Outline Introduction Regulatory mechanism of acute and late phase reactions in mast cells.

ls. Early phase type I hypersensitivity reaction in mast cells, not dependent of NFkB pathway, through SNAP-23 phosphorylation by IKK2. Late phase reaction, dependent of NFkB pathway, through again SNAP-23 phosphorylation by IKK2 remains unknown.

Severe Impairment of Anaphylactic Responses in the Absence of IKK2 They used mast cell knock-in mouse model by Galli. o FLMCs were generated from Days 11.512.5 embryonic liver of wildtype (WT) or IKK2-deficient (IKK2-/-) mice because of the lethality in the IKK2-/- embryos at E14. Culture was made with 100 ng/ml IL-3. After 4 weeks, more than 98% of cells were c-kit positive, and the number of mast cells (c-kit+IgE+ cells) recovered per mouse was indistinguishable between these mice. (WT [4.65 0.34 x 105] versus IKK2-/- [4.24 0.44 x 105]; n = 10). Staining was made with Anti-c-kit APC and IgE FITC. WT [40.4 5.4] versus IKK2-/- [41.3 4.4]; n = 40) and the mean of granule intensity (WT [93.4 22.4] versus IKK2 -/- [92.1 18.6]; n = 40). The number of granules in each mast cell was counted and the density of individual granules was measured and analyzed with Image J software (NIH, Bethesda, MD).

PCA and PSA show decreased anaphylactic response in IKK2 deficient mice than in WT.

IKK2 Plays Critical Roles in Mast Cell Degranulation Showed assays in IKK2 deficient cells with B-Hexosaminidase and histamine. There is a less annexin-V binding in IKK2 deficient cells, after exposure of granules. Finally they transfected a GFP, GFPIKK2 and GFP-IKK2M, RBL2H3 cells and performed the same assay with anti TNP and then TNP-BSA, overexpressing and showed that IKK2 and not IKK2M was the one that increased degranulation.

IKK2 Is Recruited into the Lipid Raft Fractions upon Fc3RI Stimulation and Associates with SNAP-23

Coimmunoprecipitation with SNAP23, syntaxin 4 and IKK2. Understand the assay and know how the cells where made.

IKK2 Phosphorylates SNAP-23 on Ser120 and Ser95 They examined whether SNAP-23 is a substrate to IKK2. In vitro assay showed that when changing the Ser120 and Ser95 to Ala, the new SNAP23 mutant phosphorylation was attenuated. In vivo they used phosphor specific antibodies. All results where positive according to WT or IKK2 deficient mice.

Phosphorylation of SNAP-23 Is Crucial for IKK2-Mediated Degranulation

Material and Methods RBL2H3 cells Equal parts minimal essential medium and Iscoves medium containing 20% fetal calf serum (HyClone Laboratories), 25 mM Hepes (pH 7.5), and 50 microg/ml gentamicin. Maintained as subconfluent monolayers at 37 C in a humidified atmosphere containing 5% CO and passaged with trypsin. Sensitization o 1 microg/ml anti-DNP-IgE for 24 h prior to stimulation

Stimulation o o 100 ng/ml DNP-BSA in phenol red-free RPMI 1640 medium Cells were mock-stimulated with 100 ng/ml BSA in the same medium.

HeLa Cells

Grown in DMEM supplemented with 10% fetal calf serum, 20 mM Hepes, and 50 _g/ml gentamicin Maintained as subconfluent monolayers at 37 C in a humidified atmosphere containing 5% CO and passaged with trypsin. Some experimentstransfected HeLa cells were stimulated using 10 nM phorbol myristate acetate and 1microM ionomycin

Plasmids and Recombinant Proteins Rat SNAP-23 was subcloned into pcDNA3 (Invitrogen) using EcoRI and XhoI sites introduced by PCR using a previously described full-length rat SNAP23 cDNA. GST-rat SNAP-23 was generated by subcloning rat SNAP-23 from pcDNA3 into pGEX-4T1. Ala mutants of all Ser and Thr residues present in rat SNAP-23 were generated with the QuikChange Site-directed Mutagenesis kit using pGEX4T1-rat SNAP-23 as the template. The double mutant of S95A/S120A was generated using rat SNAP-23 S120A DNA as a template. The double mutant replacing both Ser95 and Ser120 with Asp residues (S95D/S120D) was generated using the QuikChange Multi-Mutagenesis kit (Stratagene)

Transfection Exponentially growing RBL cells were re-suspended in serum-free and antibiotic-free DMEM and 10x 106 cells were transfected by electroporation (310 mV, 960 microfarads, Bio-Rad GenePulser) using 1020 microg of empty vector, FLAG-tagged wild-type SNAP-23, or FLAGtagged mutant rat-SNAP-23 as described previously. Transfected cells were immediately diluted in antibiotic-free medium and allowed to adhere to plastic culture dishes for 56 h before adding anti-DNP IgE to the medium overnight. The next day, cells were triggered for exocytosis as described above. HeLa cells were transiently transfected using Lipofectamine (Invitrogen) according to the manufacturers instructions.

Metabolic Labeling of Cells Cells per 10-cm dish. IgE-sensitized cells were starved the next day for 15 min in phosphate-free DMEM containing 3% dialyzed calf serum (HyClone Laboratories), 20 mM Hepes (pH 7.5), and gentamicin. Cells were labeled with 0.5 mCi of [32 P]orthophosphate (PerkinElmer Life Sci ences) for5hin1ml of 6 In Vitro Phosphorilation Reactions

GST and GST-SNAP-23 Purified from isopropyl 1-thio-B-D-galactopyranoside-induced BL21 Escherichia coli. Briefly, GST and GST-SNAP-23 were isolated from 200 microl bacteria lysates with 10 microl (1:1) of glutathione-Sepharose beads (Amersham Biosciences). GST proteins were eluted with 30 microl of10mM reduced glutathione and phosphorylated for 30 min at 30 C with 25 ng of purified PKC (Promega) in a reaction buffer of 20 mM Hepes (pH 7.4), 1.67 mM CaCl dithiothreitol, 10 mM MgCl2 ,20_M ATP, and 2 microCi of [32P]ATP. After 30 min the entire reaction mixture was boiled in SDS-PAGE sample buffer and analyzed by SDS-PAGE.