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Previous Article | Next Article
The Journal of Neuroscience, August 1, 2001, 21(15):5473-5483
Physiological Modulation of Rabphilin Phosphorylation
Davide L. Foletti, Jeremy T. Blitzer, and Richard H. Scheller Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5428
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
The dynamic modulation of protein function by phosphorylation plays an important 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 modifications has been hampered by the lack of reagents that specifically recognize the phosphorylated states of these proteins. In this study we analyze the physiological modulation of rabphilin using phosphospecific antibodies. We show that phosphorylation on serine-234 and serine-274 of rabphilin is dynamically regulated both under basal and stimulated conditions by the activity of kinases and phosphatases. The two sites are differentially phosphorylated by the stimulation of various kinases, suggesting a possible convergence 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 critically dependent on external Ca2+ and on the presence of Rab3a, a small GTPase that recruits rabphilin to synaptic vesicles. The rapid phosphorylation and dephosphorylation during and after stimulation demonstrates the transient nature of the modification. Our results indicate that rabphilin is phosphorylated on synaptic vesicles by Ca2+-dependent kinases that become active in synaptic terminals during exocytosis. We have found that phosphorabphilin has a reduced affinity for membranes; we therefore propose that the modulation of the membrane association of rabphilin has a role in the synaptic vesicle life cycle, perhaps in vesicle mobilization in preparation for subsequent rounds of neurotransmission.
Key words: rabphilin; Rab3a; phosphospecific antibodies; brain acute slices; synaptic transmission; protein kinases
INTRODUCTION
A combination of genetic, biochemical, structural, and functional studies has led to the discovery and characterization of molecules important in the Ca2+-regulated exocytosis of synaptic vesicles, the process that initiates synaptic transmission. The soluble N-ethylmaleimide-sensitive factor attachment receptor proteins syntaxin 1, SNAP-25, and vesicle-associated membrane protein-2 (VAMP-2), together with nSec1, Rab proteins and their effectors, play important roles during synaptic transmission and belong to large protein families whose members are implicated in every step of intracellular membrane trafficking in eukaryotic cells (for review, see Jahn and Südhof, 1999
; Lin and Scheller, 2000
It is well established that protein kinases and phosphatases have an important role in synaptic transmission. At the presynaptic terminal, the interval between an incoming action potential and the fusion of primed synaptic vesicles is likely too short for protein phosphorylation to have a direct effect. It is therefore in subsequent rounds of synaptic vesicle exocytosis that the activity-dependent stimulation of protein kinases and phosphatases becomes manifest. The regulation of synaptic protein function by phosphorylation-dephosphorylation is optimally situated to modulate aspects of synaptic plasticity. Several classes of proteins that function in synaptic transmission have been reported as potential targets for various kinases (for review, see Turner et al., 1999
In this report we use phosphospecific antibodies to characterize the modulation of the phosphorylation state of rabphilin, a synaptic protein that has been implicated in the life cycle of synaptic vesicles, but whose function is still unclear (Miyazaki et al., 1994
MATERIALS AND METHODS
Animals and reagents. Wild-type (WT; 129/B6; +/+) and Rab3a knock-out (KO; 129/B6;
/
Rat brain slices and sample preparation. Rat brain slices were prepared essentially as previously described (McQuinston and Madison, 1999
-glycerophosphate, 50 mM NaF, 50 mM Na-pyrophosphate, 2 µM Microcystin-LR, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 1 mM PMSF. In cases in which no further fractionation was performed, the homogenate was solubilized with 1% Triton X-100 for 1 hr at 4°C. After a centrifugation at 100,000 × g for 1 hr to pellet the insoluble fraction, the supernatant was collected and used in quantitative Western blot analysis (see below). For subfractionation into cytosol and membranes, the homogenate was first centrifuged at 1000 × g for 15 min. The resulting postnuclear supernatant was further centrifuged at 100,000 × g for 1 hr to separate the cytosolic fraction (supernatant) from the membrane fraction (pellet). For the NaCl and Triton X-100 extraction experiments, membranes prepared as above were resuspended in homogenization buffer. NaCl (1 M) or Triton X-100 (1%) were added and after a 1 hr incubation at 4°C the samples were centrifuged at 100,000 × g for 1 hr to separate the supernatant (extracted material) from the pellet (nonextracted material). For the preparation of the homogenate in the absence of phosphatase inhibitors, the following reagents were omitted:
Quantitative Western blotting. The generation and purification of antibodies specific for rabphilin phosphorylated at S234 and S274 (
S234-P and
RESULTS
In the accompanying paper, we have studied the developmental regulation and whole brain and subcellular distributions of rabphilin phosphorylated at serine-234 and serine-274 using phosphospecific antibodies. In this report, we investigated the in vivo modulation of the two phosphorylation events to gain insight into their functional significance. We again used the antibodies
Stimulation of phosphorylation on S234 and S274 of rabphilin
We first analyzed the effect of various pharmacological agents on the phosphorylation level of rabphilin at serine-234 and serine-274. Slices were incubated in Ringer's solution containing 56 mM K+ for 2 min, a condition that promotes strong membrane depolarization and Ca2+ influx into the nerve terminals, therefore generating a strong burst of synaptic vesicle exocytosis. The other incubations were performed for 30 min and included the following pharmacological reagents: 1 µM PDBu to stimulate the activity of PKC, 50 µM forskolin to activate PKA through stimulation of adenylyl cyclase, a combination of a membrane-permeable analog of cAMP (8-CPT-cAMP, used at 500 µM), and an inhibitor of cAMP phosphodiesterase (IBMX, used at 50 µM) also to stimulate PKA, 100 µM 4-AP, a treatment that delays action potential repolarization and increases firing rate by blocking potassium channels (Wu and Barish, 1992
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Figure 1. Stimulation of phosphorylation on S234 and S274 of rabphilin. Acute slices prepared from 6- to 8-week-old rats were incubated in normal Ringer's solution supplied with the indicated pharmacological agents. At the end of the incubation, the slices were flash-frozen in liquid nitrogen and processed for quantitative Western blotting with the phosphospecific antibodies against rabphilin S234-P (A) and S274-P (B). Each panel shows a representative Western blot result and summarizes the quantitative analysis of four to eight experiments (mean and SEM). PDBu, Phorbol-12,13-dibutyrate used at 1 µM; FO, forskolin used at 50 µM; 8-CPT-cAMP/IBMX, adenosine 3',5'-cyclic monophosphate, 8-(4-chlorophenylthio)/3-isobutyl-1-methylxanthine used at 500 or 50 µM, respectively; 4-AP, 4-aminopyridine used at 100 µM; sphi, sphingosine used at 30 µM; TEA, tetraethylammonium chloride used at 25 mM. All the above incubations, as well as the unstimulated condition, were for 30 min. 2'K+, After 28 min in Ringer's solution, the slices were incubated for 2 min in Ringer's solution containing 56 mM K+.
The absence of Rab3a greatly reduces the stimulus-dependent phosphorylation of rabphilin
Given the interaction between Rab3a and rabphilin, we investigated whether the absence of the small GTP-binding protein has an effect on the high K+-stimulated phosphorylation of rabphilin. Acute slices were prepared from WT and Rab3a KO mice. Stimulation with high K+ and processing for Western blotting were as described for rat slices. Figure 2A shows representative Western blots probed with
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Figure 2. The high K+-induced increase in rabphilin phosphorylation is severely reduced (S234-P) or completely abolished (S274-P) in slices prepared from Rab3a knock-out mice. Acute slices prepared from wild-type (WT) and Rab3a knock-out (KO) mice were incubated in normal Ringer's solution. Unstimulated slices and slices that were subjected to a 2 min 56 mM K+ stimulation were flash-frozen in liquid nitrogen and processed for quantitative Western blotting to detect changes in rabphilin phosphorylation at S234 and S274. A, Representative Western blot results. B, C, Quantitative analysis of four independent experiments (mean and SEM). In both WT and KO, the level of phosphorylation after stimulation is expressed relative to the level in unstimulated slices.
Both the basal and the high K+ stimulated levels of phosphorabphilin are strictly dependent on extracellular Ca2+
Because the high K+-induced depolarization promotes Ca2+ influx into nerve terminals, we investigated whether the presence of Ca2+ in the external medium is necessary for the observed increase in phosphorylation on rabphilin serine-234 and serine-274. For this experiment, slices were preincubated for 15 min in normal Ringer's solution or in Ringer's solution without Ca2+ and then flash frozen, or first stimulated for 2 min in high K+ Ringer's solution and then flash frozen before processing for quantitative Western blot. In Figure 3, the Western blots show representative results, and the graphs summarize the quantitative analysis of four to seven independent experiments (mean and SEM). As shown in Figure 3A for rabphilin S234-P and Figure 3B for rabphilin S274-P, depletion of Ca2+ completely prevented the increase in phosphorylation induced by high K+ and also reduced the basal level of phosphorylation of rabphilin (compare lanes 2-4 and 1-3, respectively). The high K+-stimulated increase in phosphorylation was not only completely prevented by the absence of Ca2+, but the level of phosphorylation after the stimulation was even lower than in unstimulated slices incubated in normal Ringer's solution: ~0.5-fold for rabphilin S234-P (p < 0.02; t test) and ~0.3-fold for rabphilin S274-P (p < 0.002; t test). The basal level of phosphorylation in the absence of Ca2+ was similarly reduced to ~0.5-fold on serine-234 (p < 0.005; t test) and to ~0.3-fold on serine-274 (p < 0.002; t test). The preincubation for 15 min in the absence of Ca2+ did not just simply damage the slices, because if we re-supplemented them with Ca2+ for an additional 15 min, both the basal level of phosphorylation and the increase in phosphorylation induced by high K+ stimulation were completely restored (compare lanes 1-5 and 2-6, respectively). These results indicate that Ca2+ is absolutely necessary for both the basal and the high K+-induced phosphorylation of rabphilin, suggesting that rabphilin is a substrate for Ca2+-dependent protein kinases that are directly or indirectly activated during synaptic vesicle exocytosis. CaMKII could fulfill this role for the phosphorylation of rabphilin at serine-274. For the phosphorylation at serine-234 we acknowledge two possibilities. Although the sequence around serine-234 matches the PKA phosphorylation site consensus motif, and recombinant rabphilin can be efficiently phosphorylated at this site by PKA in vitro (Fykse et al., 1995
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Figure 3. The high K+-induced increase in rabphilin phosphorylation at S234 and S274 is strictly dependent on extracellular Ca2+. Acute slices from 6- to 8-week-old rats were preincubated for 15 min in Ringer's solution with or without Ca2+, followed by incubation for 2 min in the presence or absence of 56 mM K+ and subsequent flash freezing. Some slices were first preincubated for 15 min in Ringer's solution without Ca2+ and then supplemented with Ca2+ for 15 min before a 2 min incubation in the presence or absence of 56 mM K+ and subsequent flash freezing. Slices were processed for quantitative Western blotting to detect changes in rabphilin phosphorylation at S234 (A) and S274 (B). Each panel shows a representative Western blot result and summarizes the analysis of four to seven independent experiments (mean and SEM, for each condition the level of phosphorylation is expressed relative to the level observed in unstimulated slices under normal Ca2+ conditions).
A large proportion of rabphilin is phosphorylated on serine-234 after a 2 min high K+ stimulation
We next examined the extent of phosphorylation of rabphilin by estimating how much of it becomes phosphorylated after stimulation. While raising the phosphospecific antibodies against rabphilin S234-P and rabphilin S274-P, we also immunized rabbits with the non-phospho version of the peptide used to generate
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Figure 4. A large proportion of rabphilin is phosphorylated on S234 after a 2 min high K+ stimulation. A, Specificity of the antibody specific for the form of rabphilin that is not phosphorylated at S234 (
We used
A prediction from this result is that the increase in phosphorylation promoted by high K+ (detected by
Considering that the high K+ stimulation promotes a sixfold to eightfold increase in phosphorylation at this site, we can estimate that ~10-15% of rabphilin is phosphorylated under basal unstimulated conditions.
Rapid phosphorylation and dephosphorylation of rabphilin during exocytosis
We next sought to determine if both the phosphorylation and dephosphorylation of rabphilin promoted by membrane depolarization occurs rapidly, because this would imply a tight coupling of this modification to membrane trafficking events mediating exocytosis and endocytosis of synaptic vesicles. First, we investigated the kinetics of phosphorylation during a high K+-induced depolarization. Figure 5 shows that high K+-induced phosphorylation at both serine-234 (Fig. 5A) and serine-274 (Fig. 5B) of rabphilin occurred rapidly with respective maximal increases reached within 2 min. Continued stimulation past this initial phase did not result in any additional phosphorylation; instead the levels of rabphilin S234-P and rabphilin S274-P slowly decreased. In Figure 5, the insets show representative Western blot results of the time courses of phosphorylation, and the graphs summarize the quantitative analysis of five or six independent experiments (mean and SEM). These results demonstrate that the increase in phosphorylation occurs rapidly after stimulation, consistent with the suggestion that kinases activated during exocytosis phosphorylate rabphilin on synaptic vesicles.
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Figure 5. The high K+ induced increase in rabphilin phosphorylation is maximal within 2 min. Acute slices prepared from 6- to 8-week-old rats were incubated in Ringer's solution containing 56 mM K+. At the indicated time points, slices were flash-frozen in liquid nitrogen and subsequently processed for quantitative Western blotting to detect changes in rabphilin phosphorylation at S234 (A) and S274 (B). The insets show representative Western blot results of the time course of phosphorylation; the graphs summarize the analysis of six (S234-P) and five (S274-P) independent experiments (mean and SEM).
We next examined the rate of dephosphorylation after terminating the high K+-induced depolarization by returning the slices to normal Ringer's solution. Figure 6, A (for rabphilin S234-P) and B (for rabphilin S274-P), shows the results of the time course of dephosphorylation. The insets are representative Western blot results, and the graphs summarize the quantitative analysis of four independent experiments (mean and SEM). In both graphs, the first point represents the level of phosphorylation in unstimulated slices. The second point is the level of phosphorylation at the end of a 2 min high K+ stimulation; this point also represents the time = 0 of the dephosphorylation reaction. For both serine-234 and serine-274, we observed a small additional increase in phosphorylation shortly after the end of the stimulation (t = 30 sec), followed by a rapid dephosphorylation reaction. Comparison of these data after normalization to their respective t = 0 levels (Fig. 6C) demonstrate that both serines share an initial phase of fast dephosphorylation over the first 2 min, after which rabphilin S234-P continued to become dephosphorylated to ~25% of the initial level after 30 min. In contrast, the phosphorylation level of rabphilin S274-P did not change significantly after the initial drop, and ~65% were still present after 30 min. The slower dephosphorylation of serine-274 suggests that a significant portion of the signal generated by this phosphorylation lasts for quite some time after the initial stimulus. The phosphorylation on serine-234 appears to be more dynamically regulated, and the faster and more robust dephosphorylation suggests that this modification may be linked to a cycle of events that accompany subsequent rounds of exocytosis. To further test the reversibility of this phosphorylation event, we subjected a pool of slices to five rounds of consecutive 2 min high K+ stimulation followed by 10 min of recovery in normal Ringer's solution to allow for dephosphorylation. Slices were collected at the end of each round of stimulation and recovery. The Western blot analysis confirmed that rabphilin could be reversibly phosphorylated at serine-234 with each incubation in high K+ (data not shown). This indicates a rapid turnover of the phosphorylation on serine-234, consistent with the idea that this phosphorylation event generates a signal important in successive rounds of exocytosis.
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Figure 6. A rapid phosphatase activity dephosphorylates rabphilin S234-P and S274-P. Acute slices prepared from 6- to 8-week-old rats were first subjected to a 2 min incubation in Ringer's solution containing 56 mM K+ and then rapidly transferred to normal Ringer's solution. Slices were flash-frozen in liquid nitrogen before stimulation (unst), at the end of the 2' high K+ stimulation (t = 0), and at the indicated time points after transfer to normal Ringer's solution. A (rabphilin S234-P) and B (rabphilin S274-P), Insets show representative Western blot results of the time course of dephosphorylation, and the graphs summarize the analysis of four independent experiments (mean and SEM). C, The graph shows the extent of dephosphorylation over time relative to the level of phosphorylation at the end of the stimulation (4 independent experiments, mean and SEM).
Inhibition of phosphatase activity increases both the basal and high K+-stimulated levels of phosphorabphilin
So far we have provided evidence for a dynamic regulation of the phosphorylation state of rabphilin, a cycle that appears to be linked to the events underlying exocytosis. Robust and rapid changes in the phosphorylation of rabphilin must be coupled to the activation of kinases (Figs. 1, 5) and phosphatases (Fig. 6). We therefore investigated whether the inhibition of phosphatase activity had an effect on both the basal and stimulated levels of phosphorylation of rabphilin. Rat brain slices were preincubated for 30 min in normal Ringer's solution with or without 1 µM OA, a concentration sufficient to inhibit both protein phosphatase 1 and protein phosphatase 2A. Control slices and slices treated with OA were flash-frozen after incubation in normal or high K+ Ringer's solution for 2 min and then processed for quantitative Western blot. Figure 7A shows a representative Western blot result, and Figure 7, B (rabphilin S234-P) and C (rabphilin S274-P), summarizes the quantitative analysis of four to eight independent experiments (mean and SEM). Inhibition of phosphatase activity resulted in increased phosphorylation levels on rabphilin serine-234 and serine-274 both under basal unstimulated conditions and after a 2 min high K+-induced depolarization. The basal level of rabphilin S234-P in the presence of okadaic acid was ~2.7-fold higher than in the absence of the inhibitor (Fig. 7A, top panel, B, compare lanes 1 and 3; p < 0.01; t test), and the phosphorylation level after a 2 min high K+ stimulation increased from ~6.7-fold over unstimulated to ~9.9-fold in the presence of the phosphatase inhibitor (Fig. 7A, top panel, B, compare lanes 2 and 4) (p < 0.05; t test). Similarly, for rabphilin S274-P the basal level of phosphorylation went up ~1.8-fold (Fig. 7A, bottom panel, C, compare lanes 1 and 3) (p < 0.02; t test), whereas the 2 min high K+-stimulated level increased from ~2.3-fold to ~3.4-fold (Fig. 7A, bottom panel, C, compare lanes 2 and 4) (p < 0.03; t test). These results, together with the kinetic data of Figure 6, implicate a phosphatase in the regulation of the phosphorylation state of rabphilin. The data also suggest that the basal level of rabphilin phosphorylation is regulatable and controlled by the dynamic activity of kinases and phosphatases.
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Figure 7. Inhibition of phosphatase activity increases both the basal and the high K+ stimulated level of rabphilin phosphorylation at S234 and S274. Acute slices prepared from 6- to 8-week-old rats were preincubated for 30 min in normal Ringer's solution with or without the addition of 1 µM OA. Subsequently, slices were either flash-frozen in liquid nitrogen or first stimulated for 2 min in Ringer's solution with 56 mM K+ and then flash-frozen. Finally, slices were processed for quantitative Western blotting to detect changes in rabphilin phosphorylation. A, Representative Western blot results. B, C, Quantitative analysis of rabphilin phosphorylation on S234 (B) and S274 (C) of four to eight independent experiments (mean and SEM).
Phosphorabphilin has reduced affinity for membranes
Rabphilin, which does not possess a transmembrane domain, has nevertheless been shown to be mainly membrane-bound through its association with synaptic vesicles. This is mediated, at least in part, through its interaction with the small GTPases Rab3a/c. Therefore, we investigated whether the phosphorylation state of rabphilin alters its membrane association. Rat brain slices were stimulated for 2 min in high K+ and then flash-frozen. Homogenization of the slices was performed in the presence of phosphatase inhibitors, and the postnuclear supernatant (PNS) was prepared by low-speed centrifugation (1000 × g). The PNS was further spun at high speed (100,000 × g) to separate the cytosol (C) from the membrane (M) fraction. Membranes were resuspended and extracted for 1 hr at 4°C either with high salt (1 M NaCl) or with 1% Triton X-100 (TX-100). At the end of the incubation, the samples were centrifuged again at 100,000 × g to separate the extracted [supernatant (S)] from the nonextracted [pellet (P)] fractions. Figure 8A shows a representative set of Western blots probed with
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Figure 8. Phosphorabphilin has reduced affinity for membranes. Acute slices were prepared from 6- to 8-week-old rats and stimulated for 2 min in Ringer's solution with 56 mM K+. The slices were homogenized, and the homogenate was spun at low speed (1000 × g) to make postnuclear supernatant (PNS). The PNS was further centrifuged at high speed (100,000 × g) to separate the cytosol (C) and membrane (M) fractions. Equal amounts of total protein for PNS, cytosol, and membranes were used for Western blotting. The membrane fraction was resuspended and extracted with either 1 M NaCl or 1% Triton X-100 (TX-100). After a high-speed spin to separate the supernatant (S, extracted proteins) from the pellet (P, not extracted proteins), equal volumes of the two samples were subjected to Western blotting. A, Representative Western blot results with samples probed with the two antibodies specific for the S234 and S274 phosphorylated forms of rabphilin, the antibody to detect total rabphilin, and an antibody against the integral synaptic vesicle protein VAMP-2 to control for the fractionation and membrane extraction protocols. B, Quantitative analysis of cytosol and membrane distributions of phosphorabphilin compared with total rabphilin (4-6 independent experiments, mean and SEM). C, Quantitative analysis of the relative amounts of phosphorabphilin and total rabphilin released into the supernatants after membrane extraction with 1 M NaCl (4-6 independent experiments, mean and SEM).
DISCUSSION
A large body of work has implicated the activity of various kinases and phosphatases in the regulation of synaptic transmission by controlling the phosphorylation state of several synaptic proteins (for review, see Turner et al., 1999
Our results show that the phosphorylation of rabphilin at serine-234 is greatly stimulated (about sevenfold over basal) by activation of PKA and high K+-induced membrane depolarization, a condition that mimics the events underlying synaptic vesicle exocytosis. Activation of PKC elicits a more modest but still significant (~2.5-fold) increase in phosphorylation. The activity-dependent modification is strictly dependent on external Ca2+, occurs rapidly, and is followed by an equally rapid and efficient dephosphorylation step mediated by phosphatases. The depolarization-promoted increase in phosphorylation at serine-234 is also critically dependent on the presence of Rab3a, because the effect is nearly abolished in acute slices prepared from Rab3a knock-out animals. An estimate of the relative proportion of rabphilin S234-P revealed that ~10-15% of rabphilin is phosphorylated at this site under basal conditions and that this value can reach 75-80% after the extensive stimulation of exocytosis by the 2 min incubation in high K+ Ringer's solution. Taken together, these results indicate that rabphilin undergoes a strong, dynamic, and activity-dependent phosphorylation at serine-234. We do not observe an increase in phosphorabphilin immunostaining in stimulated brain slices. Because phosphorabphilin is not detected by preimmune sera, and the immunoreactivity is blocked by phospho- but not nonphosphopeptides and is absent in Rab3A knock-out mice, our staining for phosphorylated rabphilin is clearly specific. Therefore, we propose that the sites that become phosphorylated after stimulation are blocked from recognition by the antibodies, perhaps because of interactions with other proteins.
The dependence on Rab3a and our detection of phosphorabphilin at synapses (see accompanying paper) suggest that phosphorylation of rabphilin occurs on synaptic vesicles and is initiated by the influx of Ca2+ triggered either by high K+ or action potential depolarization. The sequence around serine-234 (RRASE) matches the PKA phosphorylation site consensus motif (RRxS/Tx) (Fykse et al., 1995
The phosphorylation at serine-274 of rabphilin is stimulated approximately threefold by activation of PKA, PKC, and by high K+-induced membrane depolarization. As for the phosphorylation on serine-234, the activity-dependent increase in serine-274 phosphorylation is strictly dependent on external Ca2+ and on the presence of Rab3a. The kinetics of phosphorylation are fast and comparable to those for serine-234. Although phosphatases clearly regulate the phosphorylation state of serine-274, the dephosphorylation kinetics show that a first phase of rapid dephosphorylation is followed by a plateau with ~65% of the initial rabphilin S274-P still present for at least 30 min. The dephosphorylation kinetics of rabphilin S274-P are therefore significantly different from those of rabphilin S234-P, for which dephosphorylation proceeds steadily, resulting in only ~25% of it remaining after 30 min. CaMKII has been suggested by in vitro experiments to be the kinase that phosphorylates serine-274 in vivo, and the region around serine-274 (RANSV) matches the consensus sequence for CaMKII phosphorylation (RxxS/Tx) (Fykse et al., 1995
The domain structure of rabphilin consists of an N-terminal region containing the Rab3a binding site (Yamaguchi et al., 1993
Whereas there is clear evidence supporting a critical role for Rab3a in assuring the stability of rabphilin and its recruitment to synaptic vesicles, several experiments strongly suggest that rabphilin may have independent activities and not mediate the effects of Rab3a in exocytosis. In Rab3a knock-out mice, the levels of rabphilin are reduced to 40-50% (Geppert et al., 1994
However, there is evidence suggesting that rabphilin can interact with membranes in a manner independent of Rab3a, possibly through its two C2 domains. Shirataki et al. (1994)
Our membrane extraction experiments indicate that phosphorabphilin has a lower affinity for membranes. In vitro studies have shown that both PKA- and CaMKII-catalyzed phosphorylation of rabphilin do not affect its interaction with Rab3a (Kato et al., 1994
FOOTNOTES Received March 6, 2001; revised May 11, 2001; accepted May 17, 2001.
We thank Dr. Michael Finley for help with the preparation of rat brain acute slices and Dr. Cary Austin for critical reading of this manuscript.
Correspondence should be addressed to Richard H. Scheller, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990. E-mail: scheller{at}gene.com.
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