Exp Mol Med.  2002 Dec;34(6):434-443.

Phosphorylation of 46-kappa Da protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin

Affiliations
  • 1Department of Biochemistry, College of Medicine, Hallym University, Ockchon, Chunchon, Kangwon-do, Korea. jbpark@hallym.ac.kr
  • 2Department of Biochemistry, College of Medicine, University, Cheongju, Chungbuk, Korea.

Abstract

The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kappa Da protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca2+/ calmodulin (CaM), which causes the small GTP- binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM- dependnet protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca2+/CaM directly or indirectly through GTP-binding protein(s) and Ca2+/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca2+/CaM may be important for the regulation of transporters and neurosecretion.

Keyword

calcium; calmodulin; guanosine triphos-phate; phosphorylation; rho GTP-binding proteins; synapticvesicles

MeSH Terms

Animals
Calcium/*metabolism
Calmodulin/*metabolism
Carrier Proteins/*chemistry/*metabolism
Guanine Nucleotide Dissociation Inhibitors/metabolism
Guanosine Triphosphate/metabolism/*pharmacology
Molecular Weight
Monomeric GTP-Binding Proteins/metabolism
Phosphorylation/drug effects
Rats
Recombinant Fusion Proteins/*chemistry/*metabolism
Synaptic Membranes/chemistry/drug effects/*metabolism
Synaptic Vesicles/chemistry/drug effects/*metabolism
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