title: Phosphorylation and degradation of tomosyn-2 de-represses insulin secretion.
abstract: 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 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 (32)P 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 secretagogues. We identified 11 phosphorylation sites in tomosyn-2. Site-directed mutagenesis was used to generate phosphomimetic (Ser → Asp) and loss-of-function (Ser → Ala) mutants. The Ser → Asp mutant had enhanced protein turnover compared with the Ser → Ala mutant and wild type tomosyn-2. Additionally, the Ser → Asp 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.
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Sushant
authors.initials: S
authors.lastname: Bhatnagar
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Mufaddal S
authors.initials: MS
authors.lastname: Soni
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Lindsay S
authors.initials: LS
authors.lastname: Wrighton
authors.affiliation: Chemistry and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Hebert
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Amber S
authors.initials: AS
authors.lastname: Zhou
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Pradyut K
authors.initials: PK
authors.lastname: Paul
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Trillian
authors.initials: T
authors.lastname: Gregg
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Rabaglia
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Keller
authors.affiliation: Chemistry and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.affiliation: From the Departments of Biochemistry and adattie@wisc.edu.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Attie
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005947:Glucose / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis / Q000187:drug effects; D050683:R-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D007328:Insulin; D050683:R-SNARE Proteins; C492530:STXBP5L protein, mouse; D012694:Serine; C482345:SYVN1 protein, human; D044767:Ubiquitin-Protein Ligases; D005947:Glucose
keywords: Diabetes; Insulin; Insulin Secretion; SNARE Proteins; Type 2 Diabetes