Abstract
The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The former studies showed that the type I receptor was a good substrate for PKA (0.65 mol Pi incorporated/mol receptor), whereas type II and III receptors were phosphorylated relatively weakly. The latter studies showed that despite these differences, each of the receptors was phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express >/=99% type I receptor, revealed that minor increases in cAMP concentration were sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) were potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+ mobilizing agents were sufficient to cause phosphorylation. Finally, PKA enhanced InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor was predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization.
Highlights
The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined
InsP3 Receptor Phosphorylation in Intact SH-SY5Y Cells— Because the type I receptor is a good substrate for PKA in vitro (Figs. 1 and 2), we examined whether it is phosphorylated in intact SH-SY5Y cells in response to cAMP elevation using either a back-phosphorylation procedure [23, 25] or 32Pi labeling of intact cells
Agonist-induced Changes in cAMP Concentration—We examined cAMP levels in the three cell types to establish the extent to which vasoactive intestinal peptide (VIP), Pituitary adenylyl cyclase activating peptide (PACAP), and forskolin were stimulatory and whether [Ca2ϩ]i-elevating agents might raise cAMP levels, because this could account for the effects of carbachol, ionomycin and thapsigargin on phosphorylation (Fig. 3D)
Summary
InsP3, inositol 1,4,5-trisphosphate; PKA, cAMP-dependent protein kinase; IBMX, 3-isobutyl-1-methylxanthine; VIP, vasoactive intestinal peptide; PACAP, pituitary adenylyl cyclase activating peptide. Two serines within the PKA consensus sequence (R/ K)(R/K)XS [20] are present in the rat type I receptor coupling domain (serines 1589 and 1755 in the sequences RRDS and RRES, respectively), and in vitro studies on purified rat type I receptor [21] have shown that both residues can be phosphorylated. These sites are conserved in mouse and human type I receptors [4, 7], testifying to their importance. In the present study we have examined the ability of PKA to phosphorylate type I, II, and III receptors by analyzing PKA-induced phosphorylation of purified receptors and the ability of agents that raise cAMP to cause receptor phosphorylation in intact cells
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