Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.
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.
Holt
K H
KH
Department of Physiology & Biophysics, University of Iowa, Iowa City 52242-1109, USA.
Kasson
B G
BG
Pessin
J E
JE
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D018797:Cell Cycle Proteins; D006224:Cricetinae; D054638:Dual Specificity Phosphatase 1; D051380:GRB2 Adaptor Protein; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D048728:MAP Kinase Kinase Kinase 1; D008565:Membrane Proteins / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D020664:Son of Sevenless Proteins
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D017874:Immediate-Early Proteins; D007328:Insulin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D011994:Recombinant Proteins; D020664:Son of Sevenless Proteins; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases