Abstract
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 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.
Highlights
One candidate for this type of regulation is Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell cycle kinases that has recently been found to co-precipitate with Munc18a from rat brain [14]
The Munc18a1⁄7GST1⁄7syntaxin 1a complex was recovered from the cassette, and aliquots of approximately 15 g of total protein were added to 300 l of phosphorylation buffer containing 0.5 mM ATP and either Cdk5 immunoprecipitated from rat brain lysate or 0.42 g/ml protein kinase C
We previously reported that Munc18a is a phosphoprotein in situ and that it is subject to phosphorylation by Cdk5 in vitro [30]
Summary
Materials and Chemicals—Recombinant pGEX plasmid constructs containing GST-nSec (rat), GST-syntaxin 1a (rat), GST-Cdk (human), and GST-p25 (bovine) were gifts of R. 30 l of the beads (approximately 20 ng of Cdk immunoprecipitate) were added to 300 l of phosphorylation buffer containing ATP at 0.5 mM and substrate (i.e. wild type and mutant Munc18s) at 1 M. Determination of Fusion Protein Interactions and Analysis of Regulation by Cdk5—Binding relations between Munc18a or mutant Munc18s and syntaxin 1a were performed by incubation of GSTMunc18a proteins at 300 nM (600 nM for Munc18a T574A) bound to glutathione-Sepharose 4B beads with given concentrations of syntaxin 1a in binding buffer. The Munc18a1⁄7GST1⁄7syntaxin 1a complex was recovered from the cassette, and aliquots of approximately 15 g of total protein were added to 300 l of phosphorylation buffer containing 0.5 mM ATP and either Cdk immunoprecipitated from rat brain lysate or 0.42 g/ml protein kinase C (the protein kinase C reaction was conducted in the presence of 100 M CaCl2, 83.3 g/ml phosphatidylserine, and 8.3 g/ml diglyceride). Calibration pulses of 100 femtofarads and 500 k⍀ were generated and placed at the beginning of each Cm data record
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
