Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells.
Activation of protein kinase C (PKC) after robust stimulation is necessary for vesicle pool replenishment in secretory cells. Here we studied the contribution of a prominent downstream PKC target, Munc18-1, to this process in bovine chromaffin cells. In these cells, both activation of endogenous PKC and overexpressing of Munc18-1 promote vesicle pool replenishment after an extensive stimulation. In order to study the physiological relevance of PKC-dependent Munc18-1 phosphorylation, we generated two Munc18-1 phospho-mutants; one that mimics a constitutively PKC-phosphorylated Munc18-1 (i.e. a phosphomimetic mutant; Munc18-1(S313D)) and a second that cannot be PKC-phosphorylated (Munc18-1(3A)). Overexpression of Munc18-1(3A) caused a significant decrease in vesicle pool replenishment following a depleting stimulation, while Munc18-1(S313D) caused a significant increase in vesicle pool replenishment. These findings suggested that the phosphorylation of Munc18-1 by PKC potentiates vesicle pool replenishment. This hypothesis was further strengthened by the finding that overexpression of wild type Munc18-1 in the presence of a PKC inhibitor caused a significant reduction in vesicle pool replenishment, similar to that observed with Munc18-1(3A). Moreover, overexpression of Munc18-1(S313D) in the presence of the PKC inhibitor partly alleviated this attenuation, elucidating Munc18-1's unique contribution to vesicle pool replenishment. Finally, we demonstrate that Munc18-1 promotes vesicle docking in a phosphorylation-independent manner. This is deduced from the findings that both the wild type and the two Munc18-1 phospho-mutants enhanced docking to the same extent in bovine chromaffin cells. We conclude that Munc18-1 facilitates docking in a PKC phosphorylation-independent manner, and that its phosphorylation by PKC potentiates vesicle pool replenishment following a depleting stimulation, at a post-docking stage.
Nili
U
U
Department of Neurobiochemistry, Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
de Wit
H
H
Gulyas-Kovacs
A
A
Toonen
R F
RF
Sørensen
J B
JB
Verhage
M
M
Ashery
U
U
D000313:Adrenal Medulla / Q000166:cytology; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D002417:Cattle; D019439:Chromaffin Cells / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D046529:Microscopy, Electron, Transmission / Q000379:methods; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D015240:Phorbol 12,13-Dibutyrate; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D022142:Secretory Vesicles / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D012694:Serine / Q000235:genetics; D014162:Transfection / Q000379:methods
D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D051938:Munc18 Proteins; C081021:Go 6976; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D015240:Phorbol 12,13-Dibutyrate; D012694:Serine; D011493:Protein Kinase C; D002118:Calcium