Role of the tyrosine kinase pyk2 in the integrin-dependent activation of human neutrophils by TNF
Michele Fuortes, … , Gholson J. Lyon, Carl Nathan
Michele Fuortes, … , Gholson J. Lyon, Carl Nathan
Published August 1, 1999
Citation Information: J Clin Invest. 1999;104(3):327-335. https://doi.org/10.1172/JCI6018.
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Article

Role of the tyrosine kinase pyk2 in the integrin-dependent activation of human neutrophils by TNF

Abstract

Secretion of inflammatory products from neutrophils can be induced by a combination of signals from ligated integrins and receptors for soluble, physiological agonists such as TNF. Here we identify pyk2 in primary human neutrophils; localize it to focal adhesions and podosomes; and demonstrate its tyrosine phosphorylation, activation, and association with paxillin during stimulation of adherent cells by TNF. Tyrphostin A9 emerged as the most potent and selective of 51 tyrosine kinase inhibitors tested against the TNF-induced respiratory burst. Tyrphostin A9 inhibited TNF-induced tyrosine phosphorylation of pyk2 without blocking the cells’ bactericidal activity. Wortmannin, an inhibitor of phosphatidylinositol-3-kinase, potently blocked the TNF-induced respiratory burst and selectively inhibited tyrosine phosphorylation of pyk2. Thus, pyk2 appears to play an essential role in the ability of neutrophils to integrate signals from β2 integrins and TNF receptors.

Authors

Michele Fuortes, Maxine Melchior, Hyunsil Han, Gholson J. Lyon, Carl Nathan

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Figure 1

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Identification of pyk2 in human PMNs and its localization to adhesion st...
Identification of pyk2 in human PMNs and its localization to adhesion structures. (a) Pyk2 is evident only in PMNs treated with diisopropylfluorophosphate before lysis (first 2 lanes), in addition to the other protease inhibitors used in the second 2 lanes (see text). PMNs were plated on FBS-coated plates and treated with buffer alone (control; C) or TNF (250 ng/mL; T) for 60 minutes. Identical amounts of protein (100 μg) were loaded in each lane. (b) Indirect immunofluorescence. PMNs adherent to FBS-coated glass coverslips were stimulated with TNF (100 ng/mL) for 60 minutes, fixed, permeabilized, and stained with rabbit anti-pyk2 antibody (panel 1) or mouse anti-vinculin mAb (panel 2), followed by Cy3-conjugated donkey anti-rabbit (panels 1 and 3) or Cy2-conjugated donkey anti-mouse IgG (panels 2 and 4). In panel 3, primary antibody was omitted. In panel 4, an irrelevant mAb was used. The photomicrograph is focused at the plane of cell contact with the substratum. (c) Confocal microscopy of cells prepared as in b and stained with rabbit anti-pyk2 antibody (panel 5) or mouse anti-vinculin mAb (panel 6). Sum of four 0.32-μm-thick images taken from the plane of contact with the substratum.