Insulin Stimulation of a MEK-Dependent but ERK-Independent SOS Protein Kinase

Abstract

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

“…Once phosphorylated, Shc recruits Grb-2 and activates the Sos-Ras-c-Raf-Mek1/2-Erk pathway (38)(39)(40). The reduced phosphorylated state of the Erk1/2 protein supports the decrease in islet area observed in LP rats.…”

Constitutive activation of the Raf–MAPK pathway causes negative feedback inhibition of Ras–PI3K–AKT and cellular arrest through the EphA2 receptor

“…These two independent groups showed that only activated MEK1, not MEK2, could phenocopy the effects of activated Raf on skin differentiation in mice (Hobbs et al, 2004;Scholl et al, 2004). There is also evidence in the literature that MEK can signal independently of ERK to have effects in the cell (Holt et al, 1996). This is consistent with what is observed in this study in that both MEK1/2 upregulated ERK activity, but only MEK1 inhibited Ras-PI3K-AKT, suggesting MEK1 signals independent of ERK to have its effect on Ras and AKT.…”

Post-transcriptional silencing of RET occurs, but is not required, during raf-1 mediated differentiation of medullary thyroid carcinoma cells

“…These studies are consistent with the observation that moderate ERK1/2 activity is insu cient to reach the threshold required to di erentiate TT:DRaf-1:ER cells. It is also possible that some of the e ects of MEK in TT cell di erentiation are not mediated through MAPK; such MAPK-independent e ects have been seen in other systems (D'Arcangelo and Halegoua, 1993;Holt et al, 1996). Whether inducible MEK or MAPK constructs can individually trigger TT cell di erentiation or whether additional signaling components are required will need to be addressed in subsequent investigations.…”