Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1

Fred Robinson, Angelique W. Whitehurst, Malavika Raman, Melanie H. Cobb

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2·MEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding α-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding α-helix G in the interaction of ERK2 with MEK1/2.

Original languageEnglish (US)
Pages (from-to)14844-14852
Number of pages9
JournalJournal of Biological Chemistry
Volume277
Issue number17
DOIs
StatePublished - Apr 26 2002
Externally publishedYes

Fingerprint

Mitogen-Activated Protein Kinases
Point Mutation
Mitogen-Activated Protein Kinase Kinases
Proteins
Phosphotransferases
Chemical activation
Mutation
Phosphorylation
Mitogen-Activated Protein Kinase 3
HEK293 Cells
Mitogen-Activated Protein Kinase 1
Mutant Proteins
Mitogens
Epidermal Growth Factor
Gene expression
Yeast
Protein Kinases
Yeasts
Membranes
Gene Expression

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1. / Robinson, Fred; Whitehurst, Angelique W.; Raman, Malavika; Cobb, Melanie H.

In: Journal of Biological Chemistry, Vol. 277, No. 17, 26.04.2002, p. 14844-14852.

Research output: Contribution to journalArticle

Robinson, Fred ; Whitehurst, Angelique W. ; Raman, Malavika ; Cobb, Melanie H. / Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 17. pp. 14844-14852.
@article{480ded376602415aaf2a74bfe33fa80a,
title = "Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1",
abstract = "Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2·MEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding α-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding α-helix G in the interaction of ERK2 with MEK1/2.",
author = "Fred Robinson and Whitehurst, {Angelique W.} and Malavika Raman and Cobb, {Melanie H.}",
year = "2002",
month = "4",
day = "26",
doi = "10.1074/jbc.M107776200",
language = "English (US)",
volume = "277",
pages = "14844--14852",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "17",

}

TY - JOUR

T1 - Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1

AU - Robinson, Fred

AU - Whitehurst, Angelique W.

AU - Raman, Malavika

AU - Cobb, Melanie H.

PY - 2002/4/26

Y1 - 2002/4/26

N2 - Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2·MEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding α-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding α-helix G in the interaction of ERK2 with MEK1/2.

AB - Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2·MEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding α-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding α-helix G in the interaction of ERK2 with MEK1/2.

UR - http://www.scopus.com/inward/record.url?scp=0037177819&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037177819&partnerID=8YFLogxK

U2 - 10.1074/jbc.M107776200

DO - 10.1074/jbc.M107776200

M3 - Article

VL - 277

SP - 14844

EP - 14852

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 17

ER -