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PMID: 9278528 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Phosphorylation of the synaptic protein interaction site on N-type calcium channels inhibits interactions with SNARE proteins.

The Journal of neuroscience : the official journal of the Society for Neuroscience ·Vol. 17 ·No. 18 ·1997-09-15 ·Pages 6929-38

Yokoyama CT, Sheng ZH, Catterall WA

Abstract

The synaptic protein interaction (synprint) site on the N-type calcium channel alpha1B subunit binds to the soluble N-ethylmaleimide-sensitive attachment factor receptor (SNARE) proteins syntaxin and synaptosomal protein of 25 kDa (SNAP-25), and this association may be required for efficient fast synaptic transmission. Protein kinase C (PKC) and calcium and calmodulin-dependent protein kinase type II (CaM KII) phosphorylated a recombinant his-tagged synprint site polypeptide rapidly to a stoichiometry of 3-4 mol of phosphate/mol, whereas cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) phosphorylated the synprint peptide more slowly to a stoichiometry of <1 mol/mol. Two-dimensional phosphopeptide mapping revealed similar patterns of phosphorylation of synprint polypeptides and native rat brain N-type calcium channel alpha1B subunits by PKC and Cam KII. Phosphorylation of the synprint peptide with PKC or CaM KII, but not PKA or PKG, strongly inhibited binding of recombinant syntaxin or SNAP-25, even at a level of free calcium (15 microM) that stimulates maximal binding. In contrast, phosphorylation of syntaxin and SNAP-25 with PKC and CaM KII did not affect interactions with the synprint site. Binding assays with polypeptides representing the N- and C-terminal halves of the synprint site indicate that the PKC- and CaM KII-mediated inhibition of binding involves multiple, disperse phosphorylation sites. PKC or CaM KII phosphorylation of the synprint peptide also inhibited its interactions with native rat brain SNARE complexes containing syntaxin and SNAP-25. These results suggest that phosphorylation of the synprint site by PKC or CaM KII may serve as a biochemical switch for interactions between N-type calcium channels and SNARE protein complexes.

MeSH Terms
Animals Antigens, Surface/metabolism Binding Sites Calcium Channels/metabolism Cyclic AMP-Dependent Protein Kinase Type II Cyclic AMP-Dependent Protein Kinases/metabolism Cyclic GMP-Dependent Protein Kinases/metabolism Membrane Proteins/metabolism Nerve Tissue Proteins/metabolism Peptide Mapping Phosphorylation Protein Binding Protein Conformation Protein Kinase C/metabolism Rats SNARE Proteins Synaptosomal-Associated Protein 25 Syntaxin 1 Vesicular Transport Proteins
Chemicals
Antigens, Surface Calcium Channels Membrane Proteins Nerve Tissue Proteins SNARE Proteins Snap25 protein, rat Synaptosomal-Associated Protein 25 Syntaxin 1 Vesicular Transport Proteins Cyclic AMP-Dependent Protein Kinase Type II Cyclic AMP-Dependent Protein Kinases Cyclic GMP-Dependent Protein Kinases Protein Kinase C
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Yokoyama C T
Graduate Program in Neurobiology and Behavior, University of Washington, Seattle, Washington 98195, USA.
Sheng Z H
Catterall W A
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Article Info
Journal
The Journal of neuroscience : the official journal of the Society for Neuroscience
Abbr.
J Neurosci
ISSN
0270-6474
Published
1997-09-15
Pages
6929-38
Language
English
Region
United States
NLM ID
8102140
PMCID
PMC6573282
Subset
IM
Grants
NINDS NIH HHS · NS22625 · United States
NIGMS NIH HHS · T32 GM07108-19 · United States
External Links
PubMed source PMC Full Text View journal details
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