The Journal of Neuroscience, March 1995, 15(3): 2385-2395 Phosphorylation of Rabphilin-3A by Ca2+/Calmodulin- and CAMP-Dependent Protein Kinases in vitro Else Marie Fykse,” Cai Li, and Thomas C. Siidhof Department of Molecular Genetics and Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas 75235 Regulation of neurotransmitter release is thought to in- dependent protein kinase II (a-CaMK II) exhibit dramatic volve modulation of the release probability by protein changesin short-term synaptic plasticity even in the heterozy- phosphorylation. In order to identify novel targets for such gous state, which presumably only lowers the levels of enzyme regulatory processes, we have studied the phosphorylation by half (Silva et al., 1992; A. Silva, personalcommunication). of rabphilin9A in vitro. Rabphilin-3A is a synaptic vesicle LTP in mossy fiber synapses,a known presynapticform of syn- protein that interacts with rab3A in a GTP-dependent man- aptic plasticity (Zalutsky and Nicoll, 1990), is related to CAMP- ner and binds Ca*+ in a phospholipid-dependent manner. dependentprotein kinase (PKA) activation (Frey et al., 1993). Here we show that rabphilin-3A is an efficient substrate for Pharmacologicalexperiments using a variety of protein kinase Ca*+/calmodulin-dependent protein kinase II, which phos- inhibitors and stimulatorsalso suggesta role for protein kinases phorylates rat rabphilin-3A at residue 234 and 274, and for in synaptic plasticity (Zhuo et al., 1994; reviewed in Stevens, CAMP-dependent protein kinase, which phosphorylates rat 1993). Thus, there is significant evidence for a major role of rabphilin-3A at residue 234. This identifies the middle re- protein phosphorylation events in at least some forms of regu- gion of rabphilin4A situated between the N-terminal rab3A- lation of neurotransmitterrelease. binding sequences and the C-terminal &-domains involved At least two possiblemechanisms can be envisionedby which in Ca2+/phospholipid binding as a regulatory domain. Thus, protein phosphorylation could regulate neurotransmitterrelease. rabphilin9A is a second phosphoprotein on synaptic ves- First, protein kinasescould phosphorylate ion channelsand their icles that, similar to synapsin I, may integrate phosphory- associatedproteins. As a regulatory endpoint, such phosphory- lation signals from multiple protein kinase signaling path- lation events could lead to an increaseor decreasein the intra- ways in the cell. cellular Ca2+levels achieved after an action potential and there- [Key words: synaptic vesicles, synaptic plasticity, exo- by regulate release(for a review, see Nicoll et al., 1990). Sec- cytosis, protein kinases, Ca2+, rab proteins, phosphoryla- ond, protein kinasescould phosphorylate proteins involved in tion] the synaptic vesicle traffic. Thereby, they could modulate the efficiency of synaptic vesicle trafficking as a function of Ca2+. In nerve terminals, neurotransmittersare stored in synaptic ves- Although there is evidence in support of both mechanisms(Tak- icles and secretedby synaptic vesicle exocytosis. Neurotrans- euchi and Takeuchi, 1966; Man-Son-Hing et al., 1989; Dale and mitter secretionis acutely regulated by Ca2+,which triggers exo- Kandel, 1990; Scholz and Miller, 1992), the molecularbasis for cytosis in a rapid (<l msec) and cooperative manner.On a lon- these processesis largely obscure. In particular, for the synaptic ger time scale,neurotransmitter release is modulatedby a variety releaseapparatus, the mechanismsunderlying the modulationof of regulatory processes,such as occur during the different forms releaseand the relevant phosphorylation targets are mostly un- of short-term synaptic plasticity (Zucker, 1989), modulation by known. presynaptic receptors (Takeuchi and Takeuchi, 1966; Silinsky, 1984; Scholz and Miller, 1992; Weisskopf et al., 1993), and pos- Several protein kinase substratesin the nerve terminal have sibly long-term potentiation (LTP) and long-term depression been extensively studied. The synapsinsconstitute a family of (LTD) (Bekkers and Stevens, 1990; Malinow and Tsien, 1990; phosphoproteinsthat are associatedwith synaptic vesicles (De Bolshakov and Siegelbaum,1994). Theseregulatory phenomena Camilli et al., 1983a,b; Stidhof et al., 1989) and are substrates are thought to involve covalent modificationsof proteins, in par- for at least four different protein kinases,CaMK I and II, PKA, ticular, by protein phosphorylation. In support of this notion, and proline-directed kinase (reviewed in Jahn and Stidhof, mice carrying a mutant form of the gene for a-Caz+/calmodulin- 1994). The abundanceand regulatedphosphorylation of the syn- apsinssuggest that they function in regulating neurotransmitter release, but their exact functions are unknown except for the Received July 15, 1994; revised Oct. 3, 1994; accepted Oct. 5, 1994. C-terminal phosphorylation site for CaMK II in synapsin I, We thank I. Leznicki, A. Roth, and E. Borowicz for excellent technical assistance, Dr. H. Schulman (Stanford University), Dr. M. Kennedy (California which appearsto act as a negative regulator during paired-pulse Institute of Technology), and Dr. R. Jahn (Yale University) for supplying us facilitation (Rosahlet al., 1993). GAP-43 (also called B-50) rep- with purified enzymes and with antibodies; and Drs. H. McMahon and B. Davletov for advice. resentsan abundantphosphoprotein of the nerve terminal plas- Correspondence should be addressed to Thomas C. Stidhof at the above mamembrane,with a possiblerole in exocytosis but no currently address. known phosphorylation-dependent function (Dekker et al., “Present address: Norwegian Defence Research Establishment, Division of Environmental Toxicology, P.B. 25, N-2007 Kjeller, Norway. 1989). Dynamin I is a synaptic phosphoproteinwith the inter- Copyright 0 1995 Society for Neuroscience 0270-6474/95/152385-l 1$05,00/O esting property of quantitative dephosphorylationduring nerve 2386 Fykse et al. l Phosphotylation of Rabphilin-3A Anti body MS Anti-Rabphilin Cytosol + - + + + + Activator Ca - - cAMPcGMP Ca . .:i ./ ,...;..;. - 85 kDa Figure 1. Phosphorylation of rabphilin-3A by CAMP- and Ca*+-dependent protein kinases. Immunoprecipitates of rat brain proteins with preim- mune serum (PZS, lej? lane) or with polyclonal antibodies to rabphilin3A (Anti-Rabphilin) were incubated with or without cytosol as a source of kinases and with Ca*+, CAMP, and cGMP as activators. Samples were analyzed by SDS-PAGE, blotted onto nitrocellulose membranes, and exposed to film for 3 hr at -70°C with a screen. Antibody PIS Anti-Rabphilin Kinase CaMKiI - pm CaMKII Activator - Ca - - - Ca - 85 kDa - 85 kDa I - IgG Figure 2. Phosphorylation of rabphilin-3A by the catalytic subunit of CAMP-dependent protein kinase (PKA) and by Ca*+, calmodulin-dependent protein kinase II (C&X II). Rabphilin and control immunoprecipitates (see legend to Fig. 1) were incubated with the indicated protein kinases as described in Experimental Procedures. Samples were subjected to SDS-PAGE, blotted onto nitrocellulose membranes, and analyzed by autoradiog- raphy (iop panel; exposure: 5 hr at -70°C with screen) and by immunoblotting with rabphilin antibodies (bottom panel) to control for the amount of rabphilin-3A immunoprecipitated. The additional phosphoprotein bands observed in the samples incubated with CaMK II and Ca2+ are probably due to autophosphorylated CaMK II (see also Fig. 7). The Journal of Neuroscience, March 1995, f5(3) 2387 PP Rabphilin-3A NH2 COOH domain structure rab3A binding C2domains Residue Numbers GST 0 GST-RaphA 1-181 B 67-226 C 199-361 D 250-361 E 380-684 Figure 3. Schematic overview of the bacterial glutathione S-transferase (GS7’) fusion proteins used to map the F 380-531 phosphorylation sites in rabphilin3A. The diagram olt top depicts the domain structure of rabphilin-3A. The_ I__-_stmc- G 524-684 tures of GST ana of 10 GST-rabphilin PP fusion proteins used in the current study are shown in the middle, and the H 1-361 legend is shown on the bottom. The lo- cations of the phosphorylation sites 1-361 that were identified in the current study I are indicated by “P.” GST-RaphH contains the entire N-terminal half of J 1-361 rabphilin-3A. Serine-to-alanine muta- tions were introduced into serine234 (the putative PK.4 phosphorylation site) in GST-RaphI, and into serine234 and ser- Legend: ine274 (the putative CaMK II phosphor- ylation site) in GST-RaphJ. A mutant Point mutation * in which only the putative CaMK II Fhosphoryiafion site P phosphorylation site (serine*“) was 400 amino acids gggg substituted was also made but the ex- GST pressed protein was unstable in bacteria N-terrnlnal domain / and, therefore, not analyzed. However. First C2 domain m the corresponding mutant was also ex- pressed as a holo-protein in COS cells Second C2 domain (see below). terminal depolarization that probably plays a role in triggering (Frey et al., 1993; Chavez-Noriega and Stevens, 1994). Again, synaptic vesicle endocytosis (Robinson et al., 1993). the only currently known synaptic PKA substrates are synapsins These and other synaptic phosphoproteins (e.g., AP180, syn- I and II, which do not appear to account for all of the regulatory aptotagmin, and others) are candidates for mediating some reg- phenomena observed with PKA. Therefore, it would be of great ulatory functions in the synaptic vesicle cycle. However, the interest to define additional protein kinase substrates in the nerve currently known protein kinase targets at the synapse are clearly terminal