D-myo-inositol 1,4,5-trisphosphate 3-kinase A is activated by receptor activation through a calcium:calmodulin-dependent protein kinase II phosphorylation mechanism ively block epithelial Ca2 -activated chloride channels

ively block epithelial Ca2 -activated chloride channels David Communi1, Valérie Vanweyenberg (Vajanaphanich et al., 1994; Ismailov et al., 1996). Rapid and Christophe Erneux production of Ins(1,3,4,5)P4 in response to receptor activaInstitute of Interdisciplinary Research, Free University of Brussels, tion has been observed in various cell types, such as rat Campus Erasme, Building C, 808 route de Lennik, B-1070 Brussels, brain cortical slices (Batty et al., 1985; Challiss and Belgium Nahorski, 1990) and human platelets (King and 1Corresponding author Rittenhouse, 1989). There is experimental evidence for a e-mail: [email protected] number of possible second messenger roles for Ins(1,3,4,5)P4, e.g. in Ca2 homeostasis (Changya et al., D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kin1989; Irvine, 1991). Moreover, the recent cloning and ase, the enzyme responsible for production of D-myocharacterization of a specific high affinity Ins(1,3,4,5)P4 inositol 1,3,4,5-tetrakisphosphate, was activated 3to binding protein from pig platelets demonstrated that it 5-fold in homogenates of rat brain cortical slices after corresponds to a member of the GTPase activating protein incubation with carbachol. The effect was reproduced (GAP) 1 family (Ras-GAP1IP4BP). This protein showed in response to UTP in Chinese hamster ovary (CHO) GAP activity towards Ras and was specifically stimulated cells overexpressing Ins(1,4,5)P3 3-kinase A, the major by Ins(1,3,4,5)P4 (Cullen et al., 1995). isoform present in rat and human neuronal cells. In cDNAs encoding rat and human brain Ins(1,4,5)P3 3ortho-32P-labelled cells, the phosphorylated 53 kDa kinase A (50–53 kDa) have been isolated (Choi et al., enzyme could be identified after receptor activation by 1990; Takazawa et al., 1990b, 1991a). Evidence has been immunoprecipitation. The time course of phosphorylaprovided to show high expression of isoform A in neuronal tion was very similar to that observed for carbachol cells of the cortex, hippocampus and cerebellum in both (or UTP)-induced enzyme activation. Enzyme phosphorat and human (Mailleux et al., 1991, 1992). Rat and rylation was prevented in the presence of okadaic acid. human Ins(1,4,5)P3 3-kinases A show 93% amino acid Calmodulin (CaM) kinase II inhibitors (i.e. KN-93 and sequence identity and polyclonal antibodies to the purified KN-62) prevented phosphorylation of Ins(1,4,5)P3 3rat brain isoenzyme A also recognize the human isoform kinase. Identification of the phosphorylation site in A (Takazawa et al., 1991a). cDNAs encoding an isotransfected CHO cells indicated that the phosphorylenzyme, i.e. Ins(1,4,5)P3 3-kinase B, have been isolated ated residue was Thr311. This residue of the human from human and rat cDNA libraries (Takazawa et al., brain sequence lies in an active site peptide segment 1991b; Thomas et al., 1994; Vanweyenberg et al., 1995). corresponding to a CaM kinase II-mediated phosphoBoth indirect and direct evidence suggests that rylation consensus site, i.e. Arg-Ala-Val-Thr. The same Ins(1,4,5)P3 3-kinase is controlled by various mechanisms. residue in Ins(1,4,5)P3 3-kinase A was also phosphorylCa2 regulates Ins(1,3,4,5)P4 production in lysed thymoated in vitro by CaM kinase II. Phosphorylation cytes and in intact cells stimulated with concanavalin A resulted in 8to 10-fold enzyme activation and a 25(Zilberman et al., 1987). In the lacrimal acinar cells, fold increase in sensitivity to the Ca2 :CaM complex. acetylcholine activates Ca2 -dependent K channels even In this study, direct evidence is provided for a novel when Ins(1,4,5)P3 perfused into the same cells does not regulation mechanism for Ins(1,4,5)P3 3-kinase (iso(Morris et al., 1987). It was suggested that acetylcholine form A) in vitro and in intact cells. promotes the production of Ins(1,3,4,5)P4 and that possibly