Effects of granulocyte-macrophage colony-stimulating factor and tumour necrosis factor-α on tyrosine phosphorylation and activation of mitogen-activated protein kinases in human neutrophils

Abstract

The present study was undertaken to determine the identities and characteristics of proteins with molecular masses between 40 and 44 kDa whose tyrosine phosphorylation increases in human neutrophils following stimulation of these cells with tumour necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Immunoblotting results demonstrate that addition of GM-CSF to human neutrophils increases the tyrosine phosphorylation of two proteins with molecular masses of 42 and 44 kDa. However, the addition of TNF-alpha to neutrophils induces a time- and dose-dependent increase in tyrosine phosphorylation of a 40 kDa protein. Immunoprecipitation using specific mitogen-activated protein kinase (MAPK) isoform antibodies and an antibody which recognizes phosphotyrosine-containing proteins demonstrated that the 42 and 44 kDa proteins are isoforms of MAPKs. Utilizing an in situ gel kinase activity assay, GM-CSF increases the kinase activity of the 42 and 44 kDa proteins. Moreover, using immunoprecipitated p42 and p44 MAPK isoforms in this gel assay revealed activity associated with the p42 and p44 MAPK isoforms. Using the same in situ assay, TNF-alpha induces an increase in kinase activity of a 40-42 kDa protein. However, the 40 kDa protein whose phosphorylation on tyrosine residues increased in human neutrophils following stimulation with TNF-alpha is not a member of the known MAPK family, demonstrating the divergences in pathways utilized by GM-CSF and TNF-alpha. This 40 kDa protein may be related to the recently identified protein that becomes phosphorylated on tyrosine residues upon stimulation of the human epidermal carcinoma cell line KB by interleukin-1. In these cells the p40 protein is part of a protein kinase cascade which results in the phosphorylation of the small heat shock protein, hsp27.

Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF-α and GM-CSF

“…The MEK inhibitor, PD098059, showed a concentration-dependent inhibition in both of these assays. Waterman and Sha'afi [24] reported that GM-CSF induced time-and concentrationdependent tyrosine phosphorylation of p42 and p44 ERKs, whereas TNF-␣ only induced tyrosine phosphorylation of a 40-kDa protein, possibly p38 MAPK. Employing the same phosphorylation-specific antibodies used in this study, Zu et al [31] found that incubation of suspended human PMNs with 25 ng/mL TNF-␣ for 5 min failed to induce phosphorylation of ERK1/2.…”

“…As GM-CSF primes PMN functional responses [7,12] and stimulates ERK activity in PMNs [24,29,30], ERK activation by GM-CSF was compared between GM-CSF and TNF-␣. Using the in vitro kinase assay, GM-CSF stimulated maximal ERK activity at concentrations from 300 to 500 pM, and maximal stimulation occurred at 5 min and fell gradually though 30 min (data not shown).…”

“…GM-CSF stimulates a robust increase in ERK activity in human PMNs [24,29,30]. Several studies report that TNF-␣ fails to stimulate ERK activation in PMNs in suspension [24,26,31], whereas TNF-␣ increases ERK activity in adherent PMNs [26]. LPS does not stimulate ERK or JNK activity in human PMNs [28].…”

“…Although three priming agents, TNF-␣, GM-CSF, and LPS, stimulate p38 MAPK activity in human PMNs [24][25][26][27][28], they differ in their ability to stimulate ERK activation. GM-CSF stimulates a robust increase in ERK activity in human PMNs [24,29,30]. Several studies report that TNF-␣ fails to stimulate ERK activation in PMNs in suspension [24,26,31], whereas TNF-␣ increases ERK activity in adherent PMNs [26].…”

Phosphorylation and Activation of Cytosolic Phospholipase A2 by 38‐kDa Mitogen‐Activated Protein Kinase in Collagen‐Stimulated Human Platelets

“…These data demonstrate that an unidentified kinase or kinases cause cytosolic PLA, phosphorylation in platelets. Differential activation of p42/p44 MAP kinase and cytosolic PLA, phosphorylation has also been reported in macrophages [I31 and neutrophils [14]. Kramer et al…”