Sensitive Factor Attachment Protein Receptor Complex Research Articles

Role of complexin-lipid interactions in controlling snare-mediated membrane fusion.

Fast and reliable neurotransmitter release is essential for signaling fidelity at neuronal synapses. Synaptic exocytosis is a tightly controlled process accomplished through Ca2+ triggered fusion of neurotransmitter-laden vesicles with the presynaptic plasma membrane. This process is mediated by SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins that zipper into a four-helix bundle to drive membrane fusion. The neuronal SNARE complex, consisting of Syntaxin-1a, SNAP-25, and Synaptobrevin-2, is intrinsically capable of catalyzing slow and Ca2+ independent fusion in reconstituted fusion assays in vitro. A single model of how interactions with SNARE regulatory proteins impart physiological properties seen in neurons, such as Ca2+ specificity, has yet to be established. Complexin-1 (Cpx) is a small, soluble regulatory protein that binds SNAREs and lipid membranes. Cpx is proposed to facilitate fusion in the presence of Ca2+ as well as suppress fusion in the absence of Ca2+ through interactions of its central domain with SNARE complexes in their prefusion and postfusion states. Additionally, the Cpx C-terminal domain interacts with membranes, an interaction that has been proposed to facilitate function. Recent work has shown that Cpx's lipid-binding domains can differentiate between membranes of different compositions, though the functional relevance of this finding has yet to be established. Our group has previously developed a hybrid fusion assay that combines synthetic, reconstituted, and native components to reproduce key features of synaptic exocytosis. This method mimics intracellular conditions more closely than a reconstituted system with only purified components while maintaining the ability to easily alter lipid, protein, and ion concentrations. We utilize this system with wild-type and mutant Cpx, along with other protein and lipid regulators of SNAREs, to investigate how interaction with and composition of the fusing membranes controls Cpx's regulatory function.

Neuronal SNARE complex assembly guided by Munc18-1 and Munc13-1.

Neurotransmitter release by Ca2+ -triggered synaptic vesicle exocytosis is essential for information transmission in the nervous system. The soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) syntaxin-1, SNAP-25, and synaptobrevin-2 form the SNARE complex to bring synaptic vesicles and the plasma membranes together and to catalyze membrane fusion. Munc18-1 and Munc13-1 regulate synaptic vesicle priming via orchestrating neuronal SNARE complex assembly. In this review, we summarize recent advances toward the functions and molecular mechanisms of Munc18-1 and Munc13-1 in guiding neuronal SNARE complex assembly, and discuss the functional similarities and differences between Munc18-1 and Munc13-1 in neurons and their homologs in other intracellular membrane trafficking systems.

Epileptic Phenotypes Associated With SNAREs and Related Synaptic Vesicle Exocytosis Machinery.

SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptor) are an heterogeneous family of proteins that, together with their key regulators, are implicated in synaptic vesicle exocytosis and synaptic transmission. SNAREs represent the core component of this protein complex. Although the specific mechanisms of the SNARE machinery is still not completely uncovered, studies in recent years have provided a clearer understanding of the interactions regulating the essential fusion machinery for neurotransmitter release. Mutations in genes encoding SNARE proteins or SNARE complex associated proteins have been associated with a variable spectrum of neurological conditions that have been recently defined as “SNAREopathies.” These include neurodevelopmental disorder, autism spectrum disorder (ASD), movement disorders, seizures and epileptiform abnormalities. The SNARE phenotypic spectrum associated with seizures ranges from simple febrile seizures and infantile spasms, to severe early-onset epileptic encephalopathies. Our study aims to review and delineate the epileptic phenotypes associated with dysregulation of synaptic vesicle exocytosis and transmission, focusing on the main proteins of the SNARE core complex (STX1B, VAMP2, SNAP25), tethering complex (STXBP1), and related downstream regulators.

The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity.

Synaptic vesicle (SV) release probability (Pr) is a key presynaptic determinant of synaptic strength established by cell-intrinsic properties and further refined by plasticity. To characterize mechanisms that generate Pr heterogeneity between distinct neuronal populations, we examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in Pr and synaptic plasticity. We found the decoy soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing Pr to generate decreased levels of SV fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic Pr at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation.

Role of Aberrant Spontaneous Neurotransmission in SNAP25-Associated Encephalopathies

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. Here, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.

Mechanism of neurotransmitter release coming into focus.

Research for three decades and major recent advances have provided crucial insights into how neurotransmitters are released by Ca2+ -triggered synaptic vesicle exocytosis, leading to reconstitution of basic steps that underlie Ca2+ -dependent membrane fusion and yielding a model that assigns defined functions for central components of the release machinery. The soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) syntaxin-1, SNAP-25, and synaptobrevin-2 form a tight SNARE complex that brings the vesicle and plasma membranes together and is key for membrane fusion. N-ethyl maleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble the SNARE complex to recycle the SNAREs for another round of fusion. Munc18-1 and Munc13-1 orchestrate SNARE complex formation in an NSF-SNAP-resistant manner by a mechanism whereby Munc18-1 binds to synaptobrevin and to a self-inhibited "closed" conformation of syntaxin-1, thus forming a template to assemble the SNARE complex, and Munc13-1 facilitates assembly by bridging the vesicle and plasma membranes and catalyzing opening of syntaxin-1. Synaptotagmin-1 functions as the major Ca2+ sensor that triggers release by binding to membrane phospholipids and to the SNAREs, in a tight interplay with complexins that accelerates membrane fusion. Many of these proteins act as both inhibitors and activators of exocytosis, which is critical for the exquisite regulation of neurotransmitter release. It is still unclear how the actions of these various proteins and multiple other components that control release are integrated and, in particular, how they induce membrane fusion, but it can be expected that these fundamental questions can be answered in the near future, building on the extensive knowledge already available.

Ca2+-Triggered Synaptic Vesicle Fusion Initiated by Release of Inhibition.

Recent structural and functional studies of the synaptic vesicle fusion machinery suggest an inhibited tripartite complex consisting of neuronal soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), synaptotagmin, and complexin prior to Ca2+-triggered synaptic vesicle fusion. We speculate that Ca2+-triggered fusion commences with the release of inhibition by Ca2+ binding to synaptotagmin C2 domains. Subsequently, fusion is assisted by SNARE complex zippering and by active membrane remodeling properties of synaptotagmin. This additional, inhibitory role of synaptotagmin may be a general principle since other recent studies suggest that Ca2+ binding to extended synaptotagmin C2 domains enables lipid transport by releasing an inhibited state of the system, and that Munc13 may nominally be in an inhibited state, which is released upon Ca2+ binding to one of its C2 domains.

Doc2b Protects β-Cells Against Inflammatory Damage and Enhances Function.

Loss of functional β-cell mass is an early feature of type 1 diabetes. To release insulin, β-cells require soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, as well as SNARE complex regulatory proteins like double C2 domain-containing protein β (Doc2b). We hypothesized that Doc2b deficiency or overabundance may confer susceptibility or protection, respectively, to the functional β-cell mass. Indeed, Doc2b+/- knockout mice show an unusually severe response to multiple-low-dose streptozotocin (MLD-STZ), resulting in more apoptotic β-cells and a smaller β-cell mass. In addition, inducible β-cell-specific Doc2b-overexpressing transgenic (βDoc2b-dTg) mice show improved glucose tolerance and resist MLD-STZ-induced disruption of glucose tolerance, fasting hyperglycemia, β-cell apoptosis, and loss of β-cell mass. Mechanistically, Doc2b enrichment enhances glucose-stimulated insulin secretion (GSIS) and SNARE activation and prevents the appearance of apoptotic markers in response to cytokine stress and thapsigargin. Furthermore, expression of a peptide containing the Doc2b tandem C2A and C2B domains is sufficient to confer the beneficial effects of Doc2b enrichment on GSIS, SNARE activation, and apoptosis. These studies demonstrate that Doc2b enrichment in the β-cell protects against diabetogenic and proapoptotic stress. Furthermore, they identify a Doc2b peptide that confers the beneficial effects of Doc2b and may be a therapeutic candidate for protecting functional β-cell mass.

Decreased aluminium tolerance in the growth of Saccharomyces cerevisiae with SSO2 gene disruption.

Aluminium ions inhibit growth of the budding yeast Saccharomyces cerevisiae. Disruption of the SSO2 gene increased the susceptibility to aluminium. Sso2p belongs to the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family. SSO2 has one paralogue, SSO1, which encodes Sso1p. The SNARE complex containing Sso1/2p plays a role in the recognition of plasma membrane targeted vesicle transport. The susceptibility to aluminium stress was not increased in the Δsso1 strain. The phenotype of aluminium ion influx between the wild-type and Δsso2 strains was not different, suggesting that Sso2p was involved in the elimination of cellular aluminium. However, the cellular lipid constitution of Δsso2 was richer in unsaturated fatty acids than the wild type, indicating that Sso2p is associated with lipid homeostasis of the plasma membrane. Aluminium treatment increased the production of reactive oxygen species (ROS) during proliferation. ROS production was increased in the Δsso2 strain after 3h of aluminium treatment compared with the wild type. These results suggested that Sso2p plays a role in maintaining the lipid composition of the plasma membrane and the increase in unsaturated fatty acids amplified the production of ROS in the acute phase of aluminium stress. ROS derived from aluminium stress inhibited growth and resulted in the susceptibility of the Δsso2 strain.

The primed SNARE-complexin-synaptotagmin complex for neuronal exocytosis.

Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

A short region upstream of the yeast vacuolar Qa-SNARE heptad-repeats promotes membrane fusion through enhanced SNARE complex assembly.

Whereas SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) heptad-repeats are well studied, SNAREs also have upstream N-domains of indeterminate function. The assembly of yeast vacuolar SNAREs into complexes for fusion can be studied in chemically defined reactions. Complementary proteoliposomes bearing a Rab:GTP and either the vacuolar R-SNARE or one of the three integrally anchored Q-SNAREs were incubated with the tethering/SM protein complex HOPS and the two other soluble SNAREs (lacking a transmembrane anchor) or their SNARE heptad-repeat domains. Fusion required a transmembrane-anchored R-SNARE on one membrane and an anchored Q-SNARE on the other. The N-domain of the Qb-SNARE was completely dispensable for fusion. Whereas fusion can be promoted by very high concentrations of the Qa-SNARE heptad-repeat domain alone, at physiological concentrations the Qa-SNARE heptad-repeat domain alone has almost no fusion activity. The 181-198 region of Qa, immediately upstream of the SNARE heptad-repeat domain, is required for normal fusion activity with HOPS. This region is needed for normal SNARE complex assembly.

Myosin phosphatase and RhoA-activated kinase modulate neurotransmitter release by regulating SNAP-25 of SNARE complex.

Reversible phosphorylation of neuronal proteins plays an important role in the regulation of neurotransmitter release. Myosin phosphatase holoenzyme (MP) consists of a protein phosphatase-1 (PP1) catalytic subunit (PP1c) and a regulatory subunit, termed myosin phosphatase targeting subunit (MYPT1). The primary function of MP is to regulate the phosphorylation level of contractile proteins; however, recent studies have shown that MP is localized to neurons, and is also involved in the mediation of neuronal processes. Our goal was to investigate the effect of RhoA-activated kinase (ROK) and MP on the phosphorylation of one potential neuronal substrate, the synaptosomal-associated protein of 25 kDa (SNAP-25). SNAP-25 is a member of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, along with synaptobrevin and syntaxin, and the primary role of SNAP25 is to mediate vesicle fusion. We showed that MYPT1 interacts with SNAP-25, as revealed by immunoprecipitation and surface plasmon resonance based binding studies. Mass spectrometry analysis and in vitro phosphorylation/dephosphorylation assays demonstrated that ROK phosphorylates, while MP dephosphorylates, SNAP-25 at Thr138. Silencing MYPT1 in B50 neuroblastoma cells increased phosphorylation of SNAP-25 at Thr138. Inhibition of PP1 with tautomycetin increased, whereas inhibition of ROK by H1152, decreased the phosphorylation of SNAP-25 at Thr138 in B50 cells, in cortical synaptosomes, and in brain slices. In response to the transduction of the MP inhibitor, kinase-enhanced PP1 inhibitor (KEPI), into synaptosomes, an increase in phosphorylation of SNAP-25 and a decrease in the extent of neurotransmitter release were detected. The interaction between SNAP-25 and syntaxin increased with decreasing phosphorylation of SNAP-25 at Thr138, upon inhibition of ROK. Our data suggest that ROK/MP play a crucial role in vesicle trafficking, fusion, and neurotransmitter release by oppositely regulating the phosphorylation of SNAP-25 at Thr138.

Mechanistic Insights into NSF Mediated SNARE Complex Disassembly

Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with the adaptor protein SNAP (soluble NSF attachment protein), disassembles the SNARE complex, making individual SNAREs available for subsequent rounds of fusion. We determined structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE supercomplex (known as 20S particle) using single-particle cryo-electron microscopy (cryo-EM). We further dissected the mechanism of NSF mediated SNARE complex disassembly using single-molecule fluorescence microscopy based on the cryo-EM structures.

Synaptotagmin-7 Functions to Replenish Insulin Granules for Exocytosis in Human Islet β-Cells.

Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7’s precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells. Adenovirus–short hairpin RNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of predocked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in a Ca2+-dependent manner with syntaxin-3 and syntaxin-1A soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes known to mediate exocytosis of newcomer and predocked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, electron microscopy analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion, which could not be rescued by glucagon-like peptide 1 pretreatment. To assess the possibility that this new action of Syt-7 on SG recruitment may involve calmodulin (CaM), pretreatment of islets with CaM blocker calmidazolium showed effects very similar to those of Syt-7-KD. Syt-7 therefore plays a novel more dominant function in the replenishment of releasable SG pools in human β-cells than its previously purported role in exocytotic fusion per se.

8-Nitro-cGMP Enhances SNARE Complex Formation through S-Guanylation of Cys90 in SNAP25.

Nitrated guanine nucleotide 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) generated by reactive oxygen/nitrogen species causes protein S-guanylation. However, the mechanism of 8-nitro-cGMP formation and its protein targets in the normal brain have not been identified. Here, we investigated 8-nitro-cGMP generation and protein S-guanylation in the rodent brain. Immunohistochemistry indicated that 8-nitro-cGMP was produced by neurons, such as pyramidal cells and interneurons. Using liquid chromatography-tandem mass spectrometry, we determined endogenous 8-nitro-cGMP levels in the brain as 2.92 ± 0.10 pmol/mg protein. Based on S-guanylation proteomics, we identified several S-guanylated neuronal proteins, including SNAP25 which is a core member of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex. SNAP25 post-translational modification including palmitoylation, phosphorylation, and oxidation, are known to regulate neurotransmission. Our results demonstrate that S-guanylation of SNAP25 enhanced the stability of the SNARE complex, which was further promoted by Ca(2+)-dependent activation of neuronal nitric oxide synthase. Using site-directed mutagenesis, we identified SNAP25 cysteine 90 as the main target of S-guanylation which enhanced the stability of the SNARE complex. The present study revealed a novel target of redox signaling via protein S-guanylation in the nervous system and provided the first substantial evidence of 8-nitro-cGMP function in the nervous system.

Mechanistic insights into the recycling machine of the SNARE complex.

SummaryEvolutionarily conserved SNARE (Soluble N-ethylmaleimide sensitive factor Attachment protein REceptors) proteins form a complex that drives fusion between membranes in eukaryotes. SNARE complexes are disassembled by the ATPase NSF (N-ethylmaleimide Sensitive Factor), together with SNAP (Soluble NSF Attachment Protein) proteins, making individual SNAREs available for a subsequent round of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometer resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains, to pseudo four-fold symmetry of the SNARE complex. SNAPs are interacting with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins constitute the core of an ancient vesicle fusion machine that diversified into distinct sets that now function in different trafficking steps in eukaryotic cells. Deciphering their precise mode of action has proved challenging. SM proteins are thought to act primarily through one type of SNARE protein, the syntaxins. Despite high structural similarity, however, contrasting binding modes have been found for different SM proteins and syntaxins. Whereas the secretory SM protein Munc18 binds to the ‟closed conformation" of syntaxin 1, the ER-Golgi SM protein Sly1 interacts only with the N-peptide of Sed5. Recent findings, however, indicate that SM proteins might interact simultaneously with both syntaxin regions. In search for a common mechanism, we now reinvestigated the Sly1/Sed5 interaction. We found that individual Sed5 adopts a tight closed conformation. Sly1 binds to both the closed conformation and the N-peptide of Sed5, suggesting that this is the original binding mode of SM proteins and syntaxins. In contrast to Munc18, however, Sly1 facilitates SNARE complex formation by loosening the closed conformation of Sed5.

The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes

The fusion of yeast vacuolar membranes depends on the disassembly of cis-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and the subsequent reassembly of new SNARE complexes in trans. The disassembly of cis-SNARE complexes by Sec17/Sec18p releases the soluble SNARE Vam7p from vacuolar membranes. Consequently, Vam7p needs to be recruited to the membrane at future sites of fusion to allow the formation of trans-SNARE complexes. The multisubunit tethering homotypic fusion and vacuole protein sorting (HOPS) complex, which is essential for the fusion of vacuolar membranes, was previously shown to have direct affinity for Vam7p. The functional significance of this interaction, however, has been unclear. Using a fully reconstituted in vitro fusion reaction, we now show that HOPS facilitates membrane fusion by recruiting Vam7p for fusion. In the presence of HOPS, unlike with other tethering agents, very low levels of added Vam7p suffice to induce vigorous fusion. This is a specific recruitment of Vam7p rather than an indirect stimulation of SNARE complex formation through tethering, as HOPS does not facilitate fusion with a low amount of a soluble form of another vacuolar SNARE, Vti1p. Our findings establish yet another function among the multiple tasks that HOPS performs to catalyze the fusion of yeast vacuoles.

A new role for RINT-1 in SNARE complex assembly at thetrans-Golgi network in coordination with the COG complex

Docking and fusion of transport vesicles/carriers with the target membrane involve a tethering factor-mediated initial contact followed by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-catalyzed membrane fusion. The multisubunit tethering CATCHR family complexes (Dsl1, COG, exocyst, and GARP complexes) share very low sequence homology among subunits despite likely evolving from a common ancestor and participate in fundamentally different membrane trafficking pathways. Yeast Tip20, as a subunit of the Dsl1 complex, has been implicated in retrograde transport from the Golgi apparatus to the endoplasmic reticulum. Our previous study showed that RINT-1, the mammalian counterpart of yeast Tip20, mediates the association of ZW10 (mammalian Dsl1) with endoplasmic reticulum-localized SNARE proteins. In the present study, we show that RINT-1 is also required for endosome-to-trans-Golgi network trafficking. RINT-1 uncomplexed with ZW10 interacts with the COG complex, another member of the CATCHR family complex, and regulates SNARE complex assembly at the trans-Golgi network. This additional role for RINT-1 may in part reflect adaptation to the demand for more diverse transport routes from endosomes to the trans-Golgi network in mammals compared with those in a unicellular organism, yeast. The present findings highlight a new role of RINT-1 in coordination with the COG complex.

IκB kinase phosphorylation of SNAP-23 controls platelet secretion

AbstractPlatelet secretion plays a key role in thrombosis, thus the platelet secretory machinery offers a unique target to modulate hemostasis. We report the regulation of platelet secretion via phosphorylation of SNAP-23 at Ser95. Phosphorylation of this t-soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) occurs upon activation of known elements of the platelet signaling cascades (ie, phospholipase C, [Ca2+]i, protein kinase C) and requires IκB kinase (IKK)-β. Other elements of the nuclear factor κB/IκB cascade (ie, IKK-α,-β,-γ/NEMO and CARMA/MALT1/Bcl10 complex) are present in anucleate platelets and IκB is phosphorylated upon activation, suggesting that this pathway is active in platelets and implying a nongenomic role for IKK. Inhibition of IKK-β, either pharmacologically (with BMS-345541, BAY11-7082, or TPCA-1) or by genetic manipulation (platelet factor 4 Cre:IKK-βflox/flox), blocked SNAP-23 phosphorylation, platelet secretion, and SNARE complex formation; but, had no effect on platelet morphology or other metrics of platelet activation. Consistently, SNAP-23 phosphorylation enhanced membrane fusion of SNARE-containing proteoliposomes. In vivo studies with IKK inhibitors or platelet-specific IKK-β knockout mice showed that blocking IKK-β activity significantly prolonged tail bleeding times, suggesting that currently available IKK inhibitors may affect hemostasis.