THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 12, Issue of April 25, pp. 6944-6948,199O Printed in L? S.A.

Phosphorylation of Synapsin I at a Novel Site by Proline-directed Protein Kinase*

(Received for publication, September 26, 1989)

Frederick L. Hal@, Jeffrey P. Mitchell& and P. Richard Vullietgll From the SDivision of Orthopaedic Surgery, University of Southern California School of Medicine, Children’s Hospital, Los Angeles. California 90054 and the SDeoartment of Veterinary Pharmacology and Toxicology, University of California, Da&, &difoka 95616

- _

Previous studies identified synapsin I as a potential substrate for a newly discovered growth factor-sensi- tive, proline-directed protein kinase originally isolated from rat pheochromocytoma. The present study de- scribes the site-specific phosphorylation of synapsin I by highly purified preparations of proline-directed protein kinase. The incorporation of [32P]phosphate into bovine brain synapsin I was dependent upon both the amount of kinase present and the time of incuba- tion. The maximum stoichiometry of phosphorylation approached 1 mol of phosphate/m01 of synapsin I pro- tein. When analyzed by sodium dodecyl sulfate-gel electrophoresis and autoradiography, [32P]phosphate was found to be incorporated into both synapsin Ia and Ib. Phosphoamino acid analysis demonstrated that serine residues were phosphorylated exclusively. Digestion of phosphorylated synapsin I with trypsin followed by high performance liquid chromatography (HPLC) phosphopeptide analysis indicated that the tryptic peptide containing the major phosphorylation site eluted as a single peak at approximately 17% ace- tonitrile. The primary structure of this phosphopep- tide, determined by gas-phase sequencing, was found to be Gln-Ser-Arg-Pro-Val-Ala-Gly-Gly-Pro-Gly-Ala- Pro-Pro-Ala-Thr-Arg-Pro-Pro-Ala-Ser-Pro-Ser-Pro- Gln-Arg. Sequential Edman degradation of this HPLC- purified tryptic phosphopeptide revealed that serine 20 of this peptide was the major phosphorylated resi- due. This phosphoacceptor site is immediately flanked by a carboxyl-terminal proline residue, an observation that further verifies the proline-directed nature of this protein kinase. The tryptic phosphopeptide corre- sponds exactly to a sequence in the collagenase-sensi- tive, proline-rich “tail” region of bovine synapsin I. This novel phosphorylation site is close to but distinct from phosphorylation sites 2 and 3, which are known to be phosphorylated by calcium/calmodulin-depend- ent protein kinase II and are considered to be of regu- latory importance.

Synapsin was first identified in the mammalian nervous system by its ability to serve as a substrate for cyclic AMP-

* This research was supported by grants from the United States Air Force Office of Scientific Research and the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 Established Investigator of the American Heart Association. To whom correspondence should be sent. Tel.: 916-752-7464.

dependent protein kinase (CAMP-PK)’ (1, 2). Subsequent. studies have established that this protein is found exclusively in the nervous system (3,4), is enriched in presynaptic struc- tures (5-7), is correlated developmentally with synaptogenesis (8, 9), and is induced in PC12 pheochromocytoma cells that have been treated with nerve growth factor (10). This neuron- specific protein has been purified to homogeneity and char- acterized extensively (see 11 for review). Synapsin I exists in two forms, designated synapsin Ia (Mr = 86,000) and synapsin Ib (n/i, = 80,000). The protein exhibits an asymmetric struc- ture (12) and may be divided operationally into a relatively hydrophobic, collagenase-resistant “head” region and a pro- line-rich, collagenase-sensitive “tail.” Synapsin I has been identified as an in uiuo as well as an in vitro substrate for calcium and calmodulin-dependent protein kinase II (CaM- PK II) and will serve as an in vitro substrate of several other known protein kinases (11, 13).

Substantial evidence suggests that synapsin I plays an important role in governing the release of neurotransmitters and that the phosphorylation of synapsin I represents a major regulatory mechanism. The phosphorylation of synapsin I is increased in a variety of neuronal preparations by physiolog- ical and pharmacological agents that are known to regulate neurotransmitter release (11, 13). Studies by Llinas and co- workers (14) further supported the role of synapsin I phos- phorylation in the physiological release of neurotransmitters (15). Microinjection of dephosphorylated synapsin I into the squid giant synapse decreased the amplitude and rate of rise of the postsynaptic potential, whereas the injection of syn- apsin I that had been phosphorylated by CaM-PK II did not. When CaM-PK II was injected directly into the synapse, the amplitude of the postsynaptic potential and the rate of rise were actually increased. Under well defined in vitro condi- tions, it has been demonstrated that the binding of synapsin I to both synaptic vesicles (13, 16, 17) and cytoskeletal com- ponents (E-20) is dependent upon the state of its phosphoryl- ation. From these types of studies, a model has emerged in which synapsin I forms a functional link between synaptic vesicles and the neuronal cytoskeleton, the regulation of which governs vesicular trafficking in coordination with neu- rotransmitter release (11-15).

Specific phosphorylation sites on synapsin I which are selective for CAMP-PK, CaM-PK I, and CaM-PK II have been identified, and the locations of these sites have been assigned to functional domains within the molecule. Cyclic

’ The abbreviations used are: CAMP-PK, CAMP-dependent protein kinase; CaM-PK, calcium/calmodulin-dependent protein kinase; NGF, nerve growth factor; PDPK, proline-directed protein kinase; HPLC, high performance liquid chromatography; EGTA, [ethylene- bis(oxyethylenenitrilo)]tetraacetic acid; TPCK, L-l-tosylamido-2- phenylethyl chloromethyl ketone; SDS, sodium dodecyl sulfate.

6944

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Site-specific Phosphorylation of Synapsin I 6945

AMP-dependent protein kinase and CaM-PK I both phos- phorylate the same serine residue (site 1) located in the collagenase-resistant head region of the molecule (21, 22). Calcium and calmodulin-dependent protein kinase II phos- phorylates 2 serine residues (sites 2 and 3) in the collagenase- sensitive, proline-rich tail (23). Recent studies by Roman0 et al. (24) have established that synapsin I is phosphorylated in PC12 pheochromocytoma cells following treatment with nerve growth factor (NGF). Employing selective proteolysis and phosphopeptide fingerprint analysis, it was determined that the site phosphorylated following NGF treatment was differ- ent from any of the known phosphorylation sites and, further, that the kinase responsible for this phosphorylation was dis- tinct from any known synapsin I kinase.

Meanwhile, studies in this laboratory led to the identifica- tion and characterization of a novel protein kinase in rat pheochromocytoma which exhibits a unique phosphorylation site specificity and appears to be activated by growth factors, including NGF (25-27). This novel protein kinase, designated proline-directed protein kinase (PDPK), exhibits a minimal recognition sequence of -X-Ser/Thr-Pro-X. Since a prelimi- nary screening of potential substrates for this novel protein kinase identified synapsin I as a potential substrate, it was of interest to characterize this relationship further. In this paper, we describe the phosphorylation of bovine brain synapsin I by PDPK in additional detail. Furthermore, we identify and determine the sequence of a novel phosphorylation site, the location of which is established in relationship to the other known phosphorylation sites within the synapsin I protein.

EXPERIMENTAL PROCEDURES

Purification of Synopsin I-Synapsin I was purified from bovine brain by a modification of the acid extraction method described by Ueda and Greengard (3) as modified by Czernik et al. (23). Basically, 500 g of bovine cerebral cortex was homogenized in 5 volumes of 10 mM Tris-HCl, pH 8.2, containing 0.32 M sucrose, 4 mM EDTA, 1 mM EGTA, 0.1% mercaptoethanol, and 50 pM phenylmethylsulfonyl flu- oride. Following centrifugation at 9000 X g for 30 min, the supernatant was decanted and the pellet resuspended in 10 mM phosphoric acid, pH 2.4, containing 0.5 mM EDTA. After stirring, the pH was adjusted to pH 3.0 with NaOH, and stirring was continued for an additional 30 min. Following centrifugation at 9000 X g, the supernatant was adjusted to pH 6.0 with 1 N NaOH and then stirred for 30 min. Following centrifugation at 9000 x g, the supernatant was adjusted to pH 8.2 with 1 M Tris-HCl, pH 8.9, and then loaded onto a carboxymethylcellulose column equilibrated in 10 mM Tris-HCl, pH 8.2, 1.5 mM EDTA, and 0.1% mercaptoethanol. The protein was eluted with a linear O-400 mM ammonium chloride gradient prepared in equilibration buffer. The elution fractions were analyzed by SDS- gel electrophoresis; the fractions containing synapsin were pooled, diluted with 10 mM sodium phosphate buffer, pH 7.2, containing 1.5 mM EDTA, and 0.1% mercaptoethanol, and then loaded onto a hydroxylapatite column equilibrated in this same buffer. After exten- sive column washing (i.e. after the absorbance at 280 nm decreased to less than 0.1 absorbance unit), the protein was eluted with a linear gradient of O-250 mM ammonium chloride. The synapsin-containing fractions were identified by SDS-gel electrophoresis, pooled, and then concentrated by ammonium sulfate (50% saturation) precipitation. The resulting pellet was resuspended in sodium phosphate buffer and dialyzed overnight. The preparation was then diluted to a final concentration of approximately 3.0 mg/ml and stored at -70 “C until use. The actual concentration of synapsin J protein was confirmed by analysis of amino acid composition.

PUFifiCatiOn of the Protein Kinase-The proline-directed protein kinase was prepared from FM3A mouse mammary carcinoma cells by the method of Vulliet et al. (27) with some modifications. The cytosolic fraction (1000 X g supernatant) prepared from cellular lysates was subjected to ammonium sulfate fractionation (O-42% saturation) followed by DEAE-cellulose column chromatography. The DEAE column was equilibrated in 50 mM potassium phosphate buffer, pH 7.2, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 pg/ml leupeptin, and 0.1% mercaptoethanol. The column was washed exten-

sively, including 2 column volumes of buffer containing 10 FM CAMP used to dissociate the catalytic subunit of CAMP-PK from its regu- latory subunit and thus facilitate its elution. The proline-directed protein kinase activity was then eluted in a single step with 200 mM NaCl prepared in equilibration buffer. The protein-containing frac- tions of the DEAE eluent were pooled, diluted with an equal volume of equilibration buffer, and then loaded onto a heparin-agarose affin- ity column equilibrated in 20 mM potassium phosphate buffer, pH 7.4, 1 mM ETDA, and 1 mM dithiothreitol. The column was washed with equilibration buffer containing 100 mM KCl, and the kinase was eluted with 300 mM KC1 in equilibration buffer. Following concentra- tion by ammonium sulfate precipitation (45% saturation), resuspen- sion, and dialysis, the proline-directed protein kinase was further purified by fast protein liquid chromatography using a Pharmacia LKB Biotechnology Inc. Superose 6 sizing column equilibrated in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 150 mM NaCl. The active fractions were concentrated in Centricon 30 microfiltration devices (Amicon) and stored in small aliquots at -70 “C. Throughout the purification, the activity of the protein kinase was monitored using the selective synthetic peptide assay as described in Vulliet et al. (27). The protein kinase preparations employed in the present studies displayed the same chromatographic properties, substrate specificity, and physical characteristics as the enzyme pu- rified from rat pheochromocytoma. The addition of molecular sizing to the purification scheme served to increase the specific activity of the final preparations to approximately 5000 units/mg of protein (1 unit of PDPK activity is defined as the amount of kinase necessary to transfer 1 pmol of phosphate to TH2-16 peptide in 1 min at 30 “C under standard assay conditions: 50 mM Tris-HCl, pH 7.6, 100 FM TH2-16, 100 pM ATP (specific activity, 500-1000 cpm/pmol), and 10 mM magnesium acetate).

Analysis of Synapsin I Phosphotylation-Synapsin I was phos- phorylated by PDPK under standard assay conditions followed by precipitation with trichloroacetic acid and analysis by SDS-gel elec- trophoresis, performed by the method of Laemmli (28). Phosphoa- mino acid analysis was performed by resuspending the washed tri- chloroacetic acid pellets in 200 ~1 of 5.7 M HCl followed by incubation in a sealed container for 2 h at 110 “C. After hydrolysis, the HCl was removed by evaporation, the amino acids were resuspended in 10 ~1 of pH 1.9 electrophoresis buffer (88% formic acid:glacial acetic acid:water, 50:156:1794), and the resulting samples were spotted onto a Kodak thin-layer cellulose plate. These samples were subjected to horizontal electrophoresis, as described by Cooper et al. (29), at 1000 volts for 1 h at 10 “C, after which the plate was sprayed with 0.2% ninhydrin in acetone and developed at 65 “C.

Site-specific analysis was initiated by incubating phosphorylated synapsin I in the presence of TPCK-treated trypsin (at a ratio of 1 part of trypsin to 10 parts of synapsin I) overnight at 37 “C in 100 ~1 of 0.1 M N-methylmorpholine acetate, pH 8.3. Following centrifuga- tion and filtration, the samples were analyzed by high performance liquid chromatography (HPLC) employing a Vydac Cl8 column equil- ibrated in 0.1% trifluoroacetic acid in water. The phosphopeptides were eluted with a linear gradient (from 0 to 35% acetonitrile, also containing 0.1% trifluoroacetic acid) and collected in l-ml fractions. The fractions containing the major tryptic phosphopeptide were transferred to a siliconized microcentrifuge tube, concentrated by evaporative centrifugation (SpeedVac), and subjected to either iso- electric focusing, amino acid analysis, gas-phase sequencing, or radi- osequencing by automated sequential Edman degradation. Isoelectric focusing was performed in polyacrylamide gels with Pharmalyte car- rier ampholytes (Pharmacia) on a LKB Multiphor II horizontal electrophoresis unit. Gas-phase sequencing was performed on an Applied Biosystems model 470A sequenator using the manufacturer’s recommended methods. Radiosequencing was performed on a Beck- man model 890C liquid-phase sequenator.

RESULTS

Synapsin I is known to be a substrate for at least three protein kinases including CAMP-PK, CaM-PK I, and CaM- PK II. To assess the phosphorylation of bovine brain synapsin I by the newly discovered, proline-directed protein kinase, highly purified synapsin I was incubated in the presence of differing concentrations of PDPK for 20 min at 30 “C. As illustrated in Fig. 1, the amount of phosphate incorporated into synapsin I was dependent upon the amount of PDPK present in the reaction mixture. At higher PDPK concentra-

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Site-specific Phosphorylation of Synapsin 1

_- 0-O Synapsin + PDPK 0-o

25. O-O PDPK Only

Volume of PDPK Added (~1)

FIG. 1. Enzyme concentration curve for the phosphoryla- tion of synapsin I by the proline-directed protein kinase. Seven micrograms of purified bovine synapsin I were incubated in the presence of varying amounts of PDPK in a reaction mixture contain- ing 50 mM Tris-HCl, pH 7.6, 100 pM [r-“P]ATP (specific activity, 630 cpm/pmol), and 10 mM magnesium acetate for 20 min at 30 “C in a final volume of 50 ~1. The activity of the kinase preparation, determined under standard assay conditions, was approximately 1 unit/pi; the specific activity was approximately 5,000 units/mg of protein. The reaction was stopped by the addition of 1 ml of 25% trichloroacetic acid and 100 pg of bovine serum albumin. After vortexing, the tubes were centrifuged at 13,000 X g for 5 min. The supernatant was removed and the pellet washed three times with 25% trichloroacetic acid. The amount of phosphate incorporated into the washed pellets was quantified by Cerenkov counting. The stoichi- ometry of the phosphotransferase reaction was then calculated (in- set).

tions, the amount of phosphate incorporated reached a plateau at approximately 0.65 mol of phosphate/m01 of synapsin I. When assayed at a fixed concentration of PDPK, the amount of phosphate incorporated into synapsin I was also dependent upon the time of incubation (data not shown). The highest stoichiometric ratio observed in any of these experiments was approximately 1 mol of phosphate/m01 of synapsin I.

To investigate further PDPK phosphotransferase activity toward synapsin I, the purified protein was incubated in the presence of PDPK and [Y-~‘P]ATP for 1 h at 30 “C; the proteins were then precipitated with trichloroacetic acid and analyzed by SDS-gel electrophoresis and autoradiography. As shown in Fig. 2, A and B, appreciable [“PIphosphate was incorporated into both the 86- and the 80-kDa proteins that comprise synapsin Ia and Ib, respectively. Acid hydrolysis and phosphoamino acid analysis were then performed on the phosphorylated synapsin I protein. Upon one-dimensional electrophoresis and autoradiography (Fig. 2C), the radioactiv- ity was found to co-migrate exclusively with the phosphoser- ine standards, establishing serine residues as the only phos- phoacceptor sites.

The number of PDPK phosphorylation sites was analyzed by digesting ‘“P-labeled synapsin I with TPCK-treated tryp sin and applying the resulting tryptic peptides to a reversed- phase Cl8 HPLC column. As shown in Fig. 3, the bulk of the radioactivity eluted as a single peak at approximately 17% acetonitrile. The major tryptic phosphopeptide was collected from the HPLC column in two fractions and further analyzed by isoelectric focusing on a polyacrylamide gel followed by autoradiography (Fig. 4). Under these conditions, the radio- labeled material in each of the two fractions migrated identi- cally and focused in each case as a single spot. These results indicate that a single basic phosphopeptide was present in each of the HPLC fractions and that the two fractions con- tained the same phosphopeptide.

To determine the exact phosphorylation site(s) within the synapsin I molecule, the major tryptic phosphopeptide iden- tified by HPLC phosphopeptide mapping was collected and

A 6 kDa 205-

116- 97.4-

1 2 3

-P-SW

1- -P-Thr

-P-Tyr

FIG. 2. SDS-gel electrophoresis and phosphoamino acid analysis of synapsin I phosphorylated by the proline-directed protein kinase. Fifteen micrograms of synapsin I were incubated in 50 mM Tris-HCl, pH 7.6, 100 gM [-y-“LP]ATP (specific activity, 500 cpm/pmol), and 10 mM magnesium acetate for 60 min at 30 “C in the presence or absence of PDPK. The reaction was terminated by the addition of 1 ml of 25% trichloroacetic acid. The reaction vessel was centrifuged at 13,000 x g for 5 min. The pellets were washed three times with 25% trichloroacetic acid, twice with water, and then suspended in 50 ~1 of SDS sample buffer. After heating for 5 min at 100 “C, the samples were loaded onto a 10% polyacrylamide gel and electrophoresed as described by Laemmli (28). Following electropho- resis, the gel was stained with Coomassie Blue, destained, dried, and analyzed by autoradiography. In a parallel series of experiments, the trichloroacetic acid precipitates were subjected to acid hydrolysis and phosphoamino acid analysis, as described under “Experimental Pro- cedures.” A, the Coomassie Blue-stained gel: lane 1, synapsin I; lane 2, synapsin I plus PDPK; lane 3, PDPK alone. B, autoradiograph of the gel shown in A. C, autoradiograph of the phosphoamino acid electrophoresis, indicating the location of the stained phosphoamino acid standards.

120 80 *-a SYNAPSIN + PDPK

0-0 POPK ONLY 70 100

60 g

8.0 50 3 62

6.0 40 Lj

30 40 g 20 2

20 10

00 0 0 10 20 30 40 50 60 70 60 so

FRACTION NUMBER

FIG. 3. HPLC phosphopeptide map of synapsin I phos- phorylated by the proline-directed protein kinase. Fifteen mi- crograms of synapsin I were incubated in the presence or absence of the proline-directed protein kinase for 60 min at 30 “C. The reaction was terminated by the addition of 25% trichloroacetic acid, and the pellets were washed three times with 25% trichloroacetic acid, twice with water, and then resuspended in 0.25 ml of 0.1 M N-methylmor- pholine acetate buffer, pH 8.3. Under these conditions, phosphate was incorporated into synapsin I at a ratio of 0.9 mol of phosphate/ mol of synapsin I. Freshly prepared TPCK-treated trypsin was added (l:lO, w/w), and the mixture was vortexed several times and incubated overnight at 37 “C. Following centrifugation at 13,000 X g and filtra- tion, the supernatant was analyzed by HPLC using a Vydac Cl8 column equilibrated with 0.1% trifluoroacetic acid. Following an initial 10.min wash, the column was developed with a O-35% aceto- nitrile gradient. The bulk of the phosphate incorporated into synapsin I eluted as a single peak at approximately 17% acetonitrile.

analyzed by gas-phase sequencing methods. A single amino acid residue was detected in each cycle, demonstrating that a single polypeptide was purified. The following sequence was obtained: Gln-Ser-Arg-Pro-Val-Ala-Gly-Gly-Pro-Gly-Ala- Pro-Pro-Ala-Thr-Arg-Pro-Pro-Ala-Ser-Pro-Ser-Pro-Gln- Arg. No detectable amino acids were recovered beyond cycle 25 (arginine), indicating that the carboxyl terminus of the tryptic peptide had been reached. Since this peptide contained

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Site-specific Phosphorylation of Synapsin I 6947

I+1 Amino Acid Sequence

n^ 3.0 QSRPVAGGPGAPPATRPPASPSPQR b - 2.5

+3.75

ee 49 50

I-f FIG. 4. Isoelectric focusing of the major HPLC peak. Two

consecutive fractions spanning the peak of radioactivity observed during HPLC phosphopeptide mapping were dried down by evapo- rative centrifugation (SpeedVac), and the peptides were resuspended in water. The samples were subjected to isoelectric focusing on a polyacrylamide gel prepared with a mixture of Pharmalytes 2.5-5 and 3-10 (in a 4:3 ratio). The gel was dried and subjected to autoradiog- raphy with a Quanta III intensifying screen to visualize the radiola- beled peptides. Left lane, HPLC fraction 49, 12,600 cpm loaded; right lane, HPLC fraction 50, 13,200 cpm loaded. The positions of the anode and the cathode are marked by + and -, respectively. The position of the methyl red marker dye (PI 3.75) is as indicated. Fractions 49 and 50 both yielded a single spot with the same very basic isoelectric point. This indicates that a single phosphopeptide was eluted in both fractions and that fractions 49 and 50 contained the same phosphopeptide. The highly basic isoelectric point is in agreement with the predicted p1 of the amino acid sequence obtained for this peptide, 12.8 (for the unphosphorylated peptide, phosphoryl- ation would lower the PI).

3 serine residues, it was important to identify which of these residues actually served as the phosphorylation site. Employ- ing sequential Edman degradation, the amino acid released from each successive cycle was collected and the radioactivity monitored by liquid scintillation counting. From these data (presented in Fig. 5, with the corresponding sequence over- laid) it is clear that serine 20 represents the major phosphoryl- ation site. Although incorporation of [32P]phosphate into serine 22 is not at all evident, the release of a minor amount of “‘P could conceivably be obscured by the “bleed-through” resulting from the serine 20 peak. This bleed-through detected in successive cycles, which is commonly observed in liquid- phase radiosequencing, may have been augmented in this case by the presence of numerous proline residues that are gener- ally cleaved at a slightly lower efficiency. Therefore, although the possibility of a secondary (-X-Ser-Pro-X-) phosphoryla- tion site at serine 22 cannot be ruled out at this time, the major (X-Ser-Pro-X-) phosphorylation site at serine 20 can be established with certainty.

DISCUSSION

A functional role for synapsin I in the regulation of neuro- transmitter release has been well established. It is also clear that the site-specific phosphorylation of synapsin I is impor- tant in mediating the physiological actions of this key regu- latory protein. In this paper, we demonstrate that a newly

Cycle Number

FIG. 5. Radiosequencing of the major synapsin I tryptic phosphopeptide by sequential Edman degradation. Synapsin I, incubated in the presence of [r-“‘P]ATP and PDPK, was phosphoryl- ated to a stoichiometry of approximately 0.9 mol of phosphate/m01 of synapsin I. After precipitation with 25% trichloroacetic acid, washing of the pellet, and digestion with TPCK-treated trypsin, the preparation was applied to a reversed-phase HPLC system; the re- sulting tryptic phosphopeptides were eluted (in 0.1% trifluoroacetic acid) with a linear O-35% acetonitrile gradient. The fraction contain- ing the major tryptic phosphopeptide was transferred to a siliconized microcentrifuge tube, taken to dryness by evaporative centrifugation, and subjected to automated sequential Edman degradation performed on a Beckman model 890C liquid-phase sequenator. The butyl chlo- ride extract from each successive cycle was dried, mixed with scintil- lation fluid, and quantified by liquid scintillation counting. As shown in the graph, a prominent burst of radioactivity was released at cycle 20 followed by a characteristic decline to baseline. A comparison of these results with the amino acid sequence determined for this HPLC- purified tryptic phosphopeptide (displayed along the upper x axis) indicates that the major phosphorylation site is located at serine 20.

discovered growth factor-sensitive protein kinase phosphoryl- ates synapsin I at a novel site. The amino acid sequence of the tryptic phosphopeptide containing this novel phosphoryl- ation site was determined to be Gln-Ser-Arg-Pro-Val-Ala- Gly-Gly-Pro-Gly-Ala-Pro-Pro-Ala-Thr-Arg-Pro-Pro-Ala- Ser-Pro-Ser-Pro-Gln-Arg, which can be mapped exactly to a region (amino acids 532-556) in the sequence reported for bovine brain synapsin I (23, 30). This novel phosphorylation site is located in the proline-rich collagenase-sensitive tail region of synapsin I (see Fig. 6), close to but distinct from those sites known to be phosphorylated by CaM-PK II (sites 2 and 3).

The actual serine residue that is phosphorylated by PDPK (serine 551) was found to have a proline residue immediately adjacent on the carboxyl-terminal side, which is in agreement with the reported consensus sequence (-X-Ser/Thr-Pro-X-) and further supports the proline-directed nature of this par- ticular protein kinase (27). The sequence of amino acids immediately surrounding this novel phosphorylation site is also conserved in the reported synapsin I sequence of the rat (23, 30). Although it is clear that the first serine of the Ser- Pro-Ser-Pro sequence is the major phosphorylation site, the data do not enable the determination as to whether the second serine is also phosphorylated but to a much lesser extent. However, since isoelectric focusing of the HPLC-purified tryptic phosphopeptide demonstrated the presence of only a single phosphopeptide, it is likely that only a monophospho- rylated form of the peptide was present. The exclusion of a secondary phosphorylation site is also supported by the ob- served maximal stoichiometric ratio of 1 mol of phosphate/ mol of synapsin I protein.

The dephosphorylated form of synapsin I is known to bind tightly to synaptic vesicles with a dissociation constant of approximately 10 nM (13, 15-17). Phosphorylation of site 1 by either CAMP-PK or CaM-PK I does not change the dis- sociation constant appreciably. However, phosphorylation of sites 2 and 3 by CaM-PK II results in a 5-fold increase in the

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6948 Site-specific Phosphorylation of Synupsin I

Site 1 Ir

Site 2

Collagenase . Insensitive Head Region

Collagenase . Sensitive Tail Region

QAGPPQATRQTSVSGQAPPKASGVPP _..__.____ 1 557 568 582

[ ! !

GGQQRQGPPQKPPGPAGPTRQA6i5QAGP . . . . .._... i 583 609

FIG. 6. Mapping of the PDPK phosphorylation site to the tail region of synapsin I. The location of the novel phosphorylation site identified in the present study is shown diagrammatically in relation to other known phosphorylation sites. The precise numbering of amino acids corresponds to the predicted amino acid sequence reported for bovine brain synapsins Ia and Ib (30). The carboxyl- terminal truncation that defines synapsin Ib is indicated. Actual phosphorylation sites are identified by a circled P. The sequences of amino acids surrounding site 1 (phosphorylated by both CAMP-PK and CaM-PK I), site 2 (phosphorylated by CaM-PK II), and site 3 (phosphorylated by CaM-PK II) were identified previously (23). The tryptic phosphopeptide isolated and sequenced in the present study is underlined, and the major PDPK phosphorylation site is identified as serine 551. The serine 551 phosphorylation site of bovine brain synapsin I corresponds to serine 549 in the predicted amino acid sequence of the rat (30).

dissociation constant (16). Although an examination of vesic- ular binding was beyond the scope of the present study, it is intriguing to note the close proximity of this novel site to site 2 (within 17 amino acids), the phosphorylation of which is reported to have demonstrable effects upon the subcellular trafficking and release of neurotransmitters.

The previous observation by Roman0 et al. (24) that syn- apsin I is phosphorylated at a novel site in PC12 cells that have been treated with NGF is interesting and perhaps rele- vant to the present studies. Although Roman0 et al. did not sequence this novel site (designated peptide N) or establish its location within the synapsin I molecule, it is feasible that the so-called peptide N contains serine 551, the novel phos- phorylation site identified in the present studies. Both peptide N (24) and the tryptic peptide identified in these studies are phosphorylated on serine residues. The proline-directed pro- tein kinase was originally identified, characterized, and puri- fied from solid rat pheochromocytoma (27, 31, 32). We have demonstrated previously that this kinase exists in PC12 cells and, further, that the activity of this kinase is increased following treatment of PC12 cells with NGF (25, 27). The growth factor-sensitive, proline-directed protein kinase de- scribed herein displays a unique phosphorylation site specific- ity and does indeed phosphorylate synapsin I at a novel site. Therefore, the activation of this particular protein kinase by NGF may well have consequences in terms of regulating neuronal differentiation, neurite outgrowth, synaptogenesis, and/or neurotransmitter release. However, we have also de- tected growth factor-sensitive PDPK activity in nonneuronal cells’ and presently foresee a more global role for this newly

’ F. L. Hall and P. R. Vulliet, unpublished observations.

discovered protein kinase in the physiological regulation of cytoskeletal components.

Acknowledgments-We would like to thank Drs. Andrew Czernik, Michael Browning, and Paul Greengard for providing valuable infor- mation concerning the purification of synapsin I and also for provid- ing stimulating discussion.

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5. Fried, G., Nestler, E. J., De Camilli, p., Stiarne, L., olson, L., Lundberg, J. M.; Hokfelt, T., Ouimet, c. c., and Greengard, P. (1982) Proc. Natl. Acad. Sci. il. S. A. 79, 2717-2721

6. De Camilli, P., Cameron, R., and Greengard, P. (1983) J. Cell Biol. 96,1337-1354

7. De Camilli, P., Harris, S. M., Huttner, W. B., and Greengard, P. (1983) J. Cell Biol. 96, 1355-1373

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F L Hall, J P Mitchell and P R VullietPhosphorylation of synapsin I at a novel site by proline-directed protein kinase.

1990, 265:6944-6948.J. Biol. Chem. 

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