Phospholipase D (PLD) activity has been shown to be GTP-dependent both in vivo and in vitro. One protein that confers GTP sensitivity to PLD activity in vitro is the low-molecular-mass G-protein ADP-ribosylation factor (Arf). However, members of the Rho family and protein kinase C (PKC) have also been reported to activate PLD in various cell systems. We have characterized the stimulation of PLD in HL60 cell membranes by these proteins. The results demonstrate that a considerable proportion of HL60 PLD activity is located in a detergent-insoluble fraction of the cell membrane that is unlikely to be a caveolae-like domain, but is probably cytoskeletal. This PLD activity required the presence of Arf1, a Rho-family member and PKC for efficient catalysis of the lipid substrate, suggesting that the activity represents PLD1. We show that recombinant human PLD1b is regulated in a similar manner to HL60-membrane PLD, and that PKCα and PKCδ are equally effective PLD activators. Therefore maximum PLD activity requires Arf, a Rho-family member and PKC, emphasizing the high degree of regulation of this enzyme.
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Research Article|
March 25 1999
Characterization of the regulation of phospholipase D activity in the detergent-insoluble fraction of HL60 cells by protein kinase C and small G-proteins
Matthew N. HODGKIN;
Matthew N. HODGKIN
1
1Birmingham Institute of Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TA, U.K.
1To whom correspondence should be addressed (e-mail m.hodgkin@bham.ac.uk).
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Joanna M. CLARK;
Joanna M. CLARK
1Birmingham Institute of Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TA, U.K.
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Sally ROSE;
Sally ROSE
1Birmingham Institute of Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TA, U.K.
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Khalid SAQIB;
Khalid SAQIB
1Birmingham Institute of Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TA, U.K.
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Michael J. O. WAKELAM
Michael J. O. WAKELAM
1Birmingham Institute of Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TA, U.K.
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Publisher: Portland Press Ltd
Received:
July 09 1998
Revision Received:
December 02 1998
Accepted:
January 19 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 339 (1): 87–93.
Article history
Received:
July 09 1998
Revision Received:
December 02 1998
Accepted:
January 19 1999
Citation
Matthew N. HODGKIN, Joanna M. CLARK, Sally ROSE, Khalid SAQIB, Michael J. O. WAKELAM; Characterization of the regulation of phospholipase D activity in the detergent-insoluble fraction of HL60 cells by protein kinase C and small G-proteins. Biochem J 1 April 1999; 339 (1): 87–93. doi: https://doi.org/10.1042/bj3390087
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