Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase

Kathleen H. Holt, Barry G. Kasson, Jeffrey E. Pessin

Research output: Contribution to journalArticlepeer-review

66 Scopus citations

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 dues 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 fur SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.

Original languageEnglish (US)
Pages (from-to)577-583
Number of pages7
JournalMolecular and cellular biology
Volume16
Issue number2
DOIs
StatePublished - Feb 1996
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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