Faculty Bibliography

Stimulation of the insulin receptor (IR) results in tyrosine phosphorylation of the intermediate molecules insulin receptor substrate-1 (IRS-1), IRS-2, and Shc, which then couple the IR to downstream signaling pathways by serving as binding sites for signaling molecules with SH2 domains. It has been proposed that direct binding of IRS-1, IRS-2, and Shc to an NPX-Tyr(P) motif in the juxtamembrane region of the IR is required for tyrosine phosphorylation of these molecules by the IR. In this regard, Shc and IRS-1 contain domains that are distinct from SH2 domains, referred to as the phosphotyrosine binding (PTB) or phosphotyrosine interaction (PI) domains, which bind phosphotyrosine in the context of an NPX-Tyr(P) motif. To further clarify the role of the Shc PTB/PI domain, we identified a mutation in this domain that abrogated binding of Shc to the IR in vitro. Interestingly, this mutation completely abolished Shc phosphorylation by the IR in vivo whereas mutation of the arginine in the FLVRES motif of the Shc SH2 domain did not affect Shc phosphorylation by insulin. In addition, we identified specific amino acids on the IR that are required for the IR to stimulate Shc but not IRS-1 phosphorylation in vivo. As with the PTB/PI domain Shc mutant, the ability of these mutant receptors to phosphorylate Shc correlates with the binding of the PTB/PI domain of Shc to similar sequences in vitro. These findings support a model in which binding of the PTB/PI domain of Shc directly to the NPX-Tyr(P) motif on the IR mediates Shc phosphorylation by insulin

Tumor necrosis factor (TNF) activates both p42 and p44 mitogen-activated protein kinases (MAPK) in human FS-4 fibroblasts, cells for which TNF is mitogenic. We now show that TNF activates p42 MAPK in two cell lines whose growth is inhibited by TNF. A mutant TNF that binds only to the p55 TNF receptor (TNFR) produced a similar degree of activation as wild-type TNF in FS-4 fibroblasts, indicating that the p55 TNFR is sufficient to mediate p42/p44 MAPK activation. The upstream intracellular signals that couple the TNFR to MAPK activation are still poorly defined. We now show that neither phorbol ester-sensitive protein kinase C nor Gialpha link TNF to p42/p44 MAPK activation, because pretreatment of FS-4 cells with phorbol ester to down-regulate protein kinase C or pretreatment with pertussis toxin to block Gialpha does not inhibit p42/p44 MAPK activation by TNF. To further analyze MAPK activation in FS-4 cells, we compared p42/p44 MAPK activation by TNF and epidermal growth factor (EGF). While tyrosine phosphorylation of p42/p44 MAPK was detected almost immediately (30 s) after stimulating cells with EGF, TNF-induced tyrosine phosphorylation was detected only after a more prolonged time interval (initially detected at 5 min and peaking at 15-30 min). In addition, the anti-inflammatory drug sodium salicylate, previously demonstrated to inhibit NF- kappaB activation by TNF, blocked the activation of p42/p44 MAPK in response to TNF but not in response to EGF. These findings demonstrate that the TNF and EGF receptors utilize distinct signaling molecules to couple to MAPK activation. Elucidation of the mechanism whereby sodium salicylate blocks TNF-induced p42/p44 MAPK activation may help to clarify TNF-activated signaling pathways

Adipocytes produce a variety of molecules that are capable of functioning in both a paracrine and autocrine fashion. Tumor necrosis factor (TNF) is one of the proteins produced by adipocytes that has been shown to regulate adipocyte function. Interestingly, adipocyte expression of TNF increases with increasing adipocyte mass and expression of TNF is increased in adipocytes isolated from several genetic models of rodent obesity and from obese humans. This finding has led to the idea that TNF produced by adipocytes functions as a local 'adipostat' to limit fat accumulation. Increased production of TNF by adipocytes, however, may contribute to insulin resistance in obesity and in non-insulin-dependent diabetes mellitus (NIDDM). TNF has been shown to inhibit insulin-simulated tyrosine phosphorylation of both the insulin receptor (IR) and insulin receptor substrate (IRS)-1 and to stimulate downregulation of the insulin-sensitive glucose transporter, GLUT4, in adipocytes. These findings raise the possibility that pharmacological inhibition of TNF may provide a novel therapeutic target to treat patients with NIDDM

Nck is an adaptor protein composed of a single SH2 domain and three SH3 domains. Upon growth factor stimulation, Nck is recruited to receptor tyrosine kinases via its SH2 domain, probably initiating one or more signaling cascades. In this report, we show that Nck is bound in living cells to the serine-threonine kinase Pak1. The association between Nck and Pak1 is mediated by the second SH3 domain of Nck and a proline-rich sequence in the amino terminus of Pak1. We also show that Pak1 is recruited by activated epidermal growth factor (EGF) and platelet-derived growth factor receptors. Moreover, Pak1 kinase activity is increased in response to EGF in HeLa cells transfected with human Pak1, and the kinase activity was enhanced when Nck was co-transfected. It is concluded that Nck links receptor tyrosine kinases with Pak1 and is probably involved in targeting and regulation of Pak1 activity

Insulin binding results in rapid phosphorylation of insulin receptor substrate-1 to activate p21ras and mitogen-activated protein kinase. Insulin also activates the ribosomal protein S6 kinase (pp70 S6 kinase) independently of the Ras pathway. Chronic (18 h) treatment of L6 muscle cells with insulin increases glucose transport activity severalfold due to biosynthetic elevation of the GLUT1 and GLUT3 but not the GLUT4 glucose transporters. Here we investigate the roles of p21ras and pp70 S6 kinase in the insulin-mediated increases in GLUT1 and GLUT3 expression. L6 cells were transfected with the dominant negative Ras(S17N) under the control of a dexamethasone-inducible promoter. Induction of Ras(S17N) failed to block the insulin-mediated increase in GLUT1 glucose transporter protein and mRNA; however, it abrogated the insulin-mediated increase in GLUT3 glucose transporter protein and mRNA. Inhibition of pp70 S6 kinase by rapamycin, on the other hand, eliminated the insulin-mediated increase in GLUT1 but had no effect on that of GLUT3 in both parental and Ras(S17N) transfected L6 cells. These results suggest that the biosynthetic regulation of glucose transporters is differentially determined, with pp70 S6 kinase and p21ras playing active roles in the insulin-stimulated increases in GLUT1 and GLUT3, respectively.

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)