Activation of RBL-2H3 mast cells is dependent on tyrosine phosphorylation of phospholipase D2 by Fyn and Fgr.
Both phospholipase D1 (PLD1) and PLD2 regulate degranulation when RBL-2H3 cells are stimulated via the immunoglobulin E receptor, Fc epsilon RI. 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, Na(3)VO(4). 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 Fc epsilon RI-mediated signaling.
Choi
Wahn Soo
WS
Laboratory of Molecular Immunology, National, Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA. beavenm@nhlbi.nih.gov
Hiragun
Takaaki
T
Lee
Jun Ho
JH
Kim
Young Mi
YM
Kim
Hyoung-Pyo
HP
Chahdi
Ahmed
A
Her
Erk
E
Han
Jeung Whan
JW
Beaven
Michael A
MA
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D008407:Mast Cells / Q000166:cytology / Q000276:immunology / Q000502:physiology*; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011743:Pyrimidines / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000737:chemistry / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
C412373:AG 1879; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D011743:Pyrimidines; D034741:RNA, Small Interfering; D017455:Receptors, IgE; D014638:Vanadates; D014443:Tyrosine; C494957:FYN protein, human; C494961:Fyn protein, rat; D051076:Proto-Oncogene Proteins c-fyn; C060902:lyn protein-tyrosine kinase; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1