Abstract
The ribosomal S6 kinase p90(rsk) was studied in mature and proliferating hemopoietic cells in response to the human cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). In neutrophils, GM-CSF induced time-dependent electrophoretic mobility shifts in immunoreactive p90(rsk). Although these shifts suggested changes in the phosphorylation status of the molecule, a kinase assay with whole cell lysates detected minimal (1.5-fold) increments in enzymatic activity. Only immunoprecipitation followed by immune complex kinase assay or in-gel kinase assay performed against the RSK substrate RRLSSLRA evidenced an increase in p90(rsk) activity (3.4-fold). p90(rsk) was also detected in the GM-CSF-dependent erythroleukemia cell line TF-1. Normally cultured, cytokine-supplemented cells did not respond to further GM-CSF stimulation. However, the activity of p90(rsk) in cytokine-starved cells increased dramatically in response to short term GM-CSF challenge. This effect was readily observable in total cell lysates (6.6-fold increase over controls) and was paralleled by changes in mitogen-activated protein kinase activity (a substrate of p90(rsk)). Thus, p90(rsk) is present in mature hemopoietic cells, but the extent of the enzymatic response to GM-CSF is significantly lower than that seen in proliferative cells.
Highlights
The granulocyte-macrophage colony-stimulating factor (GMCSF),1 like other related cytokines, has multiple functions in cells of the immune system, namely, proliferation, differentiation, end-cell activity, and regulation of apoptosis
Because we were able to detect a significant amount of p90rsk in neutrophils, we further studied whether the kinase enzymatic activity was regulated upon cell stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and how this effect compared with the situation in proliferating hematopoietic cells
Antibodies, and Cell Lines rhGM-CSF and rhTNF-␣ were purchased from R&D Systems (Minneapolis, MN); lipopolysaccharide was from Difco (Detroit, MI); fMetLeu-Phe, PKA inhibitor TTYADFIASGRTGRRNAIHD, goat anti-rabbit and anti-mouse IgG, and myelin basic protein were from Sigma; diisopropyl fluorophosphate was from Aldrich; Immobilon polyvinylidene difluoride membranes were from Millipore Corporation (Bedford, MA); electrophoresis chemicals were from Bio-Rad; [␥-32P]ATP (3000 Ci/mmol) was from Amersham Life Sciences, Inc.; ion exchange chromatography cellulose
Summary
Ʈ To whom correspondence should be addressed. Tel.: 513-873-2360; Fax: 513-873-3769. Antibodies, and Cell Lines rhGM-CSF and rhTNF-␣ were purchased from R&D Systems (Minneapolis, MN); lipopolysaccharide was from Difco (Detroit, MI); fMetLeu-Phe, PKA inhibitor (rabbit sequence) TTYADFIASGRTGRRNAIHD, goat anti-rabbit and anti-mouse IgG (whole molecule agarose beads), and myelin basic protein were from Sigma; diisopropyl fluorophosphate was from Aldrich; Immobilon polyvinylidene difluoride membranes were from Millipore Corporation (Bedford, MA); electrophoresis chemicals were from Bio-Rad; [␥-32P]ATP (3000 Ci/mmol) was from Amersham Life Sciences, Inc.; ion exchange chromatography cellulose. The cells were harvested, and the relative levels of the DNA-incorporated radioactivity was determined by liquid scintillation counting
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