Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic nucleotide-dependent kinases in vitro and in rat cerebellar slices in situ

We have examined cyclic nucleotide-regulated phosphorylation of the neurona l type I inositol 1,4,5-trisphosphate (IP3) receptor immunopurified from ra t cerebellar membranes in vitro and in rat cerebellar slices in situ, The i solated IP(3)eceptor protein was phosphorylated by both cAMP- and cGMP-depe ndent protein kinases on two distinct sites as determined by thermolytic ph osphopeptide mapping, phosphopeptide 1, representing Ser-1589, and phosphop eptide 2, representing Ser-1756 in the rat protein (Ferris, C, D,, Cameron, A M,, Bredt, D, S,, Huganir, R, L,, and Snyder, S, H. (1991) Biochem. Biop hys, Res. Commun. 175, 192-198), Phosphopeptide maps show that cAMP-depende nt protein kinase (PKA) labeled both sites with the same time course and sa me stoichiometry, whereas cGMP-dependent protein kinase (PKG) phosphorylate d Ser-1756 with a higher velocity and a higher stoichiometry than Ser-1589, Synthetic decapeptides corresponding to the two phosphorylation sites (pep tide 1, AARRD (S) under bar VLAA (Ser-1589), and peptide 2, SGRRE (S) under bar LTSF (Ser-1756)) were used to determine kinetic constants for the phos phorylation by PKG and PKA, and the catalytic efficiencies were in agreemen t with the results obtained by in vitro phosphorylation of the intact prote in. In cerebellar slices prelabeled with [P-32]orthophosphate, activation o f endogenous kinases by incubation in the presence of cAMP/cGMP analogues a nd specific inhibitors of PKG and PKA induced in both cases a 3-fold increa se in phosphorylation of the IP(3)eceptor. Thermolytic phosphopeptide mappi ng of in situ labeled IP(3)eceptor by PKA showed labeling on the same sites (Ser-1589 and Ser-1756) as in vitro. In contrast to the findings in vitro, PKG preferentially phosphorylated Ser-1589 in situ, Because both PKG and t he IP(3)eceptor are specifically enriched in cerebellar Purkinje cells, PKG may be an important IP(3)eceptor regulator irt vivo.