Regulation of Exocytosis by Cyclin-dependent Kinase 5 via Phosphorylation of Munc18
Munc18a, a mammalian neuronal homologue of Saccharomyces 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 prot...
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Veröffentlicht in: | The Journal of biological chemistry 1999-02, Vol.274 (7), p.4027-4035 |
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Zusammenfassung: | Munc18a, a mammalian neuronal homologue of Saccharomyces 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
Munc18a in 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 Thr 574 . 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.274.7.4027 |