Cyclin-dependent Kinase 5 Associated with p39 Promotes Munc18-1 Phosphorylation and Ca2+-dependent Exocytosis

Lilja, Lena; Johansson, Jenny U.; Gromada, Jesper; Mandic, Slavena A.; Fried, Gabriel; Berggren, Per Olof; Bark, Christina

doi:10.1074/jbc.m312711200

Cited by 72 publications

(78 citation statements)

References 63 publications

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“…Electrophysiology. Exocytosis was monitored in single ␤-cells as changes in cell capacitance using an EPC-9 patch-clamp amplifier Pulse software (HEKA Elektronik, Lambrecht/Pfalz, Germany), as described previously (32). In Fig.…”

Section: Methodsmentioning

confidence: 99%

Tomosyn Is Expressed in β-Cells and Negatively Regulates Insulin Exocytosis

et al. 2006

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E xocytosis is delicately regulated via dynamic protein-protein interactions between different protein components localized to the plasma membrane, the secretory vesicle membrane, and the cytoplasm. According to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) hypothesis (1,2), the vesicular-SNARE vesicleassociated membrane protein (also called synaptobrevin) interacts with the cognate target-SNAREs syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) to form a core complex (also called SNARE complex) (1). The assembly of SNARE proteins between two opposing membranes and the formation of a core complex have been shown to be the key events that initiate membrane fusion and predict the specificity of vesicle fusion (1,2). That the compartmental specificity of cellular membrane fusion is encoded in SNARE proteins is further provided by the observation that these proteins have distinct localization in a cell (3). However, almost any combination of several members of vesicular-and target-SNARE proteins can form a SDS-resistant protein complex (4,5), suggesting that the interactions between SNARE proteins cannot provide all information for vesicle targeting. Additional specificity may be provided by other molecules that interact with SNARE proteins. An example of such a protein is the well-conserved syntaxin-binding protein Sec1/mammalian homolog of the Caenorhabditis elegans unc-18 gene (Munc-18). There are several Munc-18 isoforms in mammals, which are believed to support different vesicular trafficking events (rev. in 6). Munc-18-1 holds syntaxin in a closed conformation, thereby preventing the binding of SNAP-25 and vesicle-associated membrane protein to syntaxin (7). Moreover, each Munc-18 protein interacts more or less exclusively with one or two syntaxin isoforms, thereby providing further vesicle-targeting specificity (8 -13).The existence of another syntaxin-binding protein, designated tomosyn (tomo ϭ friend in Japanese, syn ϭ syntaxin), has been reported (14). Besides the original tomosyn protein, which has been named m-tomosyn, two further splice variants of tomosyn, designated big (b) and small (s) tomosyn, have been identified (15). The m-and s-tomosyn variants are mainly expressed in the brain, whereas b-tomosyn is found ubiquitously (15). More recently, two distinct genes that drive the expression of seven tomosyn isoforms in the mammalian brain have been described (16). Tomosyn is capable of dissociating Munc-18 from syntaxin 1 and thereby forming a novel complex with syntaxin 1, and synaptotagmin (14). The COOH-terminal domain of tomosyn spans a SNARE motif that allows tomosyn to form a stable complex with syntaxin 1A and SNAP-25 (17,18). Endogenous expression or overexpression of tomosyn has been shown to cause a reduction of Ca 2ϩ -dependent exocytosis (14,19 -23). The structural basis for the inhibitory role of tomosyn in exocytosis has recently been presented (24).In this study, we have investigated isoform expression and cellular localization of tomo...

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Section: Methodsmentioning

confidence: 99%

Tomosyn Is Expressed in β-Cells and Negatively Regulates Insulin Exocytosis

et al. 2006

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“…After transfection with pCMV5-hGH (29) and either empty vector pcDNA3 or plasmid of interest, INS-1E cells were seeded in 48-well plates (2 ϫ 10 5 cells per well) and cultured for 48 h. Incubation and secretion experiments were performed as described in ref. 29. hGH levels in the various samples were measured by using ELISA (Roche).…”

Section: Methodsmentioning

confidence: 99%

Neuronal calcium sensor-1 potentiates glucose-dependent exocytosis in pancreatic β cells through activation of phosphatidylinositol 4-kinase β

Gromada

Bark

Smidt

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Cytosolic free Ca 2؉ plays an important role in the molecular mechanisms leading to regulated insulin secretion by the pancreatic ␤ cell. A number of Ca 2؉ -binding proteins have been implicated in this process. Here, we define the role of the Ca 2؉ -binding protein neuronal Ca 2؉ sensor-1 (NCS-1) in insulin secretion. In pancreatic ␤ cells, NCS-1 increases exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in the readily releasable pool. The effect of NCS-1 on exocytosis is mediated through an increase in phosphatidylinositol (PI) 4-kinase ␤ activity and the generation of phosphoinositides, specifically PI 4-phosphate and PI 4,5-bisphosphate. In turn, PI 4,5-bisphosphate controls exocytosis through the Ca 2؉ -dependent activator protein for secretion present in ␤ cells. Our results provide evidence for an essential role of phosphoinositide synthesis in the regulation of glucose-induced insulin secretion by the pancreatic ␤ cell. We also demonstrate that NCS-1 and its downstream target, PI 4-kinase ␤, are critical players in this process by virtue of their capacity to regulate the release competence of the secretory granules.insulin ͉ phosphoinositides ͉ islet ͉ secretion ͉ Ca 2ϩ -dependent activator protein for secretion N euronal Ca 2ϩ sensor-1 (NCS-1) belongs to the family of EF-hand Ca 2ϩ -binding proteins and is mainly expressed in neuronal and neuroendocrine cells, where it enhances neurotransmission and Ca 2ϩ -dependent exocytosis (1-5). NCS-1 interacts with and regulates the activity of phosphatidylinositol 4-kinase ␤ (PI4K␤) (6-9). The members of the PI4K family catalyze the first step in the synthesis of PI 4,5-bisphosphate [PI(4,5)P 2 ], which has recently emerged as an important regulator of Ca 2ϩ -dependent secretion (10-12). Both a PI transfer protein (13) and a PI4P 5-kinase (14) are required for regulated exocytosis of dense core granules. In addition, the presence of synaptic vesicle and dense core granule-associated PI4K activity is essential for exocytosis (15,16). These data imply that generation of PI(4,5)P 2 is an important step in the event of secretion.In pancreatic ␤ cells, glucose dose-dependently increases ATP levels and decreases ADP levels (16). The resulting rise in the ATP at the expense of ADP is an important regulator of the two major signaling pathways involved in glucose-induced insulin secretion. The first of these pathways uses ATP-sensitive K ϩ channels to couple glucose metabolism with electrical activity, Ca 2ϩ influx, and initiation of insulin secretion. The second pathway is exerted at the level of granule priming and regulates the ␤ cell secretory capacity by modulation of the granules' release competence (17). The ␤ cell contains Ϸ10,000 insulincontaining secretory granules (18). Interestingly, as many as Ϸ5% of the granules are docked below the membrane. The readily releasable pool (RRP), defined by functional measurements, represents a subset (50-100 granules) of the docked pool (19). Granules belongin...

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“…In general, this process is mediated by proteins of the Rab complex (Rabs), SNARES and SM protein families (Jahn et al, 2003). Many proteins involved in membrane fusion are made active by intracellular signaling, involving phosphorylation and protease cleavage (Rutledge and Whiteheart, 2002;Lilja et al, 2004;Huyng et al, 2004;Hepp et al, 2005). Calcium binding proteins, such as calmodulin, are known to work as calcium sensors, leading to activation of kinases that in turn activate membrane fusion proteins (Hilfiker et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Calcium-regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixusStahl

Ramos

Miranda

Souza

et al. 2007

Journal of Experimental Biology

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SUMMARY This study examined the process of membrane fusion of yolk granules (YGs)during early embryogenesis of Rhodnius prolixus. We show that eggs collected at days 0 and 3 after oviposition contain different populations of YGs, for example day-3 eggs are enriched in large YGs (LYGs). Day-3 eggs also contain the highest free [Ca2+] during early embryogenesis of this insect. In vitro incubations of day-0 YGs with [Ca2+]similar to those found in day-3 eggs resulted in the formation of LYGs, as observed in vivo. Fractionation of LYGs and small YGs (SYGs) and their subsequent incubation with the fluorescent membrane marker PKH67 showed a calcium-dependent transference of fluorescence from SYGs to LYGs, possibly as the result of membrane fusion. Acid phosphatase and H+-PPase activities were remarkably increased in day-3 LYGs and in calcium-treated day-0 LYGs. Both fractions were found to contain vitellins as major components, and incubation of YGs with calcium induced yolk proteolysis in vitro. Altogether, our results suggest that calcium-induced membrane fusion events take part in yolk degradation, leading to the assembly of the yolk mobilization machinery.

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Cyclin-dependent Kinase 5 Associated with p39 Promotes Munc18-1 Phosphorylation and Ca2+-dependent Exocytosis

Cited by 72 publications

References 63 publications

Tomosyn Is Expressed in β-Cells and Negatively Regulates Insulin Exocytosis

Tomosyn Is Expressed in β-Cells and Negatively Regulates Insulin Exocytosis

Neuronal calcium sensor-1 potentiates glucose-dependent exocytosis in pancreatic β cells through activation of phosphatidylinositol 4-kinase β

Calcium-regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixusStahl

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