Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis.
Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.
Barclay
Jeff W
JW
Physiological Laboratory, University of Liverpool, Crown Street, United Kingdom.
Craig
Tim J
TJ
Fisher
Richard J
RJ
Ciufo
Leonora F
LF
Evans
Gareth J O
GJ
Morgan
Alan
A
Burgoyne
Robert D
RD
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D005089:Exocytosis; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D011506:Proteins / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D050765:Qa-SNARE Proteins; D033921:Vesicular Transport Proteins; D012694:Serine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium