RESUMO
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.
Assuntos
Animais , Ratos , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Transporte/química , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Peso Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Membranas Sinápticas/química , Vesículas Sinápticas/químicaRESUMO
Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.
Assuntos
Bovinos , Ratos , Envelhecimento , Animais , Encéfalo/metabolismo , Cálcio/farmacologia , Estudo Comparativo , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Peso Molecular , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Membranas Sinápticas/metabolismo , Sinaptossomos/metabolismo , Proteína cdc42 de Ligação ao GTP/biossíntese , Proteína rab3A de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/biossínteseRESUMO
Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.