Phosphorylation of the Inositol 1,4,5-Trisphosphate Receptor by Cyclic Nucleotide-dependent Kinases in Vitroand in Rat Cerebellar Slices in Situ*
- From the Neurochemical Laboratory, P. O. Box 1115 Blindern and the‡Department of Neurophysiology, P. O. Box 1104 Blindern, Department Group of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
Abstract
We have examined cyclic nucleotide-regulated phosphorylation of the neuronal type I inositol 1,4,5-trisphosphate (IP3) receptor immunopurified from rat cerebellar membranes in vitro and in rat cerebellar slices in situ. The isolated IP3 receptor protein was phosphorylated by both cAMP- and cGMP-dependent protein kinases on two distinct sites as determined by thermolytic phosphopeptide mapping, phosphopeptide 1, representing Ser-1589, and phosphopeptide 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. Biophys. Res. Commun. 175, 192–198). Phosphopeptide maps show that cAMP-dependent protein kinase (PKA) labeled both sites with the same time course and same stoichiometry, whereas cGMP-dependent protein kinase (PKG) phosphorylated Ser-1756 with a higher velocity and a higher stoichiometry than Ser-1589. Synthetic decapeptides corresponding to the two phosphorylation sites (peptide 1, AARRDSVLAA (Ser-1589), and peptide 2, SGRRESLTSF (Ser-1756)) were used to determine kinetic constants for the phosphorylation by PKG and PKA, and the catalytic efficiencies were in agreement with the results obtained by in vitro phosphorylation of the intact protein. In cerebellar slices prelabeled with [32P]orthophosphate, activation of endogenous kinases by incubation in the presence of cAMP/cGMP analogues and specific inhibitors of PKG and PKA induced in both cases a 3-fold increase in phosphorylation of the IP3 receptor. Thermolytic phosphopeptide mapping of in situ labeled IP3 receptor 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 the IP3receptor are specifically enriched in cerebellar Purkinje cells, PKG may be an important IP3 receptor regulator in vivo.
Footnotes
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↵* This study was supported by the Norwegian Research Council (to L. S. H.), by grants from Nordic Insulin Foundation (Gentofte, Denmark), and by the Jahre Foundation (Oslo, Norway).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed. Tel.: 47-22-85-10-97; Fax: 47-22-85-14-36; E-mail: annecari{at}basalmed.uio.no.
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↵2 Student’s t test may not be used when variances in the two groups are significantly different, as they were for 8-pCPT-cGMP-stimulation. Student’s t test with Welch’s approximation: p = 0.057.
- Abbreviations:
- IP3
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inositol 1,4,5-trisphosphate
- PAGE
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polyacrylamide gel electrophoresis
- PKA
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cyclic AMP-dependent protein kinase
- PKG
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cyclic GMP-dependent protein kinase
- ACSF
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artificial cerebrospinal fluid
- 8-pCPT
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8-(4-para-chlorophenylthio)
- DARPP-32
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dopamine- and cAMP-regulated phosphoprotein of 32 kDa
- EPPS
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N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid
- LTD
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long term depression
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- Received September 29, 1998.
- Revision received December 17, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
