Abstract
Insulin activation of Ras is mediated by the plasma membrane targeting of the guanylnucleotide exchange factor SOS associated with the small adapter protein Grb2. SOS also lies in an insulin-stimulated feedback pathway in which the serine/threonine phosphorylation of SOS results in disassociation of the Grb2-SOS complex thereby limiting the extent of Ras activation. To examine the relative role of the mitogen-activated protein kinases in the feedback phosphorylation of SOS we determined the signaling specificity of insulin, osmotic shock, and anisomycin to activate the ERK (extracellular-signal regulated kinase) and JNK (c-Jun kinase) pathways. In Chinese hamster ovary cells expressing the human insulin receptor and murine 3T3L1 adipocytes, insulin specifically activated ERK with no significant effect on JNK, whereas anisomycin specifically activated JNK but was unable to activate ERK. In contrast, osmotic shock was equally effective in the activation of both kinase pathways. Insulin and osmotic shock, but not anisomycin, resulted in SOS phosphorylation and disassociation of the Grb2-SOS complex, demonstrating that the JNK pathway was not involved in the insulin-stimulated feedback uncoupling of the Grb2- SOS complex. Both the insulin and osmotic shock-induced activation of ERK was prevented by treatment of cells with the specific MEK inhibitor (PD98059). However, expression of dominant-interfering Ras (N17Ras) inhibited the insulin- but not osmotic shock-stimulated phosphorylation of ERK and SOS. These data demonstrate that activation of the ERK pathway, but not JNK, is responsible for the feedback phosphorylation and disassociation of the Grb2-SOS complex.
Highlights
The mitogen-activated or extracellular-signal regulated kinases (ERK11 and ERK2) are proline-directed serine/threonine kinases that phosphorylate a number of cytosolic and nuclear transcription factors [1]
Effect of Insulin, Osmotic Shock, and Anisomycin on SOS, p90rsk, and ERK Phosphorylation in CHO/IR and 3T3L1 Adipocytes—Previous studies have demonstrated that various stress-related stimuli activate the Jun kinase (JNK) pathway [18, 23]
In contrast to insulin and osmotic shock, the stress inducing agent anisomycin had no significant effect on SOS, p90rsk, or ERK phosphorylation (Fig. 1C)
Summary
The mitogen-activated or extracellular-signal regulated kinases (ERK11 and ERK2) are proline-directed serine/threonine kinases that phosphorylate a number of cytosolic and nuclear transcription factors [1]. The tyrosine phosphorylation of transmembrane receptors and/or Shc results in the formation of a ternary complex (i.e. Shc-Grb2-SOS) that targets SOS to the plasma membrane location of Ras [8, 9] In this manner, SOS can effect the exchange of GDP for GTP on Ras. Once in the activated GTP-bound state, Ras associates with members of the Raf family of serine/threonine kinases (10 –12). In addition to the ERK pathway, mammalian cells contain two related signal transduction systems that function in response to proinflammatory cytokines and various states of stress including ultraviolet irradiation, osmotic, and heat shock [15,16,17,18] These stimuli lead to the threonine/tyrosine phosphorylation and activation of the c-Jun kinase (JNK) and the HOG1/p38 MAP kinase. These data demonstrate that, in contrast to insulin, osmotic shock activates ERK via a MEKdependent but Ras-independent pathway
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