Physiological modulation of rabphilin phosphorylation

The dynamic modulation of protein function by phosphorylation plays an impo rtant role in regulating synaptic plasticity. Several proteins involved in synaptic transmission have been shown to be targets of protein kinases and phosphatases. A thorough analysis of the physiological role of these modifi cations has been hampered by the lack of reagents that specifically recogni ze the phosphorylated states of these proteins. In this study we analyze th e physiological modulation of rabphilin using phosphospecific antibodies. W e show that phosphorylation on serine-234 and serine-274 of rabphilin is dy namically regulated both under basal and stimulated conditions by the activ ity of kinases and phosphatases. The two sites are differentially phosphory lated by the stimulation of various kinases, suggesting a possible converge nce of different pathways to modulate the function of the protein. Maximal stimulation was observed under plasma membrane-depolarizing conditions that trigger synaptic vesicle exocytosis. The increase in phosphorylation was c ritically dependent on external Ca2+ and on the presence of Rab3a, a small GTPase that recruits rabphilin to synaptic vesicles. The rapid phosphorylat ion and dephosphorylation during and after stimulation demonstrates the tra nsient nature of the modification. Our results indicate that rabphilin is p hosphorylated on synaptic vesicles by Ca2+-dependent kinases that become ac tive in synaptic terminals during exocytosis. We have found that phosphorab philin has a reduced affinity for membranes; we therefore propose that the modulation of the membrane association of rabphilin has a role in the synap tic vesicle life cycle, perhaps in vesicle mobilization in preparation for subsequent rounds of neurotransmission.