Translocation of the 85-kDa Phospholipase A
from Cytosol to the Nuclear Envelope in Rat Basophilic Leukemia Cells Stimulated with Calcium Ionophore or IgE/Antigen (*)
- Sarah Glover(1)(2),
- Timothy Bayburt(2),
- Mechthild Jonas(3),
- Emil Chi(3) and
- Michael H. Gelb(1)(2)(§)
- From the (1)Departments of Chemistry,
- (2)Biochemistry, and
- (3)Pathology, University of Washington, Seattle, Washington 98195
- § To whom correspondence should be addressed: Depts. of Chemistry and Biochemistry, University of Washington, Mail Stop BG-10, Seattle, WA 98195. Tel.: 206-543-7142; Fax 206-685-8665.
Abstract
The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic phospholipase A2 (cPLA2) that is very likely involved in liberating arachidonate from membrane phospholipid for the synthesis of eicosanoids following stimulation with either calcium ionophore or IgE/antigen. In this study, the intracellular location of cPLA2 was determined using immunofluorescence microscopy and immuno-gold electron microscopy. In nonstimulated cells, cPLA2 is distributed throughout the cytosol and is excluded from the nucleoplasm. Following cell activation with calcium ionophore, most of the cPLA2 translocates to the nuclear envelope, and the enzyme remains there during the entire period that ionophore is present. With IgE/antigen stimulation for 5 min, approximately 20-30% of the cPLA2 translocates to the nuclear envelope, and after 30 min of stimulation, most of the enzyme returns to the cytosol. Measurement of intracellular calcium using the dye Fura-2/AM shows that the level of calcium rises immediately after antigen is added, remains high for about 30 s, and then declines back to resting levels. Activation with calcium ionophore produces a 10-fold larger release of arachidonate than does stimulation with IgE/antigen. Thus, the results suggest that the extent of membrane binding of cPLA2 correlates with the release of arachidonate and that the site of arachidonate liberation is the nuclear envelope where many of the enzymes that oxygenate this fatty acid are located.
Footnotes
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↵* This work was supported by Grant HL50040 and Research Career Development Award GM562 (to M. H. G.) and Grants AI17758 and AI34578 (to E. C.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- cPLA2
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85-kDa cytosolic phospholipase A2 found in RBL-2H3.1 and other mammalian cell types
- BSA
-
bovine serum albumin
- BSS
-
balanced salt solution
- DNP-HSA
-
dinitrophenol conjugated to human serum albumin
- PBS
-
phosphate-buffered saline (pH 7.2)
- PAGE
-
polyacrylamide gel electrophoresis
- FITC
-
fluorescein isothiocyanate.
