Physiological regulation of Munc18/nSec1 phosphorylation on serine-313.
Increased protein phosphorylation enhances exocytosis in most secretory cell types, including neurones. However, the molecular mechanisms by which this occurs and the specific protein targets remain unclear. Munc18-1/nSec1 is essential for exocytosis in neurones, and is known to be phosphorylated by protein kinase C (PKC) in vitro at Ser-313. This phosphorylation has been shown to decrease its affinity for syntaxin, and to alter the kinetics of exocytosis in chromaffin cells. However, there are no data on the physiological regulation of Ser-313 phosphorylation. Using phospho-Ser-313-specific antisera, we demonstrate here that Ser-313 is phosphorylated in intact and permeabilized chromaffin cells in response to histamine and Ca2+ respectively. Furthermore, Ser-313 is rapidly and transiently phosphorylated in intact synaptosomes in response to depolarization by KCl treatment or by 4-aminopyridine, and by the metabotropic glutamate receptor agonist dihydroxyphenylglycine. PKC was identified as the kinase, and PP1 and PP2B as the phosphatases responsible for regulating Ser-313 phosphorylation. As phosphorylation of nSec1 on Ser-313 affects the rate of transmitter release in chromaffin cells, the demonstration here that this phosphorylation event occurs in neurones suggests that synaptic neurotransmitter release may be similarly regulated by nSec1 phosphorylation. Furthermore, such changes in release kinetics are associated with long-term potentiation and depression, thus implicating nSec1 phosphorylation as a potential regulatory mechanism underlying presynaptic plasticity.
Craig
Tim J
TJ
Department of Physiology, University of Liverpool, Liverpool, UK.
Evans
Gareth J O
GJ
Morgan
Alan
A
D000818:Animals; D000918:Antibody Specificity; D001923:Brain Chemistry; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000378:metabolism*; D013574:Synaptosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*
D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; C497939:Stxbp1 protein, rat; D033921:Vesicular Transport Proteins; D012694:Serine; D006632:Histamine; D011493:Protein Kinase C; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium