Nuclear localization of Munc18-1 (p67) in the adult rat brain and PC12 cells

Munc18-1, also referred to as p67, co-purifies with Cdk5 and has an important role in neurotransmitter release. The role of Munc18-1 for functional connectivity of the nervous system was demonstrated by gene knockout experiments in mice, wherein accumulation of neurotransmitter and silencing of synaptic activity was observed. Our earlier studies have shown that both Munc18-1 and Cdk5 co-purify and co-localize with cytoskeletal components, implying that apart from having a regulatory role in vesicle docking and fusion, Munc18-1 could also affect the dynamics of neuronal cytoskeleton. In the present study we have shown the presence of Munc18-1 in nuclear rich fraction from rat brain and confirmed the nuclear localization of this protein in PC12 cells and adult rat brain neurons by immunofluorescence and immunoelectron microscopy. We also demonstrate the binding of Munc18-1 to double stranded (ds) DNA. The ability of Munc18-1 to bind dsDNA, albeit the lack of DNA binding domains, suggests that the binding may be mediated through protein-protein interaction through some other DNA-binding proteins. The presence of both nuclear import and export signals in Munc18-1 primary structure corroborates its nuclear localization and makes it a putative shuttle protein between nuclear and cytoplasmic compartments, the precise physiological relevance of which needs to be elucidated.     

Cyclin-dependent kinase 5 associated with p39 promotes Munc18-1 phosphorylation and Ca(2+)-dependent exocytosis

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase that requires association with a regulatory protein, p35 or p39, to form an active enzyme. Munc18-1 plays an essential role in membrane fusion, and its function is regulated by phosphorylation. We report here that both p35 and p39 were expressed in insulin-secreting beta-cells, where they exhibited individual subcellular distributions and associated with membranous organelles of different densities. Overexpression of Cdk5, p35, or p39 showed that Cdk5 and p39 augmented Ca(2+)-induced insulin exocytosis. Suppression of p39 and Cdk5, but not of p35, by antisense oligonucleotides selectively inhibited insulin exocytosis. Transient transfection of primary beta-cells with Munc18-1 templates mutated in potential Cdk5 or PKC phosphorylation sites, in combination with Cdk5 and the different Cdk5 activators, suggested that Cdk5/p39-promoted Ca(2+)-dependent insulin secretion from primary beta-cells by phosphorylating Munc18-1 at a biochemical step immediately prior to vesicle fusion.     

Regulation of the fusion pore conductance during exocytosis by cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase involved in synaptogenesis and brain development, and its enzymatic activity is essential for slow forms of synaptic vesicle endocytosis. Recent work also has implicated Cdk5 in exocytosis and synaptic plasticity. Pharmacological inhibition of Cdk5 modifies secretion in neuroendocrine cells, synaptosomes, and brain slices; however, the specific mechanisms involved remain unclear. Here we demonstrate that dominant-negative inhibition of Cdk5 increases quantal size and broadens the kinetics of individual exocytotic events measured by amperometry in adrenal chromaffin cells. Conversely, Cdk5 overexpression narrows the kinetics of fusion, consistent with an increase in the extent of kiss-and-run exocytosis. Cdk5 inhibition also increases the total charge and current of catecholamine released during the amperometric foot, representing a modification of the conductance of the initial fusion pore connecting the granule and plasma membrane. We suggest that these effects are not attributable to an alteration in catecholamine content of secretory granules and therefore represent an effect on the fusion mechanism itself. Finally, mutational silencing of the Cdk5 phosphorylation site in Munc18, an essential protein of the late stages of vesicle fusion, has identical effects on amperometric spikes as dominant-negative Cdk5 but does not affect the amperometric feet. Cells expressing Munc18 T574A have increased quantal size and broader kinetics of fusion. These results suggest that Cdk5 could, in part, control the kinetics of exocytosis through phosphorylation of Munc18, but Cdk5 also must have Munc18-independent effects that modify fusion pore conductance, which may underlie a role of Cdk5 in synaptic plasticity.     

Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology

The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurons. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP-dependent manner, causing hyperphosphorylation and down-regulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones, Pak1 phosphorylated on Thr-212 localized to actin and tubulin-rich areas, suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganization. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurons, membrane-associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation, which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodeling.     

Evidence for amylase release by cyclin-dependent kinase 5 in the rat parotid

Cyclin-dependent kinase 5 (Cdk5) plays no apparent role in cell cycle regulation, and Cdk5 is not activated by cyclins but only p35 or p39. Although the enzymatic activity of Cdk5 is highest in the central nervous system, recent reports indicate that it also has important functions in non-neuronal cells. In the present study, we investigated whether Cdk5 and its activators are expressed in rat parotid acinar cells, whether a β-adrenergic agonist enhances the expression of Cdk5, and whether Cdk5 mediates amylase release. We found that Cdk5 and its activator, cyclin I, were expressed in rat parotid acinar cells, and that the expression of Cdk5 was enhanced by treatment of the cells with isoproterenol. Amylase release stimulated by isoproterenol was depressed by the addition of olomoucine, a Cdk5 inhibitor, or by the introduction of an anti-Cdk5 antibody. Cdk5 activity was enhanced by treatment with isoproterenol and this enhanced activity was attenuated by the addition of olomoucine. Olomoucine also attenuated both phosphorylation of Munc18c and translocation of Munc18c from the plasma membrane induced by isoproterenol. These results indicated that β-stimulation of rat parotid acinar cells enhanced the expression of Cdk5, and that this Cdk5 activation may mediate amylase release through phosphorylation of Munc18c.     

Munc 18-1 and granuphilin collaborate during insulin granule exocytosis

Munc 18-1 is a member of the Sec/Munc family of syntaxin-binding proteins known to bind to the plasma membrane Q-SNARE syntaxin1 and whose precise role in regulated exocytosis remains controversial. Here, we show that Munc 18-1 plays a positive role in regulated insulin secretion from pancreatic beta cells. Munc 18-1 depletion caused a loss in the secretory capacity of both transiently transfected INS 1E cells and a stable clone with tetracycline-regulated Munc 18-1 RNA interference. In addition, Munc 18-1-depleted cells exhibited defective docking of insulin granules to the plasma membrane and accumulated insulin in the trans Golgi network. Furthermore, glucose stimulation after Munc 18-1 depletion resulted in the rapid formation of autophagosomes. In contrast, overexpression of Munc 18-1 had no effect on insulin secretion. Although there was no detectable interaction between Munc 18-1 and Munc-18-interacting protein 1 or calcium/calmodulin-dependent serine protein kinase, Munc 18-1 associated with the granular protein granuphilin. This association was regulated by glucose and was required for the specific interaction of insulin granules with syntaxin1. We conclude that Munc 18-1 and granuphilin collaborate in the docking of insulin granules to the plasma membrane in an initial fusion-incompetent state, with Munc 18-1 subsequently playing a positive role in a later stage of insulin granule exocytosis.     

WNK1 is a novel regulator of Munc18c-syntaxin 4 complex formation in soluble NSF attachment protein receptor (SNARE)-mediated vesicle exocytosis

Defects in soluble NSF attachment protein receptor (SNARE)-mediated granule exocytosis occur in islet beta cells, adipocytes, and/or skeletal muscle cells correlate with increased susceptibility to insulin resistance and diabetes. The serine/threonine kinase WNK1 (with no K (lysine)) has recently been implicated in exocytosis and is expressed in all three of these cell types. To search for WNK1 substrates related to exocytosis, we conducted a WNK1 two-hybrid screen, which yielded Munc18c. Munc18c is known to be a key regulator of accessibility of the target membrane (t-SNARE) protein syntaxin 4 to participate in SNARE core complex assembly, although a paucity of Munc18c-binding factors has precluded discovery of its precise functions. To validate WNK1 as a new Munc18c-interacting partner, the direct interaction between WNK1 and Munc18c was confirmed using in vitro binding analysis, and endogenous WNK1-Munc18c complexes were detected in the cytosolic and plasma membrane compartments of the islet beta cell line MIN6. This binding interaction is mediated through the N-terminal 172 residues of Munc18c and the kinase domain residues of WNK1 (residues 159-491). Expression of either of these two minimal interaction domains resulted in inhibition of glucose-stimulated insulin secretion, consistent with a functional importance for the endogenous WNK1-Munc18c complex in exocytosis. Interestingly, Munc18c failed to serve as a WNK1 substrate in kinase activity assays, suggesting that WNK1 functions in SNARE complex assembly outside its role as a kinase. Taken together, these data support a novel role for WNK1 and a new mechanism for the regulation of SNARE complex assembly by WNK1-Munc18c complexes.     

A gain-of-function mutant of Munc18-1 stimulates secretory granule recruitment and exocytosis and reveals a direct interaction of Munc18-1 with Rab3

Munc18-1 plays a crucial role in regulated exocytosis in neurons and neuroendocrine cells through modulation of vesicle docking and membrane fusion. The molecular basis for Munc18 function is still unclear, as are the links with Rabs and SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor-attachment protein) receptor] proteins that are also required. Munc18-1 can bind to SNAREs through at least three modes of interaction, including binding to the closed conformation of syntaxin 1. Using a gain-of-function mutant of Munc18-1 (E466K), which is based on a mutation in the related yeast protein Sly1p, we have identified a direct interaction of Munc18-1 with Rab3A, which is increased by the mutation. Expression of Munc18-1 with the E466K mutation increased exocytosis in adrenal chromaffin cells and PC12 cells (pheochromocytoma cells) and was found to increase the density of secretory granules at the periphery of PC12 cells, suggesting a stimulatory effect on granule recruitment through docking or tethering. Both the increase in exocytosis and changes in granule distribution appear to require Munc18-1 E466K binding to the closed form of syntaxin 1, suggesting a role for this interaction in bridging Rab- and SNARE-mediated events in exocytosis.     

Cortical development: Cdk5 gets into sticky situations

Cyclin-dependent kinase 5 (Cdk5) is much more than its name implies; it plays a role in neuronal migration, neurite outgrowth and degeneration. Recent evidence suggests that Cdk5 regulates neuronal adhesion and cytoskeletal dynamics.     

Phosphorylation of Munc18-1 by Dyrk1A regulates its interaction with Syntaxin 1 and X11α

J. Neurochem. (2012) 122, 1081-1091. ABSTRACT: Dual-specificity tyrosine(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) is a protein kinase that might be responsible for mental retardation and early onset of Alzheimer's disease in Down's syndrome patients. Dyrk1A plays a role in many cellular pathways through phosphorylation of diverse substrate proteins; however, its role in synaptic vesicle exocytosis is poorly understood. Munc18-1, a central regulator of neurotransmitter release, interacts with Syntaxin 1 and X11α. Syntaxin 1 is a key soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein involved in synaptic vesicle docking/fusion events, and X11α modulates amyloid precursor protein processing and β amyloid generation. In this study, we demonstrate that Dyrk1A interacts with and phosphorylates Munc18-1 at the Thr(479) residue. The phosphorylation of Munc18-1 at Thr(479) by Dyrk1A stimulated binding of Munc18-1 to Syntaxin 1 and X11α. Furthermore, the levels of phospho-Thr(479) -Munc18-1 were enhanced in the brains of transgenic mice over-expressing Dyrk1A protein, providing in vivo evidence of Munc18-1 phosphorylation by Dyrk1A. These results reveal a link between Munc18-1 and Dyrk1A in synaptic vesicle trafficking and amyloid precursor protein processing, suggesting that up-regulated Dyrk1A in Down's syndrome and Alzheimer's disease brains may contribute to some pathological features, including synaptic dysfunction and cognitive defect through abnormal phosphorylation of Munc18-1.     

Phosphorylation of protein phosphatase inhibitor-1 by Cdk5

Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation of N-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation.     

Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner

Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon–synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.     

Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.     

Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells

Although Munc18-1 was originally identified as a syntaxin1–interacting protein, the physiological significance of this interaction remains unclear. In fact, recent studies of Munc18-1 mutants have suggested that Munc18-1 plays a critical role for docking of secretory vesicles, independent of syntaxin1 regulation. Here we investigated the role of Munc18-1 in syntaxin1 localization by generating stable neuroendocrine cell lines in which Munc18-1 was strongly down-regulated. In these cells, the secretion capability, as well as the docking of dense-core vesicles, was significantly reduced. More importantly, not only was the expression level of syntaxin1 reduced, but the localization of syntaxin1 at the plasma membrane was also severely perturbed. The mislocalized syntaxin1 resided primarily in the perinuclear region of the cells, in which it was highly colocalized with Secretogranin II, a marker protein for dense-core vesicles. In contrast, the expression level and the plasma membrane localization of SNAP-25 were not affected. Furthermore, the syntaxin1 localization and the secretion capability were restored upon transfection-mediated reintroduction of Munc18-1. Our results indicate that endogenous Munc18-1 plays a critical role for the plasma membrane localization of syntaxin1 in neuroendocrine cells and therefore necessitates the interpretation of Munc18-1 mutant phenotypes to be in terms of mislocalized syntaxin1.     

Involvement of Munc18 isoforms in the regulation of granule exocytosis in neutrophils

Human neutrophil granule exocytosis mobilizes a complex set of secretory granules. This involves different combinations of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins to facilitate membrane fusion. The control mechanisms governing the late fusion steps are still poorly understood. Here, we have analyzed SNARE-interacting Sec1/Munc18 (SM) family members. We found that human neutrophils express Munc18-2 and Munc18-3 isoforms and that Munc18-2 interacts with the target-SNARE syntaxin 3. Munc18-2 was associated preferentially with primary granules but could also be found with secondary and tertiary granules, while Munc18-3 was majorily associated with secondary and tertiary granules. Ultrastructural analysis showed that both Munc18-2 and Munc18-3 were often located in close proximity to their respective SNARE-binding partners syntaxin 3 and syntaxin 4. Both isoforms were also found in plasma membrane fractions and in the cytosol, where they associate with cytoskeletal elements. Upon stimulation, Munc18-2 and Munc18-3 redistributed and became enriched on granules and in the plasma membrane. Munc18-2 primary granule exocytosis can be blocked by introduction of Munc18-2-specific antibodies indicating a crucial role in primary granule fusion. Our results suggest that Munc18-2 acts as a regulator of primary granule exocytosis, while Munc18-3 may preferentially regulate the fusion of secondary granules.     

GTP-dependent secretion from neutrophils is regulated by Cdk5

We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35(-/-) mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils.     

Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites

Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.