InsP₃-induced Ca²⁺ signals regulate many cellular processes from fertilisation to cell death. In addition to regulation by InsP₃ and Ca²⁺, InsP₃R activity is modulated by protein-protein interactions and post-translational modifications. Using the Scansite motif-searching algorithm (1) to interrogate the InsP₃R1 sequence for consensus sites for protein phosphorylation, we have identified a consensus site for phosphorylation by protein kinase B (PKB/Akt) at its COOH-terminus. PKB is a serine/threonine kinase that is recruited to the plasma membrane upon growth factor/agonist stimulation where it subsequently activated. A number of substrates for PKB have been identified, many of which are important in cell proliferation, cell survival and glucose metabolism. Here, we investigated whether the InsP₃R is a bona-fide substrate for PKB and whether PKB affects InsP₃-mediated calcium signals. To test whether InsP₃Rs were phosphorylated by PKB, InsP₃Rs were immunoprecipitated from HeLa cells that that had been maintained in ³²P orthophosphate and in which PKB activity had been modified using pharmacological or molecular approaches. Stimulation of serum-starved HeLa cells with either Insulin (1 µg/ml, 5 min) or FBS (10% v/v) resulted in an LY294002 (10 µM) sensitive phosphorylation of InsP₃Rs. No phosphorylation was detected in non-stimulated cells. Furthermore, InsP₃Rs isolated from HeLa cells grown under serum replete conditions were also phosphorylated in an LY294002-sensitive manner (n=3). By immunoprecipitation of overexpressed InsP₃R1, in which the candidate phosphorylatable serine was mutated to an alanine, we determined that the predicted PKB consensus site was susceptible to phosphorylation by PKB. The effect of InsP₃R phosphorylation upon InsP₃-induced Ca²⁺ release was next investigated in HeLa stable cell lines inducibly overexpressing either constitutively active (CA)-PKB, kinase dead (KD)-PKB or YFP. Experiments were performed on three separate days on three cover slips per day. Statistical significance was determined by ANOVA. Using fura-2 imaging, we found that the percentage of cells responding to 0.5 µM histamine (4.01±2.7%, p<0.05) was significantly inhibited by PKB overexpression when compared to cells expressing KD-PKB (42.71±3.73) or YFP (54.18±8.77). This effect was due to a decrease in InsP₃R sensitivity, since the amplitude of the Ca²⁺ signal induced by a cell permeant InsP₃-ester were also significantly inhibited by PKB overexpression (404.7±25.54 nM in controls vs 278.3±15.4 nM in CA-PKB expressing cells, p<0.05). Taken together, we have shown a novel role for PKB in regulating calcium signalling via a direct interaction with the InsP₃R. We propose that this interaction decreases InsP₃R sensitivity to agonist stimulation and creates a link between growth factor signalling, the InsP₃R/CaCa²⁺-signalling cascade and apoptosis.