Regulation of Exocytosis by Cyclin-dependent Kinase 5 via Phosphorylation of Munc18

doi:10.1074/jbc.274.7.4027

Regulation of Exocytosis by Cyclin-dependent Kinase 5 via Phosphorylation of Munc18*

From the Departments of Physiology and of^‡Pharmacology, University of Michigan, Ann Arbor, Michigan 48109

Abstract

Munc18a, a mammalian neuronal homologue ofSaccharomyces cerevisiae Sec1p protein, is essential for secretion, likely as a result of its high affinity interaction with the target SNARE protein syntaxin 1a (where SNARE is derived from SNAP receptor (the soluble N-ethylmaleimide-sensitive fusion protein)). However, this interaction inhibits vesicle SNARE interactions with syntaxin that are required for secretory vesicles to achieve competency for membrane fusion. As such, regulation of the interaction between Munc18a and syntaxin 1a may provide an important mechanism controlling secretory responsiveness. Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division kinases, co-purifies with Munc18a from rat brain, interacts directly with Munc18a in vitro, and utilizes Munc18a as a substrate for phosphorylation. We have now demonstrated that Cdk5 is capable of phosphorylating Munc18ain vitro within a preformed Munc18a·syntaxin 1a heterodimer complex and that this results in the disassembly of the complex. Using site-directed mutagenesis, the Cdk5 phosphorylation site on Munc18a was identified as Thr⁵⁷⁴. Stimulation of secretion from neuroendocrine cells produced a corresponding rapid translocation of cytosolic Cdk5 to a particulate fraction and an increase of Cdk5 kinase activity. Inhibition of Cdk5 with olomoucine decreased evoked norepinephrine secretion from chromaffin cells, an effect not observed with the inactive analogue iso-olomoucine. The effects of olomoucine were independent of calcium influx as evidenced by secretory inhibition in permeabilized chromaffin cells and in cells under whole-cell voltage clamp. Furthermore, transfection and expression in chromaffin cells of a neural specific Cdk5 activator, p25, led to a strong increase in nicotinic agonist-induced secretory responses. Our data suggest a model whereby Cdk5 acts to regulate Munc18a interaction with syntaxin 1a and thereby modulates the level of vesicle SNARE interaction with syntaxin 1a and secretory responsiveness.

Footnotes

↵* This work was supported by National Institutes of Health Grants NS36227 (to E. L. S.) and DK50127 (to Ronald Holz and M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: Dept. of Physiology, 7804 Medical Sciences II Bldg., University of Michigan, Ann Arbor, MI 48109-0622. Tel.: 734-763-4477; Fax: 734-936-8813; E-mail:esterm{at}umich.edu.
Abbreviations:

SNARE

SNAP receptor

NSF

N-ethylmaleimide-sensitive factor

SNAP

soluble NSF attachment protein

Cdk5

cyclin-dependent kinase 5

PAGE

polyacrylamide gel electrophoresis

GST

glutathioneS-transferase

ds

double-stranded

PIPES

1,4-piperazinediethanesulfonic acid

hGH

human growth hormone

DMPP

1,1-dimethyl-4-phenylpiperazinium iodide

Cm

membrane capacitance.
- Received August 31, 1998.
- Revision received November 9, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.