Munc18-1 PHOSPHORYLATION BY PROTEIN KINASE C POTENTIATES VESICLE POOL REPLENISHMENT IN BOVINE CHROMAFFIN CELLS U. NILI, a1 H. DE WIT, b1 A. GULYAS-KOVACS, c1 R. F. TOONEN, b1 J. B. SØRENSEN, c * M. VERHAGE b * AND U. ASHERY a * a Department of Neurobiochemistry, Wise Faculty of Life Sciences Tel Aviv University, Tel Aviv 69978, Israel b The Center for Neurogenomics and Cognitive Research, Vrije Uni- versiteit, Amsterdam, The Netherlands c Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany Abstract—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 prom- inent 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-phosphor- ylated (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 signifi- cant 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 con- tribution 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 phosphory- lation by PKC potentiates vesicle pool replenishment following a depleting stimulation, at a post-docking stage. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: Munc18, PKC, exocytosis, syntaxin, vesicle re- plenishment, chromaffin cells. In neurons and neuroendocrine cells, the amount of trans- mitter that is released per stimulation is determined by the number of fusion-competent vesicles that reside in the readily releasable pool of vesicles (RRP). During repetitive stimulation, the RRP is depleted, and consequently syn- aptic transmission is attenuated (Zucker, 1999). Fast re- plenishment of vesicles is thus necessary to allow the neuron to sustain neurotransmitter release during high frequency stimulation (Kuromi and Kidokoro, 2002; Stevens and Sullivan, 1998). Protein kinase C (PKC) ac- tivation has been shown to cause enhancement of secre- tion from neurons and neuroendocrine cells by accelerat- ing the rate of vesicle recruitment and vesicular pools replenishment during and after stimulation (Gillis et al., 1996; Majewski and Iannazzo, 1998; Smith et al., 1998; Stevens and Sullivan, 1998; Smith, 1999). However, the molecular pathway by which PKC influences recruitment of vesicles into the releasable pools is still largely unknown. The most likely targets of PKC-phosphorylation during this process are proteins involved in synaptic vesicle exocyto- sis. A former study in chromaffin cells showed that PKC is activated following a rise in [Ca 2+ ] i and phosphorylates synaptosome-associated protein of 25 kDa (SNAP-25), and that this phosphorylation contributes to the recruit- ment of vesicles to the releasable pools following in- tense stimulation. That study also concluded that SNAP- 25 is not the only molecular PKC target necessary for PKC-dependent enhancement of vesicle replenishment (Nagy et al., 2002). Munc18-1 belongs to the SM family of proteins, which are essential for neurotransmitter secretion in Drosophila, C. elegans and mice, and for vesicle fusion in yeast (re- viewed in Rizo and Sudhof, 2002; Toonen and Verhage, 2003). Deletion of Munc18-1 in chromaffin cells, or UNC-18 (its C. elegans’ homologue) in neurons, results in a significant decrease in the number of docked vesicles, suggesting a positive role for Munc18-1 in regulation of vesicle docking (Voets et al., 2001; Weimer et al., 2003). Munc18-1 binds tightly to syntaxin-1a (Hata et al., 1993; Pevsner et al., 1994b), which is a member of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment pro- tein receptor) family of proteins that participate in nearly all eukaryotic membrane fusion events. Regulated exocytosis is mediated by the plasma membrane SNAREs syn- taxin-1a and SNAP-25, and the vesicle-associated mem- brane protein SNARE VMAP/synaptobrevin. These pro- teins form a stable ternary complex, the SNARE complex, 1 All four authors contributed equally to this work. *Corresponding author. Tel: +972-3-6409827; fax: +972-3-6407643 (U. Ashery). E-mail address: uria@post.tau.ac.il (U. Ashery); matthijs@cncr.vu.nl (M. Verhage); jsoeren@gwdg.de (J. B. Sørensen). Abbreviations: cDNA, complementary DNA; DMSO, dimethyl sulfox- ide; GFP, green fluorescent protein; HEK cells, human embryonic kidney cells; LDCVs, large dense core vesicles; PBS, phosphate- buffered saline; PKC, protein kinase C; RRP, readily releasable pool of vesicles; RT, room temperature; SDS, sodium dodecyl sulfate; S.E.M., standard error of measurement; SNAP-25, synaptosome-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor. Neuroscience 143 (2006) 487–500 0306-4522/06$30.00+0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.08.014 487