Bhatnagar S, Soni MS, Wrighton LS, Hebert AS, Zhou AS, Paul PK, Gregg T, Rabaglia ME, Keller MP, Coon JJ, Attie AD

The abundance and functional activity of proteins involved in the formation of the SNARE complex are tightly regulated for efficient exocytosis. Tomosyn proteins are negative regulators of exocytosis. Tomosyn causes an attenuation of insulin secretion by limiting the formation of the SNARE complex. We hypothesized that glucose-dependent stimulation of insulin ...

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secretion from β-cells must involve reversing the inhibitory action of tomosyn. Here we show that glucose increases tomosyn protein turnover. Within 1 h of exposure to 15 mM glucose, ~50% of tomosyn was degraded. The degradation of tomosyn in response to high glucose was blocked by inhibitors of the proteasomal pathway. Using 32P-labeling and mass spectrometry, we showed that tomosyn-2 is phosphorylated in response to high glucose, phorbol esters, and analogs of cAMP, all key insulin secretagouges. We identified eleven phosphorylation sites in tomosyn-2. Site-directed mutagenesis was used to generate phosphomimetic (S→D) and loss-of-function (S→A) mutants. The S→D mutant had enhanced protein turnover compared to the S→A mutant and wild type tomosyn-2. Additionally, the S→D tomosyn-2 mutant was ineffective at inhibiting insulin secretion. Using a proteomic screen for tomosyn-2 binding proteins we identified Hrd-1, an E3-ubiquitin ligase. We showed that tomosyn-2 ubiquitination is increased by Hrd-1 and knockdown of Hrd-1 by short-hairpin RNA resulted in increased abundance in tomosyn-2 protein levels. Taken together, our results reveal a mechanism by which enhanced phosphorylation of a negative regulator of secretion, tomosyn-2, in response to insulin secretagogues targets it to degradation by the Hrd-1 E3-ubiquitin ligase.

Date: Jul. 07, 2014

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