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.