PLD2 Complexes with the EGF Receptor and Undergoes Tyrosine Phosphorylation at a Single Site upon Agonist Stimulation*
- From the ‡Department of Molecular Signaling, Hagedorn Research Institute, Niels Steensens Vej 6, 2820 Gentofte, Denmark, the ¶Department of Pharmacology, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen Ø, Denmark, and the‖Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794-8651
Abstract
Mammalian phospholipase D (PLD) activity becomes up-regulated when cells are stimulated by a variety of hormones, growth factors, and other extracellular signals. Two distinct PLDs, PLD1 and PLD2, have been identified. The mechanism through which each PLD is activated, however, is poorly understood. Using transiently transfected human embryonic kidney fibroblasts (HEK293), we demonstrate here that PLD1 activity, and to a lesser extent PLD2 activity, is stimulated in response to epidermal growth factor (EGF). PLD2, but not PLD1, associates with the EGF receptor in a ligand-independent manner and becomes tyrosine-phosphorylated upon EGF receptor activation. Tyrosine 11 (Tyr-11) of PLD2 was identified as the specific phosphorylation site. Mutation of this residue to phenylalanine enhanced basal activity almost 2-fold, but did not alter the magnitude of the EGF-mediated increase in PLD2 activity. In conclusion, we show here for the first time agonist-stimulated activation of both PLD1 and PLD2 in vivo and provide evidence of a distinct type of interaction for each isoform with the EGF receptor. Moreover, our results suggest that agonist-induced tyrosine phosphorylation plays a role in PLD2 regulation.
Footnotes
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↵* This work was supported by grants from the Danish Medical Research Council (9600821 to K. S.) (to H. S. H.) and from the National Institutes of Health (GM54813 to M. A. F.). The Hagedorn Research Institute is an independent basic research component of Novo Nordisk A/S.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed. Tel.: 0045 44 43 82 30; Fax: 0045 44 43 80 00; E-mail:RiSl{at}hagedorn.dk.
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↵2 R. Slaaby and K. Seedorf, unpublished observation.
- Abbreviations:
- PLD
-
phospholipase D
- HEK
-
human embryonic kidney
- EGF
-
epidermal growth factor
- PC
-
phosphatidylcholine
- PA
-
phosphatidic acid
- LPA
-
lysophosphatidic acid
- PAP
-
phosphatidic acid phosphohydrolase
- DAG
-
diacylglycerol
- PKC
-
protein kinase C
- ARF
-
ADP-ribosylation factor
- EGFR
-
EGF receptor
- PtdBut
-
phosphatidylbutanol
- PAGE
-
polyacrylamide gel electrophoresis.
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- Received April 16, 1998.
- Revision received August 18, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
