Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin

Ah Ram Kim, Won Ho Choi, Sae Ra Lee, Jun Sub Kim, Chan Young Jeon, Jong-Il Kim, Jaebong Kim, Jae Yong Lee, Eung Gook Kim, Jae Bong Park

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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. 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 RaIA 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.

Original languageEnglish
Pages (from-to)434-443
Number of pages10
JournalExperimental and Molecular Medicine
Volume34
Issue number6
DOIs
StatePublished - 31 Dec 2002

Fingerprint

Synaptic Membranes
Phosphorylation
Synaptic Vesicles
Calmodulin
Guanosine Triphosphate
Membranes
GTP-Binding Proteins
Proteins
Phosphotransferases
rab3A GTP-Binding Protein
Neurotransmitter Agents
Neurosecretion
Membrane Fusion
Phosphoprotein Phosphatases
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Cell membranes
Electrophoresis
Phosphoric Monoester Hydrolases
Protein Kinases
Cyclosporine

Keywords

  • Binding proteins
  • Calcium
  • Calmodulin
  • Guanosine triphosphate
  • Phosphorylation
  • Rho GTP
  • Synaptic vesicles

Cite this

Kim, Ah Ram ; Choi, Won Ho ; Lee, Sae Ra ; Kim, Jun Sub ; Jeon, Chan Young ; Kim, Jong-Il ; Kim, Jaebong ; Lee, Jae Yong ; Kim, Eung Gook ; Park, Jae Bong. / Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin. In: Experimental and Molecular Medicine. 2002 ; Vol. 34, No. 6. pp. 434-443.
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title = "Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin",
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. 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 RaIA 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.",
keywords = "Binding proteins, Calcium, Calmodulin, Guanosine triphosphate, Phosphorylation, Rho GTP, Synaptic vesicles",
author = "Kim, {Ah Ram} and Choi, {Won Ho} and Lee, {Sae Ra} and Kim, {Jun Sub} and Jeon, {Chan Young} and Jong-Il Kim and Jaebong Kim and Lee, {Jae Yong} and Kim, {Eung Gook} and Park, {Jae Bong}",
year = "2002",
month = "12",
day = "31",
doi = "10.1038/emm.2002.61",
language = "English",
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Kim, AR, Choi, WH, Lee, SR, Kim, JS, Jeon, CY, Kim, J-I, Kim, J, Lee, JY, Kim, EG & Park, JB 2002, 'Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin', Experimental and Molecular Medicine, vol. 34, no. 6, pp. 434-443. https://doi.org/10.1038/emm.2002.61

Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin. / Kim, Ah Ram; Choi, Won Ho; Lee, Sae Ra; Kim, Jun Sub; Jeon, Chan Young; Kim, Jong-Il; Kim, Jaebong; Lee, Jae Yong; Kim, Eung Gook; Park, Jae Bong.

In: Experimental and Molecular Medicine, Vol. 34, No. 6, 31.12.2002, p. 434-443.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin

AU - Kim, Ah Ram

AU - Choi, Won Ho

AU - Lee, Sae Ra

AU - Kim, Jun Sub

AU - Jeon, Chan Young

AU - Kim, Jong-Il

AU - Kim, Jaebong

AU - Lee, Jae Yong

AU - Kim, Eung Gook

AU - Park, Jae Bong

PY - 2002/12/31

Y1 - 2002/12/31

N2 - 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 RaIA 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.

AB - 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 RaIA 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.

KW - Binding proteins

KW - Calcium

KW - Calmodulin

KW - Guanosine triphosphate

KW - Phosphorylation

KW - Rho GTP

KW - Synaptic vesicles

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U2 - 10.1038/emm.2002.61

DO - 10.1038/emm.2002.61

M3 - Article

VL - 34

SP - 434

EP - 443

JO - Experimental & molecular medicine

JF - Experimental & molecular medicine

SN - 1226-3613

IS - 6

ER -