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  • ibotenic acid mg br What are the local

    2021-09-02


    What are the local exocytotic protein targets of PKC? A direct method for PKC to potentiate insulin release would be to phosphorylate and activate components of the exocytotic machinery. Bulk measurements show that potentiation occurs by enhancement of the calcium sensitivity of exocytosis [56], [57], [58], [59]. At a basic level this results in more insulin released per calcium stimulus. Several possible pathways could work to achieve this: Many exocytotic proteins are targets of post-translational modification by a host of kinases [15], [108], [109], [110]. In vitro, PKC phosphorylates syntaxin [108], [110]. Also, PKC can phosphorylate VAMP [108], [111], though other kinases are likely to be more relevant [15]. NSF is a PKC target and phosphorylation appears to reduce its affinity for SNARE complexes [112]. There is some evidence in neurons that NSF and PKC-ε control trafficking of receptors to the cell surface [113]. However, although many of these proteins can be phosphorylated in vitro, there remains little direct evidence for these phosphorylation events playing a ibotenic acid mg role in insulin secretion. In contrast, the most well studied PKC targets in the exocytotic machinery are munc18, synaptotagmin, and SNAP25 [15]. Munc18 is a syntaxin-binding protein that plays a critical docking and priming role in exocytosis [114] (Fig. 1B1-3). Munc18 binds to the closed form of syntaxin, but also interacts with the ternary SNARE complex via a binding site on syntaxin’s N-terminus [114]. Munc18 is phosphorylated by PKC at residues Ser306 and Ser313 [115], [116]. In vivo munc18 phosphorylation has been observed in many cell systems: islets, chromaffin cells, neurons, and others [117], [118], [119], [120], [121], [122]. In neuronal synapses, munc18 phosphorylation is kept low by phosphatases, such that upon PKC activation a large dynamic change in phosphorylation state can be achieved [118], [123]. Munc18 appears to be important in RRP refilling, which could be attributed to a syntaxin trafficking role or to an increase in SNARE ternary complex assembly [119], [120]. Overall there is good evidence that munc18 is regulated by PKC in vivo and that the protein’s phosphorylation state is dynamically controlled, suggesting it could be an important regulatory module. How does munc18 phosphorylation relate to insulin release? A longstanding hypothesis is that phosphorylation of munc18 causes opening of a closed form of syntaxin and allows for more vesicles to dock to the plasma membrane [115]. This would increase the number of calcium-sensitive vesicles at the plasma membrane. Another hypothesis comes from work by Mandic et al. who looked more closely at munc18-1 and munc18-2, two isoforms expressed in beta cells, and their function in insulin secretion [124]. Interestingly, these authors show that munc18-1 and munc18-2 support exocytosis with distinct kinetics and that the two isoforms affect the calcium sensitivity of vesicles at the membrane. Phosphorylation of both is important for their function. Differential phosphorylation and activation of these isoforms could be the molecular correlate of the PKC-dependent calcium sensitivity changes of exocytosis in beta cells. Ultimately, the mechanistic details of munc18 phosphorylation with respect to insulin release remain unclear. Synaptotagmin is the primary calcium sensor of exocytosis, making it an ideal candidate for PKC-mediated calcium sensitization of the exocytotic apparatus [125] (Fig. 1B3). However, phosphorylation of synaptotagmin does not appear to directly affect its calcium affinity. The protein is phosphorylated by PKC at Thr112, and phosphorylation levels increase when PKC is activated by PMA [126]. There are many synaptotagmin isoforms, with Syt-1 and Syt-7 playing major roles in neuronal and endocrine secretion, respectively [125]. Synaptotagmin isoforms have different affinities for calcium, suggesting that they could mediate exocytosis of distinct vesicle pools in vivo[125]. Sorting of Syt-1 and Syt-7 onto distinct vesicle pools occurs in chromaffin cells and affects exocytotic behavior [127].