Phosphorylation of Syntaphilin by cAMP-dependent Protein Kinase Modulates Its Interaction with Syntaxin-1 and Annuls Its Inhibitory Effect on Vesicle Exocytosis

Abstract

cAMP-dependent protein kinase (PKA) can modulate synaptic transmission by acting directly on the neurotransmitter secretory machinery. Here, we identify one possible target: syntaphilin, which was identified as a molecular clamp that controls free syntaxin-1 and dynamin-1 availability and thereby regulates synaptic vesicle exocytosis and endocytosis. Deletion mutation and site-directed mutagenesis experiments pinpoint dominant PKA phosphorylation sites to serines 43 and 56. PKA phosphorylation of syntaphilin significantly decreases its binding to syntaxin-1A in vitro. A syntaphilin mutation of serine 43 to aspartic acid (S43D) shows similar effects on binding. To characterize in vivo phosphorylation events, we generated antisera against a peptide of syntaphilin containing a phosphorylated serine 43. Treatment of rat brain synaptosomes or syntaphilintransfected HEK 293 cells with the cAMP analogue BIMPS induces in vivo phosphorylation of syntaphilin and inhibits its interaction with syntaxin-1 in neurons. To determine whether PKA phosphorylation of syntaphilin is involved in the regulation of Ca 2؉ -dependent exocytosis, we investigated the effect of overexpression of syntaphilin and its S43D mutant on the regulated secretion of human growth hormone from PC12 cells. Although expression of wild type syntaphilin in PC12 cells exhibits significant reduction in high K ؉ -induced human growth hormone release, the S43D mutant fails to inhibit exocytosis. Our data predict that syntaphilin could be a highly regulated molecule and that PKA phosphorylation could act as an "off" switch for syntaphilin, thus blocking its inhibitory function via the cAMP-dependent signal transduction pathway.Neurotransmitter release involves a series of protein interactions between the membranes of synaptic vesicles and presynaptic terminals, culminating in the calcium-dependent fusion of the two membranes (for reviews see Refs. 1-4). The synaptic vesicle-associated protein synaptobrevin interacts with two membrane-associated proteins, SNAP 1 -25 and syntaxin, to form a stable SNARE complex (5-12). Formation of the SNARE complex has been proposed to bring the synaptic vesicle and plasma membranes into close apposition and provide the energy that drives the mixing of the two lipid bilayers (6, 7, 10). Calcium influx into the presynaptic terminal triggers the calcium sensor of the fusion machinery, upon which the SNARE core complex binding matures from a trans-state to a cis-state, resulting in the complete fusion of the two membranes and the release of neurotransmitter. The very stable cis-SNARE core complex is then dissociated by the action of ␣-SNAP and the ATPase N-ethylmaleimide-sensitive factor (10). The assembly and disassembly of the SNARE complex must be highly regulated to gain plasticity in neurotransmitter release. Recent studies have made significant progress in understanding this regulatory mechanism by the isolation of the SNARE complex interacting proteins, including Munc18/nSec1/rbSec1, complexins, Munc-13, tomosyn, cysteine st...

cAMP‐dependent protein kinase A and the dynamics of epithelial cell surface domains: Moving membranes to keep in shape

“…In polarized neurons for example, these include syntaphilin, a protein interacting with dynamin-1 and syntaxin-1 which, in turn, regulate the scission and fusion of secretory vesicles, respectively, at the axonal synapse (the neuronal equivalent of the epithelial apical plasma membrane domain (74) ). The phosphorylation of syntaphilin by PKA on Ser43 in isolated rat brain synaptosomes or syntaphilin-transfected HEK293 cells inhibits its interaction with dynamin-and syntaxin-1 (75) , and annuls its inhibitory effect on synaptic vesicle exocytosis ( figure 2B). In cultured superior cervical ganglion neurons, PKA phosphorylates tomosyn which, like synthaphilin, is a member of SNARE regulatory protein family that limits synaptic transmission.…”

Interaction Between Munc13-1 and RIM Is Critical for Glucagon-Like Peptide-1–Mediated Rescue of Exocytotic Defects in Munc13-1–Deficient Pancreatic β-Cells

“…Although Rim-Munc13-1 binding is important for maintenance of normal RRP, it may not be the case for recovery of vesicles (26), implying that upregulation of RimMunc13-1 interactions in Munc13-1 ϩ/Ϫ ␤-cells may not be sufficient to fully account for the rescue of the exocytotic defects by PKA activation. PKA potentiation of exocytosis is contributed by phosphorylation of other exocytotic proteins, including SNAP25, ␣1.2-subunit of voltage-dependent Ca 2ϩ channels, K ATP channel (18), ␣-SNAP (40), synapsin I (41), snapin (42), cysteine string protein (43), tomosyn (44), and syntaphilin (45), the majority of which are also involved in insulin secretion (46 -48). Although few studies have attempted to explain the precise functional consequences of PKA-catalyzed phosphorylation of these substrates on insulin and neurotransmitter release (49,50), much is still unknown and remains to be further studied, including the possibility that they could functionally interact with Munc13-1.…”

Protein kinase A-dependent phosphorylation of B/K protein

“…Extensive analysis of the phosphorylation of exocytotic proteins has yielded many substrates of PKA such as synapsin (Fiumara et al, 2004), cysteine string protein (Evans and Morgan, 2003), syntaphilin (Boczan et al, 2004), SNAP-25 (Nagy et al, 2004), and Snapin (Thakur et al, 2004). However, physiological effects attributed to their phosphorylation as well as exact molecular mechanisms still remain unexplained.…”