Physiological regulation of Munc18/nSec1 phosphorylation on serine‐313

Abstract

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 Ca 2+ 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.

A Random Mutagenesis Approach to Isolate Dominant-Negative Yeastsec1Mutants Reveals a Functional Role for Domain 3a in Yeast and Mammalian Sec1/Munc18 Proteins

“…As discussed above, the Y337L Munc18-1 mutation affects exocytotic release kinetics, consistent with an effect on the late stages of the membrane fusion process. Interestingly, S313 in Munc18-1, which is very close to Y337 in domain 3a, has been shown to be regulated physiologically by PKC phosphorylation, resulting in altered exocytotic release kinetics (Barclay et al 2003;Craig et al 2003). Taken together, it seems likely that domain 3a is a functionally important region in SM proteins that is involved in the membrane fusion process.…”

Syntaxin/Munc18 Interactions in the Late Events during Vesicle Fusion and Release in Exocytosis

“…The R39C mutation or a phosphomimetic mutant (S306E,S313E) of Munc18-1 reduced the affinity of binding to syntaxin, and in amperometric measurements of release their expression resulted in reduced quantal size (possibly because of a switch to kiss and run exocytosis) and faster release kinetics. These effects may reflect physiological regulation of exocytosis via protein kinase C activation (29,51). The effect of these Munc18-1 mutants in transfected cells could potentially arise from more ready release of bound syntaxin into the open conformation or alternatively from syntaxin-independent effects of released Munc18-1 mutants that are not bound to syntaxin.…”

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCα and PKCδ

“…In many cell types, Munc-18 is crucial for the docking and priming of secretory granules, as well as for actual fusion with the plasma membrane [30]. It has been demonstrated that Munc-18 is phosphorylated by PKC in response to phorbol ester treatment [31,32].…”