INSULIN STIMULATION OF A MEK-DEPENDENT BUT ERK-INDEPENDENT SOS PROTEIN-KINASE

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosp horylation and dissociation from Grb2 following insulin receptor kinas e activation of Ras. To determine the serine/threonine kinase(s) respo nsible for SOS phosphorylation in vivo, we assessed the role of mitoge n-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 do minant-interfering MEK mutant, in which lysine 97 was replaced with ar ginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively activ e MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Alt hough expression of the mitogen-activated protein kinase-specific phos phatase (MKP-1) completely inhibited the insulin stimulation of ERK ac tivity both in vitro and in vivo, SOS phosphorylation and the dissocia tion of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specif icity of insulin for the ERK pathway. The insulin-stimulated and MKP-1 -insensitive SOS-phosphorylating activity was reconstituted in whole-c ell extracts and did not bind to a MonoQ anion-exchange column. In con trast, ERK1/2 protein was retained by the MonoQ column, eluted with ap proximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also do es not bind to MonoQ, immunodepletion analysis demonstrated that MEK i s not the insulin-stimulated SOS-phosphorylating activity. Together, t hese data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS compl ex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.