Binary Interactions of the SNARE Proteins Syntaxin-4, SNAP23, and VAMP-2 and Their Regulation by Phosphorylation

• Published in: Biochemistry (Easton) 1998-08, Vol.37 (31), p.11089-11096
• Main Authors: Foster, Leonard J, Yeung, Brian, Mohtashami, Mahmood, Ross, Kathryn, Trimble, William S, Klip, Amira
• Format: Article
• Language: English
• Subjects: Animals; Carrier Proteins - immunology; Carrier Proteins - metabolism; Casein Kinase II; Cyclic AMP-Dependent Protein Kinases - metabolism; Macromolecular Substances; Membrane Proteins - immunology; Membrane Proteins - metabolism; Mice; Nerve Tissue Proteins - metabolism; Phosphates - metabolism; Phosphorylation; Precipitin Tests; Protein Binding; Protein Kinase C - metabolism; Protein-Serine-Threonine Kinases - metabolism; Qa-SNARE Proteins; Qb-SNARE Proteins; Qc-SNARE Proteins; R-SNARE Proteins; Rats; SNARE Proteins; Sodium Dodecyl Sulfate; Syntaxin 1; Vesicular Transport Proteins
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi980253t

The SNARE hypothesis proposes that synaptic vesicles dock at presynaptic membranes via interactions among the vesicular, integral membrane proteins VAMP (vesicle-associated membrane protein) and synaptotagmin and the target membrane proteins SNAP25 (synaptosome-associated protein with an M r of 25 kDa) and syntaxin-1. Non-neuronal cells express isoforms of these proteins, believed to mediate secretory vesicle docking and/or fusion. Secretion in neuronal and non-neuronal systems differs in time course, Ca2+ dependence, and regulatory input. It is not known whether the non-neuronal protein isoforms form complexes akin to those of their neuronal counterparts. In this study, we defined the binding characteristics of three SNARE proteins:  SNAP23, VAMP-2, and syntaxin-4. Binary, saturable interactions among all three partners (VAMP-2−syntaxin-4, VAMP-2−SNAP23, and SNAP23−syntaxin-4) were measured in vitro. Unlike its neuronal counterpart, SNAP23 did not potentiate VAMP-2 binding to its putative t-SNARE partner, syntaxin-4. The susceptibility of SNARE proteins to phosphorylation by exogenous kinases and their impact on binary interactions were explored. Syntaxin-4 was efficiently phosphorylated by casein kinase II (CKII) and cAMP-dependent protein kinase (PKA) (incorporating 0.8 and 3.9 mol of phosphate/mol of syntaxin-4, respectively), while syntaxin-1 was only strongly phosphorylated by CKII. Each of the syntaxin isoforms was weakly phosphorylated by protein kinase C (PKC) (