When activated by cAMP,

type I PKA phosphorylates Csk S364, increasing Csk activity [8] and thus inducing phosphorylation of the inhibitory Y505 on the Src kinase Lck [9]. As a result, signalling downstream of the TCR and further T cell activation is downregulated [8, 10]. On this background, we wanted to investigate localization of type I PKA and Csk and the effect of modulation of these signalling molecules on DPC organization. Upon sustained activation of primary human T cells, we observed translocation of type I PKA via the IS to the DPC, where it localized with active ezrin (phosphorylated (p)ERM), EBP50, PAG, Csk, and CD43, a known negative regulator of T cell function and constituent of the DPC [1, 11]. This sequestration of negative effector molecules that are away from the TCR-proximal signalling machinery may be necessary for full T cell activation to proceed. MK-1775 chemical structure Moreover, translocation of type I PKA, ezrin, EBP50, PAG, Csk and CD43 to the DPC was inhibited by the type I PKA antagonist Rp-8-Br-cAMPS, suggesting a role learn more for type I PKA in the modulation of DPC organization. Primary

T cells were, upon approval by the Regional Ethics Review Board Southern Norway and written informed consent, isolated from buffy coats of healthy donors using the RosetteSep® Human T Cell Enrichment Cocktail (StemCell Technologies, Grenoble, France) according to the manufacturer’s instructions and cultured in RPMI 1640 GlutaMAX supplemented with 10% (v/v) foetal bovine serum, 1 mm sodium pyruvate, 1:100 MEM pentoxifylline non-essential amino acids, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Invitrogen, Carlsbad, CA, USA) (complete medium).

Over night cultures were treated with 1.0 mm Rp-8-Br-cAMPS or 0.3 mm Sp-8-Br-cAMPS or left untreated at 37 °C for 30 min prior to stimulation with Dynabeads® CD3/CD28 T Cell Expander (Invitrogen) at cell/bead ratio 1:1 for various times. Raji B cells were maintained in RPMI 1640 GlutaMAX complete medium and primed with 2 μg/ml each of staphylococcal enterotoxin (SE)A, SEB, SEC3 and SEE (Toxin Technology, Sarasota, FL, USA) at 37 °C for 15 min. Over night cultures of primary human T cells were stimulated with SE-primed Raji B cells at a 2:1 T cell/antigen-presenting cell ratio at 37 °C for 30 min. For immunofluorescence analysis, cell samples were attached to poly-(L-lysine) (Sigma-Aldrich, St. Louis, MO, USA)-coated coverslips on ice and fixed with 3% paraformaldehyde/PBS. After permeabilization with 0.1% nonyl phenoxylpolyethoxylethanol/PBS for 5 min and blocking in 2% BSA/0.01% Tween 20/PBS for 30 min, cells were incubated with primary antibodies against β-tubulin (TUB2.