Incubation of human neutrophils with 500 pM granulocyte-macrophage colony-stimulating factor (GM-CSF) results in a rapid and time-dependent increase in the phosphorylation of cytosolic phospholipase A2 (cPLA2), which was reflected in a slower electrophoretic mobility of the enzyme. The GM-CSF-induced phosphorylation of cPLA2 was accompanied by a parallel and time-dependent increase in the enzyme activity. Preincubation of neutrophils with the tyrosine kinase inhibitor genistein caused inhibition of the GM-CSF-stimulated phosphorylation and activity of cPLA2. Immunoprecipitation of the enzyme following incubation of neutrophils with [32P]Pi shows that cPLA2 is phosphorylated by GM-CSF. Potato acid phosphatase caused dephosphorylation of the enzyme, indicating that cPLA2 is indeed phosphorylated by GM-CSF. The subcellular distribution of cPLA2 in GM-CSF-stimulated neutrophils revealed that the enzyme resides almost completely in the cytosolic fraction. Addition of Ca2+ to the lysis buffer before homogenization results in the translocation of the phosphorylated and the dephosphorylated forms of the enzyme to the membranes. Translocation of cPLA2 was also achieved after incubation with 0.1 μM N-formylmethionyl-leucyl-phenylalanine (fMLP) after GM-CSF stimulation and when neutrophils were challenged with the Ca2+ ionophore A23187. EDTA and EGTA were unable to solubilize the translocated enzyme from the neutrophil membranes, indicating that cPLA2 is attached to the membranes by strong bonds and not merely due to ionic forces exerted by Ca2+. The inability of GM-CSF to promote arachidonic acid mobilization is probably due to the fact that GM-CSF does not cause an increase in intracellular Ca2+, which is necessary for the translocation of the enzyme to the membranes where its substrate(s) reside.
Skip Nav Destination
Follow us on Twitter @Biochem_Journal
Article navigation
January 1996
-
Cover Image
Cover Image
- PDF Icon PDF LinkTable of Contents
Research Article|
January 15 1996
Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes phosphorylation and an increase in the activity of cytosolic phospholipase A2 in human neutrophils Available to Purchase
Nabeel NAHAS;
Nabeel NAHAS
*
*To whom correspondence should be sent, at the U.S.A. address. Permanent address: Department of Biology, Bir-Zeit University, occupied West-Bank.
Search for other works by this author on:
Waltraut H. WATERMAN;
Waltraut H. WATERMAN
1Department of Physiology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3505 U.S.A.
Search for other works by this author on:
Ramadan I. SHA'AFI
Ramadan I. SHA'AFI
1Department of Physiology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3505 U.S.A.
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
April 03 1995
Revision Received:
August 10 1995
Accepted:
September 06 1995
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 313 (2): 503–508.
Article history
Received:
April 03 1995
Revision Received:
August 10 1995
Accepted:
September 06 1995
Citation
Nabeel NAHAS, Waltraut H. WATERMAN, Ramadan I. SHA'AFI; Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes phosphorylation and an increase in the activity of cytosolic phospholipase A2 in human neutrophils. Biochem J 15 January 1996; 313 (2): 503–508. doi: https://doi.org/10.1042/bj3130503
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Biochemical Society Member Sign in
Sign InSign in via your Institution
Sign in via your InstitutionGet Access To This Article
Follow us on Twitter @Biochem_Journal
Open Access for all
We offer compliant routes for all authors from 2025. With library support, there will be no author nor reader charges in 5 journals. Check here |
![]() View past webinars > |