Technical Reference #1668

1668.

The Beta and Gamme Isoforms of Type I PIP5k Regulate Distinct Stages of Calcium 2+ Signaling in Mast cells Lavanya Vasudevan; Andreas Jeromin; Laura Volpicelli-Daley; Pietro De Camilli; David Holowka and Barbara Baird, Cornell University, Journal of Cell Science, 122(1668), (2009)
Link To Paper

Abstract:
Crosslinking of IgE receptors by antigen initiates Ca2+ mobilization in mast cells by activating phospholipase-C -mediated hydrolysis of phosphatidylinositol-45-bisphosphate [PtdIns(45)P2]. The resulting inositol 145-trisphosphatemediated Ca2+ release from the endoplasmic reticulum (ER) activates store-operated Ca2+ entry which is necessary for exocytotic release of inflammatory mediators. To investigate roles for PtdIns(45)P2-synthesizing isozymes of the type I phosphatidylinositol 4-phosphate 5-kinase family (PIP5K-I) in mast cell signaling we compared the ectopic expression of wildtype and catalytically inactive PIP5K-I in RBL-2H3 mast cells. Surprisingly both antigen and thapsigargin-stimulated Ca2+ influx were reduced by overexpression of active PIP5K-I whereas antigen-stimulated Ca2+ release from ER stores was unaffected. Consistent with these results Ca2+ entry stimulated by antigen or thapsigargin was enhanced by expression of a plasma-membrane-associated inositol polyphosphate 5 - phosphatase whereas antigen-stimulated Ca2+ release from stores was reduced. To investigate the role of PIP5K-I in antigen-stimulated Ca2+ mobilization we used bone-marrowderived mast cells from PIP5K-I –/– mice. Antigen-stimulated Ca2+ release from ER stores was substantially reduced in the absence of PIP5K-I but thapsigargin-mediated Ca2+ entry was unaffected. In summary PIP5K-I positively regulates antigenstimulated Ca2+ release from ER stores whereas PIP5K-I negatively regulates store-operated Ca2+ entry suggesting that these different PIP5K-I isoforms synthesize functionally distinct pools of PtdIns(45)P2 at the plasma membrane.

Keywords:
IgE receptors; Store-operated Ca2+ entry; Phosphatidylinositol 4;5-bisphosphate; Plasma membrane pools

Materials & Methods:
Immunocytochemistry Cells were plated at a subconfluent density of 0.5 106 cells/ml in 35 mm MatTek dishes (MatTek Corporation Ashland MA) and cultured overnight. Cells were fixed the next day using 3.7% formaldehyde permeabilized with 0.1% Triton X-100 and labeled for 1 hour with appropriate antibodies in PBS with 10 mg/ml BSA. Images were collected using a Leica TCS SP2 laser scanning confocal system (Leica Microsystems Exton PA) with a 63 0.9 NA HCX APO L U-V-I water-immersion objective. For detecting HA-tagged wt and mutant PIP5K-Iβ anti-HA mAb (1:100; Covance Research Products Berkeley CA) was used as the primary antibody followed by Alexa Fluor 568-anti mouse IgG1 as the secondary antibody (1:100; Invitrogen). FITC-phalloidin (1:200; Invitrogen) was used to label membrane ruffles.

Microscopic Technique
Confocal Microscopy, Real Time

Cell Type(s)
RBL-2H3