Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II Thierry Dubois 1 , Preeti Kerai 2, *, Michele Learmonth 1 , Andy Cronshaw 1 and Alastair Aitken 1 1 The University of Edinburgh, Division of Biomedical and Clinical Laboratory Sciences, UK; 2 Division of Protein Structure, National Institute for Medical Research, London, UK Casein kinases I ( CKI) are s erine/threonine protein k inases widely expressed in a range of eukaryotes including yeast, mammals and plants. They have been shown to play a role in diverse physiological events including membrane trafficking. CKIa is associated with synaptic vesicles and phosphory- lates s ome synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phospho- rylated in vitro by CKI on Thr21. Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and w e h ave i dentified Ser14 a s the CKI I phospho- rylation site, w hich is known to be phosphorylated in vivo. As syntaxin-1A p lays a k ey role in the regulation of n euro- transmitter release by forming part o f the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) c omplex, we propose that CKI may play a role in synaptic vesicle exocytosis. Keywords: CKI; CKII; syntaxin-1A; trafficking. Casein kinase I (CKI) belongs to a family of serine/ threonine protein kinases with seven isoforms identified in mammals (CKI a, b, d, e, c1, c2, and c3; reviewed in [1]). The kinase domain is highly conserved between members of the CKI family but unique N- and C-terminal tails characterize each isoform. In yeast, the functions of CKI have been much more extensively studied compared to their mammalian counterparts. Recently, many reports have linked yeast CKIs to cytokinesis and vesicle traffick- ing especially in endocytosis [2–9]. Mammalian CKIs appear to have similar functions and a lso have been involved in DNA repair, c ircadian rhythms, and wnt signaling. Like their yeast counterparts, CKIcshavebeen implicated in cytokinesis and in membrane trafficking [10]. CKIa interacts with a nd pho sphorylates t he clathrin adapter A P-3 [11] that i s involved in endocytosis. CKIa has been found to colocalize in neurones with synaptic vesicle markers an d to phosphorylate some synaptic vesicle associated proteins including SV2 [12]. More importantly, the phosphorylation of SV2 by CKI modulates its ability to interact with synaptotagmin [13]. SV2 plays a role in neurotransmitter release suggesting a role for CKI in this biological process. We have recently identified centaurin -a 1 , a p rotein shown t o associate with presynaptic vesicular structures [14], a s a novel CKI partner [15]. In this report, we have identified s yntaxin-1A as a novel substrate for CKI, which further supports a role for CKI in membrane trafficking. Indeed, the involve- ment o f syntaxin-1A in n eurotransmitter release is well documented (reviewed in [16,17]). Regulated n eurotrans- mitter secretion is the key s tep in synaptic transmission and is the basis of intercellular communication in the nervous system. Synaptic vesicle exocytosis is regulated by Ca 2+ and by a large number of proteins (reviewed in [17,18]). Syntaxin-1A is associated with the presynaptic membrane and associates with the plasma membrane protein SNAP-25 and the synaptic vesicle protein syna- ptobrevin to form a ÔSNARE complexÕ.Assemblyofthis complex is necessary and may be sufficient to trigger membrane fusion (reviewed in [17]). Syntaxin-1A has been previously shown to b e phospho- rylated in vitro by casein kinase II (CKII) [19–21]. Although the site of phosphorylation was not identified, Ser14 was speculated to be the phosphorylation site as it is present within a CKII consensus motif. Recently, it has been shown using phospho-specific antibodies that Ser14 i s phosphory- lated in vivo [22]. H owever, the kinase responsible for this was not identified. Here, we h ave identified the in v itro phosphorylation sites within syntaxin-1A that are phospho- rylated by both CKI and CKII. CKI and CKII phospho- rylate the N-terminal domain o f syntaxin-1A o n Thr21 and on Ser14, respectively. MATERIALS AND METHODS Materials [c- 32 P]ATP was from Amersham. Casein and histone H1 were purchased from Sigma. Recombinant c asein kinase II and the catalytic subunit o f protein kinase A ( PKA) were from Calbiochem-Novabiochem. The plasmids encoding the cytop lasmic domains of rat s yntaxin-1A-pGEX4T-1 (1–265, 1–190 and 191–265) were obtained from T. Abe, Niigata University, Japan. The rat munc18-1-pGEX2T Correspondence to T. Dubois, Institut Curie – Section Recherche, CNRS UMR 144, 26 rue d’Ulm, 75 248 Paris cedex 05, France. Fax: + 3 3 1 42 34 63 77, Tel.: + 33 1 42 34 6 3 67, E-mail: thierry.dubois@curie.fr Abbreviations: CKI, casein kinase I; CKII, casein kinase II; PKA, protein kinase A; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor. *Present address: Wolfson Institute for Biomedical Re search, University College London, Cruc iform Building, Gower Street, London WC1E 6BT. (Received 20 September 2001, rev ised 3 December 2001, a ccepted 4 December 2001) Eur. J. Biochem. 269, 909–914 (2002) Ó FEBS 2002 plasmid was a kind gift f rom P. DeCamilli, Yale University School of Medicine, CT, USA. Protein purification Recombinant GST, 14- 3-3 f,14-3-3c,centaurin-a 1 and CKIa were expressed and pu rified as d escribed previously [15,23]. Escherichia coli DH5 a cells containing the over- expressing plasmid w ere grown overnigh t at 37 °Cin 500 mL Luria-Bertani broth containing 100 lgÆmL )1 ampi- cillin. Cultures w ere diluted 1 0-fold with fresh medium and allowed to grow until a D 600  0.6 was reached. Expression of GST–syntaxin-1A ( 1–265, 1–190 and 191–265) was induced with 0.5 m M isopropyl thio-b- D -galactoside (IPTG, Biogene Ltd) for 4 h at 37 °C. Bacterial cells were harvested and resuspended in 150 mL sonication buffer (NaCl/P i , 1m M EDTA, 5 m M dithiothreitol, 1 m M benzamidine, 2 lgÆmL )1 aprotinin and 1 m M phenylmethanesulfonyl fluoride) and sonicated on ice at 4 lAsixtimesfor30s with 30-s r est intervals, using a MSE Soniprep 150 (9.5-mm probe). The sonicate was centrifuged at 10 000 g,for 40 min at 4 °C and the supernatant was loaded onto a NaCl/P i equilibrated glutathione–Sepharose matrix (Phar- macia). The matrix was washed with 10 bed volumes of buffer (NaCl/P i ,500m M NaCl). Thrombin cleavage of syntaxin was performed directly on the column. The column was equilibrated w ith thrombin buffer (50 m M Tris/HCl pH 8, 150 m M NaCl, 2 .5 m M CaCl 2 ). Dig estion w as allowed t o take place at room temperature for 1 h and 500 lL fractions were collected. Eluate containing syntaxin was then loaded onto a benzamidine-Sepharose matrix (Pharmacia) to remove thrombin contamination. The purity of s yntaxin was determined by analysis on 12.5% SDS/ PAGE. Syntaxin was stored in 20 m M Tris/HCl pH 7.5, 150 m M NaCl, 10% glycerol at )20 °C and protein concentration was estimated using the Bio-Rad protein assay. Cleavage of the GST fusion construct w ith thrombin resulted in an addit ional five amino acids at the N-terminus (GSPEF). Munc18-1 was purified essentially as described for syntaxin, e xcept for the following. Expression of GST– Munc18-1 was i nduced with 1 m M IPTG for 4 h at 30 °C. Munc18-1 was further purified using gel filtration on a S-200 column. The column was washed and equilibrated with 20 m M Tris/HCl pH 8, 200 m M NaCl. Munc18-1 was analysed o n 12.5% SDS/PAGE. F rac- tions containing munc18-1 were s tored in 20 m M Tris/ HClpH8,200m M NaCl, 10% glycerol at )70 °C. Cleavage of the GST fusion construct with thrombin resulted in an additional four amino acids at the N-terminus (GSPG). Kinase assays Twenty picomol of purified proteins were tested for their ability to b e phosphorylated in vitro by CKIa as described previously [15], or by C KII and PKA a ccording to t he manufacturer’s i nstructions. Alternatively, 2 lgofthe different deletion constructs of syntaxin-1A were used as potential substrates for CKI and C KII. Reactions were stopped by the addition of electrophoresis sample buffer and analyzed on SDS/P AGE. Gels were stained with Coomassie Blue and autoradiographed. Tryptic digestion of phosphorylated proteins Recombinant syntaxin-1A (5 lg) was phosphorylated by CKIa or CKII. Carrier unphosphorylated syntaxin (10 lg) was added and digested with trypsin. The peptides were purified by reverse phase HPLC on a Vydac Ôlow TFAÕ 4.6-mm column and fractions of 0.5 m L were collected. The elution po sitions of the 32 P-labelled p eptides were deter- mined by C erenkov counting and the phosphopeptide fractions were analysed by ion-trap mass spectrometry. Mass spectrometry (MS) of phosphorylated peptides Ion-trap MS of in-gel digested phosphoprotein and solid phase sequencing on arylamine membranes were carried out as described previously [24]. RESULTS AND DISCUSSION Phosphorylation a nd dephosphorylation of protein su b- strates by k inases and phosphatases are enzymatic activities that play prominent roles in many biological processes including synaptic vesicle exocytosis [17,25]. For example, the phosphorylation of synaptic vesicle-associated protein SV2 by CKI modulates its ability to interact with synap- totagmin. Phosphorylation of syntaxin-1A by CKII increa- ses its ability to i nteract with s ynaptotagmin [21]. I n addition, the phosphorylation of m unc18 by PKC [26] and cdk5 [27] leads to a significantly reduced affinity for s yntaxin-1A. A role f or CKI i n exocytosis has been proposed as it is associated with synaptic vesicles and phosphorylates SV2 [12,13]. In a ddition, we have found that CKI interacts with centaurin-a 1 [15], a protein that associates with presynaptic vesicular structures [14]. Therefore, we examined whether CKIa was capable of phosphorylating syntaxin-1A. Figure 1 shows that the cytoplasmic tail of syntaxin-1A (residues 1–265) is ph osphorylated by recombinant CKIa. Munc18, a protein which interacts with syntaxin-1A and regulates SNARE complex f ormation, is not a s ubstrate for CKIa (Fig. 1). 14-3-3 f is phosphorylated by CKI [23] and was used as a positive c ontrol. CKIa was unable to phosphorylate GST or centaurin-a 1 , which were used as negative controls [15]. T herefore, these results indicate that CKIa specifically phosphorylates syntaxin-1A. In agreement with our data in Fig. 1, CKII has been reported to phosphorylate syntaxin-1A in vitro [19–21]. GST and cas ein were used as negative and positive controls, respectively. In contrast with previous reports [20], we show that munc18 is also phosphorylated by CKII but to a much lesser extent compared to syntaxin-1A. In addition, we show that both centaurin-a 1 and 14-3-3 f are not substrates for CKII, thus confirming the specificity o f the kinase activity. Phosphorylation of syntaxin-1A and munc18 by PKA was also investigated. Our d ata in Fig. 1 supports previous reports indicating that syntaxin-1A is not a substrate for PKA [20,21,28]. Among the potential substrates tested (GST, centaurin-a 1, munc18 and 14-3-3 c), only munc18 was phosphorylated by PKA. Histone H1 was used as a positive c ontrol. In contrast to our findings, Hirling & Scheller reported that munc18 is not a substrate for PKA [20]. However, several potential PKA sites are present within the primary structure of munc18 according to the 910 T. Dubois et al. (Eur. J. Biochem. 269) Ó FEBS 2002 PHOSPHOBASE program f rom t he Center for Biological Sequence [29]. Figure 1 indicates that syntaxin-1A is phosphorylated by both CKI and CKII, but not by PKA. In addition, we show that munc18 is phosphorylated by PKA. Centaurin-a 1 ,a CKI interacting protein [15], is not phosphorylated by CKI, CKII nor PKA. We then identified the sites on syntaxin-1A that are phosphorylated by CKI a nd CKII. Initially two truncated mutants of syntaxin-1A (1–190 and 191–265) were subjected to in vitro kinase assays. Syntaxin-1A (1–190), but not syntaxin-1A (191–265), is a substrate for both kinases indicating th at the phosphorylated residue(s) a re located within t he N-terminal moiety of syntaxin-1A (Fig. 2). Our data support previous reports indicating that CKII phos- phorylates the N-terminal 75 amino acids of syntaxin-1A while a fragment containing residues 76–265 is not phos- phorylated [19]. Risinger & Bennett obtained preliminary evidence that the CKII phosphorylation s ite is within residues 8–75 [21]. This region contains three potential phosphorylation sites for CKII (Ser10, Ser14 and Thr71). These authors reported that phosphorylation occurred on both serine and threonine residues, thus suggesting that Thr71 could be one of the residues phosphorylated by CKII [21]. However our mass spectrometry results clearly show that the p eptide including this threonine is not phosphory- lated by CKII. The peptide consisting of residues 71–84 was identified by mass spectrometry exclusively as an unpho- sphorylated peptide of mass 1692.9 Da (M + H) + .No 32 P radioactivity was associated with this peptide from the HPLC (data not shown). To identify the site(s) of syntaxin-1A that a re phospho- rylated by CKIa, constructs 1–190 and 1–265 of syntaxin-1A were phosphorylated by CKI a and subjected to trypsin digestion. The tryptic peptides from both constructs were separated by HPLC and the 32 P content was measured. Most of the 32 P-labelled p eptide(s) e luted in one peak (Fig. 3; fraction 16 in syntaxin-1A). As expected, results were similar for syntaxin-1A, 1–265 (data not shown). The radioactive peptide p eaks purified by HPLC after phosphorylation by CKIa were analysed by ion-trap mass MS. We identified the presence of two doubly charged peptides (M + H) 2+ ,of masses 783.7 and 824.0. The l atter peptide is the phospho- rylated form o f t he first one. Asingly charged p eptide of mass 1566.5 was also observed. These three peptides correspond to residues 13–26 o f s yntaxin-1A [DSDDDDDVTVTVDR (13–26)]. Peptides of mass 919.3 (dephospho-form) as well as 960.0 and 1917.9 Da (phospho-forms) corresponding to residues 13–28 of syntaxin-1A [DSDDDDDVTVTVDRDR (13–28)] were also observed due to incomplete trypsin digestion. Tandem MS-MS sequencing of the doubly charged peptides confirmed their identities (data not shown). Fractions containing phosphorylated peptide(s) were ana- lysed b y solid phase sequencing. Figure 4 shows r elease of 32 P a t each cycle of Edman degradation on covalently coupled peptides a fter phosphorylation by C KIa and purification by HPLC. The radioactivity was released in Fig. 1. Phosphorylation of centaurin-a 1 , 14-3-3, syntaxin-1A, and munc18 by CKI, CKII and PKA. Casein kinase I a (CKIa,upper panel), casein kinase II (CKII, middle panel) or protein kinase A (PKA, bottom panel) were tested for their ability to phosphorylate in vitro 20 pmoles of centaurin-a 1 , munc18, s yntaxin 1 A or 14-3-3 proteins. Histone H1, 14-3-3 f and c asein were used as p ositive controls for PKA, CKI a nd CKII, respectively. The positions of p hosphory- lated m unc18 and syntaxin 1 A are in dicated. T he weak signals observed with syntaxin-1A by PKA and m unc18 b y CKI represent background and nonspecific phosphorylation. The positions of the molecular mass markers (k Da) are indicated. Fig. 2. Phosphorylation of syntaxin-1A (1–265, 1–190, 191–265) by CKI and CKII. In order to map the CKI and C KI I phosphorylation site(s) w ithin syntaxin 1 A, 2 lg of the c ytoplasmic domain (residues 1–265) or the truncated versions (1–190 and 191–265) of syntaxin 1 A were tested for their ability to be phosphorylated by CKIa (left panel) or CKII (right panel). The positions of the molecular mass markers (kDa) are indicated. Ó FEBS 2002 Phosphorylation of syntaxin-1A by CKI and CKII (Eur. J. Biochem. 269) 911 cycle 9 corresponding to residue Thr21 and this residue represents the only amino acid phosphorylated within the peptide. A total of three separate sequencing runs were carried out on phosphopeptides from the two constructs. Similarly, we identified the residue(s) of syntaxin-1A phosphorylated by CKII. Phosphorylated forms of syn- taxin-1A (1–190 and 1–265) were digested with trypsin and the peptides were separated by HPLC (data not shown). The mass spectrom etry of r adioactive peptides showed the presence of a doubly charged phosphopeptide of mass 974.0 and a singly charged phosphopeptide of mass 1946.3 both o f which correspond only to r esidues 10–26 [TAK DSDDDDDVTVTVDR(10–26)]. We also observed a dou- bly charged phosphopeptide of 1109.9 Da corresponding to residues 10–28 [TAKDSDDDDDVTVTVDRDR(10–28)]. Therefore, the s ame region o f syntaxin-1A was phosphory- lated by both CKI and CKII. However, when phosphory- lated by CKII, the p eptide bond K12-D13 was mainly uncleaved by trypsin b ecause residue 14 (see results below) was p hosphorylated, and this bond was highly resistant to trypsin cleavage. This is a consequence o f r esidues involved in the active site o f the enzyme because t rypsin readily cle aves K/R-S p /T p bonds but normally cleaves very poorly when the phosphoamino acid is two residues C-terminal to Arg or Lys (i.e. K/R-X-S p /T p bonds [30]). Release of 32 P at each cycle of Edman degradation on covalently coupled peptides after phosphorylation of syntaxin-1A (1–265) by CKII and purification by HPLC in dicates that Ser14 (corresponding to cycle 5) w as the site of phosphorylation (Fig. 5). Radioactivity associated with cycle 1 (Thr10) was due to incomplete washing of the radioactivity associated nonspe- cifically to the d isc (see Fig. 6). This result indicates th at there is only one residue (Ser14) phosphorylated within the peptide. Identical results were obtained with syntaxin-1A, 1–190 (data not shown). Two separate sequencing runs were carried out on phosphopeptides from each of the constructs. Because the site of phosphorylation by CKII was close to the N-terminus of syntaxin-1A, automated Edman degra- dation of the syntaxin ( 1–265) which had not been digested with trypsin w as carried out. Surprisingly this gave two sequences in a ratio of 2 : 1 of the shorter form (Fig. 6). This Fig. 4. CKIa phosphorylates in vitro syntaxin 1 A on Thr21. Release of 32 P at each cycle of automatic Edman degradation after phosphory- lation of syntaxin-1A by C KIa and purification by HPLC. Peptides (fraction 16 from Fig. 3) were covalently coupled to arylamine mem- brane and se quen ced i n an A BI 477 s eq uencer [24]. S eq uence d ata from one of three runs is shown. Result indicates that the radioactivity incorporated into the peptide is released in cycle 9 corresponding to Thr21. This residue was the only one to be phosphorylated within the peptide. Fig. 3. HPLC trac e (absorbance a t 210 nm) with positions of 32 P-labelled tryptic peptides of syntaxin-1A ( 1–190) phosphorylated by CKIa. A C-terminal truncated con struct of mutant of syntaxin-1A containing residues 1–190 was phosphorylated by CKIa and subjected to trypsin digestion. The r esulting peptides were separated by HPLC (see the t race at A 210 nm), and the elution position of the 32 P labelled peptide(s) was determ inated by Cerenkov co unting. Only one phos- phopeptide (fraction 16) was recovered and its position is indicated by an arrow on t he HPLC tr ace. Fig. 5. CKII phosphorylates in vi t ro syntaxin-1A on Ser14. The figure represents the r elease of 32 P at each cycle of Edman degradation on covalently coupled peptide after ph osphorylation of syntaxin-1A (1–265) by C KII and purification by HPLC. Result indicates t hat the radioactivity i ncorporated into the peptide i s re leased i n c ycle 5 corresponding to Ser14. This residue was the only one to be phosphorylated within the pe ptide. 912 T. Dubois et al. (Eur. J. Biochem. 269) Ó FEBS 2002 showed that the N-terminus was cleaved by thrombin to give the main sequence of the recombinant protein (begin- ning at residue 10; TAKDSDDDD…)aswellassome protein cleaved at the expected thrombin cleavage site (GSPEFM 1 KDR…). The sequence around this region of the N-terminus would b e exposed to the thrombin used to cleave the G ST fusion moiety and although i t may not appear so at first glance, it clearly must sufficiently resemble the consensus cleavage site to be cleaved by thrombin which has a preference for R-X peptide bonds. Thrombin cleavage site, LVPR/GSPEFM 1 KDR…; additional cleavage observed, QELR/TAK DSDDDD … (where T i s T10). The direct sequen cing b y E dman degradation of the protein showed a burst of radioactivity at cycle 5 which corresponds to Ser14 (from the additional thrombin cleavage site). No counts were observed a t cycle 15 (i.e. Thr10) verifying that the 32 P released at cycle 1 ( Fig. 5) was due to incomplete washing of nonspecific radioactivity associated with the disc. A ÔburstÕ of counts was also observed at cycle 19 corresponding to Ser14 from the sequencing of the protein without the additional cleavage site (Fig. 6 ). The sites of phosphorylation by C KIa (Thr21) and CKII (Ser14) on syntaxin-1A are summarized in Fig. 7 with the masses of the peptides identified. To summarize, we have shown that CKIa and CKII phosphorylate syntaxin-1A on Thr21 and on Ser14, respectively. Both sites are located at the N-terminal domain of syntaxin-1A. T hese sites are remote from the H3 domain required for protein–protein interactions, and known to be crucial in the formation of the four-helix bundle s tructure that it is part of the SNARE complex involved in the fusion process of synaptic vesicles with the plasma membrane. However, the phosphorylation of syntaxin-1A by CKII enhances its capacity to associate with synaptotagmin [21]. Therefore, phosphorylation of Ser14 by CKII suggests an important role for t his residue in regulating the interaction between syntaxin-1A and synaptotagmin. Consistent with our data, F oletti and coauthors have shown using specific phosphopeptide-antibodies that Ser14 is phosphorylated in vivo [22]. When phosphorylated on Ser14, syntaxin-1A is preferentially associated with SNAP-25 a nd does not localize with pools of synaptic vesicles [22]. Although we have shown that CKII phosphorylates in vitro syntaxin-1A on Ser14, and that this residue is p hosphorylated in vivo,it remains to be determined whether CKII is the kinase Fig. 7. Summary of phosphorylation sites on recombinant syntaxin-1A. The figure shows the sites o f phosphorylation by CKIa (Thr21) and C KII (Ser14). The masses of the major peptides, in the positive ion mode (M + H ) + , identified by mass spectrometry are indicated. The main peptides recovered after trypsin digestion of syntaxin-1A phosphorylated by CKIa and CKII are indicated by the bars. Where two masses are shown these are the doubly then singly charged unphosph orylated peptides followed b y the masses of the correspon ding phosphof orms (at 40 and 80 Da highe r, respectively). The theoretical mas ses are 783.8, 1566.6, 823.8, 919.4, 959.4, 1917.8, 9 73.9, 1946.8 and 1109.5, respectively. The two thrombin cleavage sites are also indicated. Fig. 6. Solid phase Edman se quencing of syntaxin-1A (1–265) after phosphorylation by CKII. The cyt oplasmic domain o f syntaxin- 1A (residues 1–265) was phosphorylated in vitro b y CKII, and au tomated Edman degradat ion was c arr ied out w ithou t prior trypsin digestion. Th e 32 P-radioactivity and the PTH-amino acids released at e ach cycle (Ôcycle numberÕ) are shown. The top sequence (ÔExpected TCÕ, for expected thrombin cleavage) c orresponds to the ÔintactÕ prot ein after thrombin digestion. The bottom sequence corresponds to the shorter protein obtained from t he additional thrombin c leavage site (ÔAdditional TCÕ). Ó FEBS 2002 Phosphorylation of syntaxin-1A by CKI and CKII (Eur. J. Biochem. 269) 913 responsible for phosphorylating this site in vivo. Risinger & Bennett proposed that both serine and threonine residues were phosphorylated by CKII within residues 8–75 of syntaxin-1A [21], but we have no evidence for any other additional phosphorylation sites apart from Ser14. Previous reports have suggested a role for CKI in synaptic vesicle exocytosis as i t i s known to associate with synaptic vesicles [12], and to phosphorylate the synaptic associated protein SV2 [12,13]. Our data further support a role for CKI in synaptic vesicle cycling. Phosphorylation o f syntaxin-1A by CKI and CKII m ay play a prominent role in the regulation of neurotransmitter release. ACKNOWLEDGEMENTS The authors would like to thank Pietro de Camilli for the munc18- pGEX2T plasm id and Teruo Abe for the various syntaxin constructs used in this study. This w ork was supported by a Medical Research Council P rogramme Grant (to A. A.) a nd by the Medical Research Council (to P . K.). REFERENCES 1. Gross, S.D. & Anderson, R.A. (1998) Casein kinase I: spatial organization an d p ositioning of a multifunctional p rotein k inase family. Cell. Signal. 10, 699–671. 2. Robinson, L .C., M enold, M .M., G arret, S. & Culbertson, M.R. 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