MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: Analysis of phosphopeptides and regulation of activity

Anne M. Gardner, Richard Vaillancourt, Carol A. Lange-Carter, Gary L. Johnson

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal- regulated kinase kinase (MEKK) independently phosphorylate and activate MEK- 1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK- 1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.

Original languageEnglish (US)
Pages (from-to)193-201
Number of pages9
JournalMolecular Biology of the Cell
Volume5
Issue number2
StatePublished - Feb 1994
Externally publishedYes

Fingerprint

MAP Kinase Kinase Kinase 1
Phosphopeptides
Mitogen-Activated Protein Kinase Kinases
Mitogen-Activated Protein Kinases
Phosphorylation
Extracellular Signal-Regulated MAP Kinases
Phosphotransferases
Threonine
raf Kinases
Phosphoamino Acids
MAP Kinase Kinase Kinases

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase : Analysis of phosphopeptides and regulation of activity. / Gardner, Anne M.; Vaillancourt, Richard; Lange-Carter, Carol A.; Johnson, Gary L.

In: Molecular Biology of the Cell, Vol. 5, No. 2, 02.1994, p. 193-201.

Research output: Contribution to journalArticle

@article{933cbe01241b4ec092f6218ec4c115fa,
title = "MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: Analysis of phosphopeptides and regulation of activity",
abstract = "MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal- regulated kinase kinase (MEKK) independently phosphorylate and activate MEK- 1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK- 1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90{\%} of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.",
author = "Gardner, {Anne M.} and Richard Vaillancourt and Lange-Carter, {Carol A.} and Johnson, {Gary L.}",
year = "1994",
month = "2",
language = "English (US)",
volume = "5",
pages = "193--201",
journal = "Molecular Biology of the Cell",
issn = "1059-1524",
publisher = "American Society for Cell Biology",
number = "2",

}

TY - JOUR

T1 - MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase

T2 - Analysis of phosphopeptides and regulation of activity

AU - Gardner, Anne M.

AU - Vaillancourt, Richard

AU - Lange-Carter, Carol A.

AU - Johnson, Gary L.

PY - 1994/2

Y1 - 1994/2

N2 - MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal- regulated kinase kinase (MEKK) independently phosphorylate and activate MEK- 1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK- 1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.

AB - MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal- regulated kinase kinase (MEKK) independently phosphorylate and activate MEK- 1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK- 1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.

UR - http://www.scopus.com/inward/record.url?scp=0028297817&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028297817&partnerID=8YFLogxK

M3 - Article

C2 - 8019005

AN - SCOPUS:0028297817

VL - 5

SP - 193

EP - 201

JO - Molecular Biology of the Cell

JF - Molecular Biology of the Cell

SN - 1059-1524

IS - 2

ER -