable complex with two tyrosine-phosphorylated proteins. One protein is the major insulin receptor substrate IRS-1 and the second is the SH2 domain-containing oncogenic protein, Shc. The interactions between GRB2 and these two proteins require ligand activation of the insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS-1 and Shc. Although GRB2 associates with IRS-1 and Shc, it is not tyrosine-phosphorylated after insulin stimulation, implying that GRB2 is not a substrate for the insulin receptor. Furthermore, we have identified a short sequence motif (YV/IN) present in IRS-1, EGFR and Shc, which specifically binds the SH2 domain of GRB2 with high affinity. Interestingly, both GRB2 and phosphatidylinositol-3 (PI-3) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS-1 molecule, suggesting a mechanism whereby IRS-1 could provide the core for a large signalling complex. We propose a model whereby insulin stimulation leads to formation of multiple protein--protein interactions between GRB2 and the two targets IRS-1 and Shc. These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules