Abstract
Regulation of phospholipase D (PLD) activity participating in signal transduction involves complex interactions with small G-proteins (ARF, Rho) and protein kinase C isoforms (PKCα). In SK-N-MC human neuroblastoma cells, phorbol ester (TPA) activation of PLD was enhanced by overexpressing myristoylated alanine-rich C kinase substrate (MARCKS). To study MARCKS interactions with PLD, we investigated PLD isoform expression and activation by TPA and GTPγS in intact and digitonin-permeabilized clones transfected with MARCKS (M22). PLD2 was in both cytosol and membrane fractions while PLD1 was primarily membrane-associated in both vector control and M22 cells; location or quantities were unaltered by TPA treatment. TPA-stimulated PLD activity was higher in both intact and digitonin-permeabilized M22 cells than in vector controls. In contrast, GTPγS-stimulated PLD activity was independent of MARCKS expression but was additive with MARCKS-PKC-dependent activation in permeabilized cells. Combinations of PKC inhibition and down- regulation in intact and permeabilized (with GTPγS present) cells indicated that a PKC-mediated phosphorylation event was necessary in intact cells without access to GTPγS, stimulation of PLD mediated by GTPγS was independent of PKC, and PLD activation by PKC in permeabilized cells was kinase-independent. Western blot analysis showed that MARCKS, PKCα, PLD1 and PLD2 were present in a detergent-insoluble fraction (DIF); GTPγS increased recovery of PLD2 in DIF. Disruption of cholesterol-rich DIFs with digitonin, cyclodextrin or filipin potentiated activation of PLD by TPA. Our studies suggest that activation of PLD by PKC requires MARCKS and can involve both phosphorylation-independent and -dependent processes. As PLD activation by GTPγS is PKC-MARCKS-independent, MARCKS may provide a fine tuning component in conjunction with G-protein-mediated mechanisms for regulation of PLD.
Original language | English |
---|---|
Pages (from-to) | 177-189 |
Number of pages | 13 |
Journal | Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids |
Volume | 1487 |
Issue number | 2-3 |
DOIs | |
Publication status | Published - Sept 27 2000 |
Bibliographical note
Funding Information:The skilled technical assistance of Anne Murphy and Anthony Ashagbley in performing experiments and Robert Zwicker and Gladys Keddy in cell culture is gratefully acknowledged. This study was supported by a Program Grant (PG-11476) from the Medical Research Council of Canada and the Lockwood Foundation (SCM). We are grateful to Dr. Sylvain Bourgoin, Centre de Recherche du CHUL, Quebec, Canada for providing PLD2 antibody.
ASJC Scopus Subject Areas
- Molecular Biology
- Cell Biology
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't