Phosphotyrosines 627 and 659 of Gab1 Constitute a Bisphosphoryl Tyrosine-based Activation Motif (BTAM) Conferring Binding and Activation of SHP2

Jess M. Cunnick, Lin Mei, Craig A. Doupnik, Jie Wu

Research output: Contribution to journalArticle

112 Citations (Scopus)

Abstract

A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, we characterized the Gab1-SHP2 interaction. We found that both Tyr-627 and Tyr-659 of Gab1 were required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine (Tyr(P))-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide sub. strates, SHP2 PTPase was activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2ΔN) could not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2ΔN chimera resulted in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 were dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggested that Gab1 was a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase.

Original languageEnglish (US)
Pages (from-to)24380-24387
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number26
DOIs
StatePublished - Jun 29 2001

Fingerprint

Phosphotyrosine
Protein Tyrosine Phosphatases
Non-Receptor Type 11 Protein Tyrosine Phosphatase
src Homology Domains
Binders
Tyrosine
Chemical activation
Extracellular Signal-Regulated MAP Kinases
Epidermal Growth Factor
Mitogen-Activated Protein Kinases
Far-Western Blotting
Phosphopeptides
Peptides
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Phosphotyrosines 627 and 659 of Gab1 Constitute a Bisphosphoryl Tyrosine-based Activation Motif (BTAM) Conferring Binding and Activation of SHP2. / Cunnick, Jess M.; Mei, Lin; Doupnik, Craig A.; Wu, Jie.

In: Journal of Biological Chemistry, Vol. 276, No. 26, 29.06.2001, p. 24380-24387.

Research output: Contribution to journalArticle

@article{b578f7d32a3f43f6a735e965407243f7,
title = "Phosphotyrosines 627 and 659 of Gab1 Constitute a Bisphosphoryl Tyrosine-based Activation Motif (BTAM) Conferring Binding and Activation of SHP2",
abstract = "A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, we characterized the Gab1-SHP2 interaction. We found that both Tyr-627 and Tyr-659 of Gab1 were required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine (Tyr(P))-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide sub. strates, SHP2 PTPase was activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2ΔN) could not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2ΔN chimera resulted in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 were dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggested that Gab1 was a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase.",
author = "Cunnick, {Jess M.} and Lin Mei and Doupnik, {Craig A.} and Jie Wu",
year = "2001",
month = "6",
day = "29",
doi = "10.1074/jbc.M010275200",
language = "English (US)",
volume = "276",
pages = "24380--24387",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "26",

}

TY - JOUR

T1 - Phosphotyrosines 627 and 659 of Gab1 Constitute a Bisphosphoryl Tyrosine-based Activation Motif (BTAM) Conferring Binding and Activation of SHP2

AU - Cunnick, Jess M.

AU - Mei, Lin

AU - Doupnik, Craig A.

AU - Wu, Jie

PY - 2001/6/29

Y1 - 2001/6/29

N2 - A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, we characterized the Gab1-SHP2 interaction. We found that both Tyr-627 and Tyr-659 of Gab1 were required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine (Tyr(P))-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide sub. strates, SHP2 PTPase was activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2ΔN) could not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2ΔN chimera resulted in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 were dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggested that Gab1 was a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase.

AB - A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, we characterized the Gab1-SHP2 interaction. We found that both Tyr-627 and Tyr-659 of Gab1 were required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine (Tyr(P))-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide sub. strates, SHP2 PTPase was activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2ΔN) could not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2ΔN chimera resulted in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 were dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggested that Gab1 was a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase.

UR - http://www.scopus.com/inward/record.url?scp=0035968279&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035968279&partnerID=8YFLogxK

U2 - 10.1074/jbc.M010275200

DO - 10.1074/jbc.M010275200

M3 - Article

VL - 276

SP - 24380

EP - 24387

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 26

ER -