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

Abstract

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 Mr 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) (<0.05 mol of phosphate/mol of syntaxin-4). Importantly, PKA but not casein kinase II phosphorylation of syntaxin-4 disrupted its binding to SNAP23. We hypothesize that PKA may modulate syntaxin-4-dependent SNARE complex formation to regulate exocytosis in non-neuronal cells.

VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts

“…Protein samples were separated by 10 or 12% (vol/vol) SDS-PAGE as indicated and electrotransferred onto polyvinylidene difluoride membranes as described previously (Foster et al, 1998). Immunoreactive bands were visualized with HRP-conjugated goat antirabbit IgG for polyclonal antibodies, as indicated, by means of an enhanced chemiluminescence detection technique.…”

t-SNARE Phosphorylation Regulates Endocytosis in Yeast

“…In particular, kinases involved in growth control are likely to modulate SNARE functions, presumably by the posttranslational modification of residues that participate in SNARE partnering or the binding of SNARE regulatory proteins (reviewed in Gerst, 1999;Turner et al, 1999;Klenchin and Martin, 2000). Numerous studies have outlined the possible involvement of kinases in the regulation of SNARE protein interactions (Hirling and Scheller, 1996;Shimazaki et al, 1996;Foster et al, 1998;Shuang et al, 1998;Risinger and Bennett, 1999;Chung et al, 2000) as well as the availability of SNAREs to participate in membrane trafficking events (Cabaniols et al, 1999;Foletti et al, 2000;Kataoka et al, 2000). Therefore, a role for signal-induced protein kinases in regulating SNARE complex assembly is likely and offers a potential mechanism for coordinating the dynamics of cell growth with cell cycle control.…”

Arginine vasopressin-induced glucagon release: interaction with glucose and cyclic AMP-dependent protein kinase

“…Syntaxin and SNAP-25 are phosphorylated by CaMK II and PKA (Trudeau et al, 1998;Foster et al, 1998). SNAP-25 has been shown to be expressed in pancreatic islets and in pancreatic aand p-cells (Sadoul et al, 1995).…”

“…Although, the precise downstream targets of PKA are currently unknown, several intracellular regulatory proteins have been shown to be substrates for PKA in vitro. These include rabphilin-3A (Lonart et al, 1998), synapsin , syntaxin (Trudeau et al, 1998) and SNAP-25 (Foster et al, 1998). Thus, it is likely that the modulation of glucagon release produced by the activated PKA could occur through a direct phosphorylation of one or more of these exocytotic proteins.…”

“…In addition, A VP-induced glucagon release is mediated largely by a Ca 2 *-dependent pathway . It is well known that Ca 2 * activates CaMK II, which may lead to phosphorylation of a set of exocytotic proteins including rabphilin-3A (Fykse et al, 1995), synaptotagmin (Popoli, 1993), syntaxin (Trudeau et al, 1998) and SNAP-25 (Foster et al, 1998).…”