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Molecular and Cellular Biology, August 2004, p. 6980-6992, Vol. 24, No. 16
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.16.6980-6992.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Activation of RBL-2H3 Mast Cells Is Dependent on Tyrosine Phosphorylation of Phospholipase D2 by Fyn and Fgr

Wahn Soo Choi,^1,2* Takaaki Hiragun,¹ Jun Ho Lee,² Young Mi Kim,^1, Hyoung-Pyo Kim,¹ Ahmed Chahdi,^1, Erk Her,² Jeung Whan Han,^1,3 and Michael A. Beaven^1*

Laboratory of Molecular Immunology, National, Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892,¹ Department of Immunology, College of Medicine, Konkuk University, Chungcheongbuk-Do 380-701,² College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea³

Received 12 September 2003/ Returned for modification 3 November 2003/ Accepted 24 May 2004

Both phospholipase D1 (PLD1) and PLD2 regulate degranulation when RBL-2H3 cells are stimulated via the immunoglobulin E receptor, FcRI. However, the activation mechanism for PLD2 is unclear. As reported here, PLD2 but not PLD1 is phosphorylated through the Src kinases, Fyn and Fgr, and this phosphorylation appears to regulate PLD2 activation and degranulation. For example, only hemagglutinin-tagged PLD2 was tyrosine phosphorylated in antigen-stimulated cells that had been made to express HA-PLD1 and HA-PLD2. This phosphorylation was blocked by a Src kinase inhibitor or by small interfering RNAs directed against Fyn and Fgr and was enhanced by overexpression of Fyn and Fgr but not by other Src kinases. The phosphorylation and activity of PLD2 were further enhanced by the tyrosine phosphatase inhibitor, Na₃VO₄. Mutation of PLD2 at tyrosines 11, 14, 165, or 470 partially impaired, and mutation of all tyrosines blocked, PLD2 phosphorylation and activation, although two of these mutations were detrimental to PLD2 function. PLD2 phosphorylation preceded degranulation, both events were equally sensitive to inhibition of Src kinase activity, and both were enhanced by coexpression of PLD2 and the Src kinases. The findings provide the first description of a mechanism for activation of PLD2 in a physiological setting and of a role for Fgr in FcRI-mediated signaling.

Present address: Department of Pharmacy, Duksung Women's University, Tobong-gu, Seoul 132-714, Korea.

Present address: Medicine (Nephrology), Medical College of Wisconsin, Milwaukee, WI 53226.

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