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KMID : 0620920020340060434
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Experimental & Molecular Medicine
2002 Volume.34 No. 6 p.434 ~ p.443
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Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin
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Jae Bong Park/Ah Ram Kim
Won Ho Choi/Sae Ra Lee/Jun Sub Kim/Chan Young Jeon/Jong Il Kim/Jae Bong Kim/Jae Yong Lee/Eung Gook Kim/Jae Bong Park
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Abstract
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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. GTPγS stimulated the phosphorylation of 46 kDa protein (p46) with pI value of 5.0-5.2, but GDPβS 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.
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KEYWORD
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