T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-γ1-binding phosphotyrosyl protein pp36/38

T. Fukazawa, K. A. Reedquist, G. Panchamoorthy, S. Soltoff, T. Trub, B. Druker, L. Cantley, S. E. Shoelson, Hamid Band

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as ζ and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-γ1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85α cDNA into Jurkat cells followed by antihemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI- 3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.

Original languageEnglish (US)
Pages (from-to)20177-20182
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number34
DOIs
StatePublished - Jan 1 1995

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Phosphatidylinositol 3-Kinase
T-cells
Type C Phospholipases
src Homology Domains
Carrier Proteins
Chemical activation
Association reactions
T-Lymphocytes
Phosphorylation
Tyrosine
Jurkat Cells
Cell membranes
T-Cell Antigen Receptor
Proteins
Cell Membrane
Phosphopeptides
Lymphocytes
5'-((N-(2-decanoylamino-3-hydroxy-3-phenylpropyloxycarbonyl)glycyl)amino)-5'-deoxyuridine
Hemagglutinins
Lymphocyte Activation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-γ1-binding phosphotyrosyl protein pp36/38. / Fukazawa, T.; Reedquist, K. A.; Panchamoorthy, G.; Soltoff, S.; Trub, T.; Druker, B.; Cantley, L.; Shoelson, S. E.; Band, Hamid.

In: Journal of Biological Chemistry, Vol. 270, No. 34, 01.01.1995, p. 20177-20182.

Research output: Contribution to journalArticle

Fukazawa, T. ; Reedquist, K. A. ; Panchamoorthy, G. ; Soltoff, S. ; Trub, T. ; Druker, B. ; Cantley, L. ; Shoelson, S. E. ; Band, Hamid. / T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-γ1-binding phosphotyrosyl protein pp36/38. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 34. pp. 20177-20182.
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abstract = "Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as ζ and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-γ1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85α cDNA into Jurkat cells followed by antihemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI- 3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.",
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T1 - T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-γ1-binding phosphotyrosyl protein pp36/38

AU - Fukazawa, T.

AU - Reedquist, K. A.

AU - Panchamoorthy, G.

AU - Soltoff, S.

AU - Trub, T.

AU - Druker, B.

AU - Cantley, L.

AU - Shoelson, S. E.

AU - Band, Hamid

PY - 1995/1/1

Y1 - 1995/1/1

N2 - Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as ζ and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-γ1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85α cDNA into Jurkat cells followed by antihemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI- 3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.

AB - Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as ζ and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-γ1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85α cDNA into Jurkat cells followed by antihemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI- 3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.

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