Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

J Cell Biol. 1994 Aug;126(4):1111-21. doi: 10.1083/jcb.126.4.1111.

Abstract

Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti-Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12-myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies
  • CD18 Antigens
  • Cell Adhesion
  • Enzyme Activation
  • Epitopes / analysis
  • Humans
  • Integrins / deficiency
  • Integrins / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • N-Formylmethionine Leucyl-Phenylalanine / pharmacology
  • Neutrophils / drug effects
  • Neutrophils / enzymology*
  • Peptides / chemical synthesis
  • Peptides / immunology
  • Phosphoproteins / blood*
  • Phosphoproteins / isolation & purification
  • Phosphorylation
  • Phosphotyrosine
  • Protein-Tyrosine Kinases / blood*
  • Proto-Oncogene Proteins / blood*
  • Proto-Oncogene Proteins / isolation & purification
  • Tetradecanoylphorbol Acetate / pharmacology
  • Tumor Necrosis Factor-alpha / pharmacology*
  • Tyrosine / analogs & derivatives*
  • Tyrosine / blood
  • src-Family Kinases

Substances

  • Antibodies
  • CD18 Antigens
  • Epitopes
  • Integrins
  • Peptides
  • Phosphoproteins
  • Proto-Oncogene Proteins
  • Tumor Necrosis Factor-alpha
  • Phosphotyrosine
  • Tyrosine
  • N-Formylmethionine Leucyl-Phenylalanine
  • Protein-Tyrosine Kinases
  • proto-oncogene proteins c-fgr
  • src-Family Kinases
  • Tetradecanoylphorbol Acetate