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

• Kim AR
• Choi WH
• Lee SR
• Kim JS
• Jeon CY
• Kim JI
• Kim JB
• Lee JY
• Kim EG
• Park JB

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