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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Wortmannin inhibits the TNF-induced respiratory burst and tyrosine phosp...
Wortmannin inhibits the TNF-induced respiratory burst and tyrosine phosphorylation of pyk2. (a) H2O2 release from PMNs measured 120 minutes after the addition of TNF (100 ng/mL; squares) or PMA (100 ng/mL; circles). Wortmannin (10 nM) was added to separate sets of cells at the indicated number of minutes after TNF or PMA. Results are mean ± SEM for triplicates in 1 experiment of 3; some error bars fall within the symbols. (b) Tyrosine phosphorylation. PMNs were plated on FBS-coated plates and treated for 60 minutes with buffer alone or TNF (100 ng/mL) in the presence or absence of wortmannin (Wort) at 10 nM or the src kinase inhibitor PP2 (2.5 μM) as indicated. Cell lysates were immunoprecipitated (IP) with anti-pyk2 antibody. Total cell lysate (top) and anti-pyk2 immunoprecipitates (bottom) were separated by reducing SDS-PAGE, transferred to nitrocellulose, and Western blotted (WB) with anti-phosphotyrosine mAb followed by ECL detection. Molecular mass markers are indicated in kilodaltons.