RESUMO
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 ß-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 â Asp) and loss-of-function (Ser â Ala) mutants. The Ser â Asp mutant had enhanced protein turnover compared with the Ser â Ala mutant and wild type tomosyn-2. Additionally, the Ser â 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.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas R-SNARE/metabolismo , Serina/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Células Cultivadas , Glucose/farmacologia , Células HEK293 , Humanos , Immunoblotting , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Modelos Moleculares , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Estrutura Terciária de Proteína , Proteólise/efeitos dos fármacos , Proteínas R-SNARE/química , Proteínas R-SNARE/genética , Interferência de RNA , Serina/química , Serina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
We previously demonstrated that micro-RNAs (miRNAs) 132 and 212 are differentially upregulated in response to obesity in two mouse strains that differ in their susceptibility to obesity-induced diabetes. Here we show the overexpression of miRNAs 132 and 212 enhances insulin secretion (IS) in response to glucose and other secretagogues including nonfuel stimuli. We determined that carnitine acyl-carnitine translocase (CACT; Slc25a20) is a direct target of these miRNAs. CACT is responsible for transporting long-chain acyl-carnitines into the mitochondria for ß-oxidation. Small interfering RNA-mediated knockdown of CACT in ß-cells led to the accumulation of fatty acyl-carnitines and enhanced IS. The addition of long-chain fatty acyl-carnitines promoted IS from rat insulinoma ß-cells (INS-1) as well as primary mouse islets. The effect on INS-1 cells was augmented in response to suppression of CACT. A nonhydrolyzable ether analog of palmitoyl-carnitine stimulated IS, showing that ß-oxidation of palmitoyl-carnitine is not required for its stimulation of IS. These studies establish a link between miRNA-dependent regulation of CACT and fatty acyl-carnitine-mediated regulation of IS.
