We show that Hrd-1 binds, ubiquitinates, and regulates the abundance of tomosyn-2, suggesting that Hrd-1 is an E3-ligase for tomosyn-2. 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 usually 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. enhances synaptic transmission (14). In bovine chromaffin cells, tomosyn-1 inhibits exocytosis by decreasing the degree of depletion and replenishment of dense core vesicles (15). Tomosyn-1 and tomosyn-2 are syntaxin-1A-binding proteins. Their inhibitory effect on exocytosis was attributed to the C-terminal R-SNARE domain name. However, this model has undergone several revisions. Rabbit Polyclonal to LMTK3 The N-terminal domain name of tomosyn-1, which UK 14,304 tartrate makes up 90% of the protein, is required for the inhibitory effects of tomosyn-1 on exocytosis (8, 11). An N-terminal region deletion mutant of tomosyn-1 was able to bind syntaxin-1A but lacked the ability to inhibit exocytosis (16). Additionally, a tomosyn-1 fragment lacking the R-SNARE domain name was able to attenuate exocytosis (16). The N-terminal domain name of tomosyn-1 binds and inhibits synaptotagmin-1-mediated neurotransmitter release (17). Recently, Williams (18) exhibited that fragments made up of loop 1 (537C578 amino acids) or loop 3 (933C955 amino acids) deletions in the N-terminal region of tomosyn-1 were able to bind syntaxin-1A by the R-SNARE domain name but failed UK 14,304 tartrate to inhibit exocytosis. Together, this suggests that the N-terminal domain name in tomosyn-1 and, by extension, tomosyn-2 plays a critical role for imparting the inhibitory effects on exocytosis. The N-terminal domain name of tomosyn-1 contains a hypervariable region (HVR),2 between amino acids 702 and 822. This is the least conserved region between isoforms of tomosyn-1 and tomosyn-2 (19). A protein kinase A (PKA) phosphorylation site at serine 724, was identified in the HVR of tomosyn-1 (20). Phosphorylation of this residue decreases the inhibitory function of tomosyn-1, leading to an increase in exocytosis (20). Additionally, tomosyn-1 is usually SUMOylated at lysine 730 in the HVR (18). SUMOylation of tomosyn-1 also reduces its ability to inhibit exocytosis. Post-translational modifications in the HVR of tomosyn-1 suggest an important regulatory role of this region. We previously positionally cloned tomosyn-2 as a gene underlying a diabetes susceptibility locus in an F2 intercross of the C57BL/6 and BTBR mouse strains (10). Islets from congenic mice carrying the BTBR allele of tomosyn-2 (harboring an S912L mutation) had an attenuated response to stimulation of insulin secretion compared with islets carrying the C57BL/6 allele. This reduction in secretion was observed in the presence of normal levels of tomosyn-1, suggesting a nonoverlapping role of tomosyn-1 and tomosyn-2 in inhibiting insulin secretion. Here, we describe a novel mechanism whereby insulin secretagogues enhance turnover of tomosyn-2 as part of a mechanism to stimulate insulin secretion. We identify phosphorylation sites in tomosyn-2 that respond to insulin secretagogues and an E3-ubiquitin ligase that targets tomosyn-2 for degradation. EXPERIMENTAL PROCEDURES Antibodies and Chemicals Insulin was measured with an in-house ELISA using an anti-insulin antibody from Fitzgerald UK 14,304 tartrate Industries. Antibodies used to perform experiments were as follows: mouse anti-Myc antibody from Millipore; mouse anti-V5 antibody.
