Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin.
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 kDa 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. Ca(2+)/calmodulin (CaM), which causes the small GTP-binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM-dependent 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 Ca(2+)/CaM directly or indirectly through GTP-binding protein(s) and Ca(2+)/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca(2+)/CaM may be important for the regulation of transporters and neurosecretion.
Kim
Ah-Ram
AR
Department of Biochemistry, College of Medicine, Hallym University, Ockchon 1 Chunchon, Kangwon-do 200-702, Korea.
Choi
Won-Ho
WH
Lee
Sae-Ra
SR
Kim
Jun-Sub
JS
Jeon
Chan-Young
CY
Kim
Jong-Il
JI
Kim
Jaebong
J
Lee
Jae-Yong
JY
Kim
Eung-Gook
EG
Park
Jae-Bong
JB
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D008970:Molecular Weight; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D009687:Nuclear Proteins; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D013570:Synaptic Membranes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D002147:Calmodulin; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D009687:Nuclear Proteins; C498298:PICK1 protein, rat; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D020559:Monomeric GTP-Binding Proteins; D002118:Calcium