Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals.
Munc18-1 is a mammalian member of the SEC1 protein family implicated in neuronal secretion. Its sequence contains several consensus sites for phosphorylation by protein kinase C (PKC), a kinase known to enhance secretion. We have characterized the phosphorylation of the synaptic munc18-1 pool by endogenous, presynaptic PKC-isoforms. In isolated rat brain nerve terminals, munc18-1 was almost completely nonphosphorylated. Its phosphorylation state increased by 250% on inhibition of endogenous phosphatases and by 1500% on additional, direct PKC activation using phorbol esters. K+-evoked depolarization also increased munc18-1 phosphorylation, by 50% within 5 s in a Ca2+-dependent manner. Munc18-1 phosphorylation in nerve terminals was blocked by PKC inhibitors. Activation of endogenous PKC in nerve terminals inhibited the interaction of synaptic munc18-1 with its binding partner syntaxin-1A by 50%. Munc18-1 antisera precipitated 80% of native, brain-derived munc18-1 from salt solutions, but only 12% from synaptosomal lysates, together with 6% synaptic syntaxin-1A/B; these amounts were not changed by PKC activation. In this 12%, the phosphate incorporation per mole of munc18 was four-fold lower than the total pool. We conclude that the synaptic munc18-1 pool can be readily and rapidly phosphorylated by endogenous presynaptic PKC isoforms. A high constitutive phosphatase activity keeps its basal phosphorylation state low so that PKC activation can increase the phosphorylation state dramatically. These phosphorylation dynamics and the effects on the interaction with syntaxin-1A make munc18-1 a prominent candidate to account for PKC-dependent enhancement of secretion.
de Vries
K J
KJ
Rudolf Magnus Institute for Neurosciences, University of Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
Geijtenbeek
A
A
Brian
E C
EC
de Graan
P N
PN
Ghijsen
W E
WE
Verhage
M
M
D000818:Animals; D016572:Cyclosporine / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D016548:Prosencephalon / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013574:Synaptosomes / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D010710:Phosphates; D011506:Proteins; D011993:Recombinant Fusion Proteins; C497939:Stxbp1 protein, rat; D033921:Vesicular Transport Proteins; D015240:Phorbol 12,13-Dibutyrate; D016572:Cyclosporine; D019311:Staurosporine