Differential phosphorylation of syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) isoforms

The synaptic plasma membrane proteins syntaxin and synaptosome-associated p rotein of 25 kDa (SNAP-25) are central participants in synaptic vesicle tra fficking and neurotransmitter release. Together with the synaptic vesicle p rotein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N-ethylmaleimide -sensitive factor (NSF) and soluble NSF attachment protein (alpha-SNAP). Co nsequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membra ne trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have i nvestigated the ability of syntaxin and SNAP-25 isoforms to serve as substr ates for a variety of serine/threonine protein kinases. Syntaxins IA and 4 were phosphorylated by casein kinase Il, whereas syntaxin 3 and SNAP-25 wer e phosphorylated by Ca2+- and calmodulin-dependent protein kinase Il and cy clic AMP-dependent protein kinase, respectively. The biochemical consequenc es of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between p hosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca2+ sensor in triggering synaptic vesicle exocytosis. No othe r effects on the formation of SNARE complexes (comprised of syntaxin, SNAP- 25, and VAMP) or interactions involving n-Sec1 or alpha-SNAP were observed. These findings suggest that although phosphorylation does not directly reg ulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.