Characterization of rabphilin phosphorylation using phospho-specific antibodies.

Rab3A is a GTP-binding protein of synaptic vesicles that regulates neurotransmitter release and cycles on and off synaptic vesicles as a function of exocytosis. Rab3A presumably functions via GTP-dependent interactions with effectors. Two putative rab3A effectors have been described in neurons, rabphilin which is a soluble protein that moves onto and off synaptic vesicles in concert with rab3A, and RIM which is an active zone protein that only binds to rab3A on docked vesicles. Rabphilin is an abundant, evolutionarily conserved protein whose function has remained enigmatic since a knockout of rabphilin does not display the functional deficiencies observed in the rab3A knockout. However, previous studies have shown that rabphilin is phosphorylated by protein kinase A and CaM Kinase II, suggesting that it may have a regulatory role. In the present study, we have examined the site and regulation of rabphilin phosphorylation in living nerve terminals using phospho-specific antibodies raised against phospho-serine234 of rabphilin. With these antibodies, we demonstrate that rabphilin is physiologically phosphorylated on serine234, and that soluble rabphilin which is not bound to rab3A on synaptic vesicles is the primary target. However, different from synapsins which are induced to dissociate from synaptic vesicles by PKA phosphorylation, phosphorylation of rabphilin is not instrumental for dissociating rabphilin from synaptic vesicles. Our data support the notion that dissociated rabphilin is a synaptic phosphoprotein in vivo that may play a role in the regulation of nerve terminal protein-protein interactions.