Dataset Information

SNAP-23 participates in SNARE complex assembly in rat adipose cells.

ABSTRACT: SNARE proteins are required for vesicle docking and fusion in eukaryotic cells in processes as diverse as homotypic membrane fusion and synaptic vesicle exocytosis [SNARE stands for SNAP receptor, where SNAP is soluble NSF attachment protein]. The SNARE proteins syntaxin 4 and vesicle-associated membrane protein (VAMP) 2/3 also participate in the insulin-stimulated translocation of GLUT4 from intracellular vesicles to the plasma membrane in adipose cells. We now report the molecular cloning and characterization of rat SNAP-23, a ubiquitously expressed homologue of the essential neuronal SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Rat SNAP-23 is 86% and 98% identical respectively to human and mouse SNAP-23. Southern blot analysis reveals that the rat, mouse and human SNAP-23 genes encode species-specific isoforms of the same protein. Co-immunoprecipitation of syntaxin 4 and SNAP-23 shows association of these two proteins in rat adipose cell plasma membranes, and insulin stimulation does not alter the SNAP-23/syntaxin 4 complex. In addition, we demonstrate for the first time the participation of SNAP-23, along with syntaxin 4 and VAMP2/3, in the formation of 20S SNARE complexes prepared using rat adipose cell membranes and recombinant alpha-SNAP and NSF proteins. The stoichiometry of the SNARE complexes formed is essentially identical using membranes from either unstimulated or insulin-stimulated adipose cells. These data demonstrate that rat SNAP-23 associates with syntaxin 4 before insulin stimulation and is present in the SNARE complexes known to mediate the translocation of GLUT4 from intracellular vesicles to the plasma membrane of rat adipose cells.

SUBMITTER: St-Denis JF

PROVIDER: S-EPMC1220107 | biostudies-other | 1999 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Reversible phosphorylation of neuronal proteins plays an important role in the regulation of neurotransmitter release. Myosin phosphatase holoenzyme (MP) consists of a protein phosphatase-1 (PP1) catalytic subunit (PP1c) and a regulatory subunit, termed myosin phosphatase targeting subunit (MYPT1). The primary function of MP is to regulate the phosphorylation level of contractile proteins; however, recent studies have shown that MP is localized to neurons, and is also involved in the mediation of neuronal processes. Our goal was to investigate the effect of RhoA-activated kinase (ROK) and MP on the phosphorylation of one potential neuronal substrate, the synaptosomal-associated protein of 25 kDa (SNAP-25). SNAP-25 is a member of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, along with synaptobrevin and syntaxin, and the primary role of SNAP25 is to mediate vesicle fusion. We showed that MYPT1 interacts with SNAP-25, as revealed by immunoprecipitation and surface plasmon resonance based binding studies. Mass spectrometry analysis and in vitro phosphorylation/dephosphorylation assays demonstrated that ROK phosphorylates, while MP dephosphorylates, SNAP-25 at Thr138. Silencing MYPT1 in B50 neuroblastoma cells increased phosphorylation of SNAP-25 at Thr138. Inhibition of PP1 with tautomycetin increased, whereas inhibition of ROK by H1152, decreased the phosphorylation of SNAP-25 at Thr138 in B50 cells, in cortical synaptosomes, and in brain slices. In response to the transduction of the MP inhibitor, kinase-enhanced PP1 inhibitor (KEPI), into synaptosomes, an increase in phosphorylation of SNAP-25 and a decrease in the extent of neurotransmitter release were detected. The interaction between SNAP-25 and syntaxin increased with decreasing phosphorylation of SNAP-25 at Thr138, upon inhibition of ROK. Our data suggest that ROK/MP play a crucial role in vesicle trafficking, fusion, and neurotransmitter release by oppositely regulating the phosphorylation of SNAP-25 at Thr138.

Dataset Information

SNAP-23 participates in SNARE complex assembly in rat adipose cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure