Synapsin I Is Phosphorylated at Ser 603 by p21-activated Kinases (PAKs) in Vitro and in PC12 Cells Stimulated with Bradykinin*

The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser 603 residue (site 3) is considered to be a pivotal site targeted by Ca 2 (cid:1) /calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser 603 residue responds to several kinds of stimuli, it is unlikely that many or all of the stimuli activate the CaMKII-involved pathway. Among the several stimulants tested in PC12 cells, bradykinin evoked the phosphorylation of Ser 603 without inducing the autophosphorylation of CaMKII, which was

Synapsin is a phosphoprotein found exclusively in neuronal presynaptic terminals and characterized as an anchoring protein between the vesicle phospholipid layer and the neuronal cytoskeleton. The cross-linking activity is regulated in a phosphorylation state-dependent manner, thereby controlling vesicle movement to active zones (1,2). It has been reported that phosphorylation of synapsins is evoked by various stimuli such as psychological or electric stress (3,4,5), ␤-adrenergic agonists (6), and protein kinase C (PKC) 1 activators (7). Isoproterenol and phorbol dibutylate produce a dose-dependent increase in the phosphorylation of synapsin I at the Ser 603 residue (site 3) (6, 7), which is recognized as the site specific for Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) and as one of the most effective sites for neurotransmitter release (1,2). However, it is unknown whether these stimuli directly or indirectly activate CaMKII through the adenylate cyclase/protein kinase A (PKA)-pathway or PKC-pathway(s). On the other hand, Hosaka et al. (8) proposed that neurotransmitter release may require phosphorylation of site 1 (Ser 9 ) in the N terminus of synapsins but not of Ser 603 , the former being recognized as a PKA and CaMKI site, the latter as a CaMKII site (1,9). Recently, Jovanovic et al. (10) reported that brain-derived neurotrophic factor induced the release of neurotransmitter from rat synaptosomes coincidentally with the activation of mitogenactivated protein kinase and concomitantly with the phosphorylation of site 4/5 and site 6, but not with autophosphorylation of CaMKII. In contrast, Liu et al. (11) reported that brainderived neurotrophic factor can promote the induction of long term potentiation and concomitant activation of CaMKII but not of mitogen-activated protein kinase in rat hippocampal slices, which probably occurred at a postsynaptic site. Furthermore, Matsubara et al. (12) reported that cyclin-dependent kinases phosphorylate the site 4 (Ser 551 ) and/or site 2 (Ser 553 ) on synapsin I in vitro and that this phosphorylation affects the affinity of synapsin I to F-actin. Although the phosphorylation site essential for the release of neurotransmitters is still controversial, the Ser 603 residue must be one of the pivotal sites for the release (1,2). However, it is unlikely that many, or all of the stimuli that induce the phosphorylation of Ser 603 activate the CaMKII pathway. Rather, it is plausible that many stimuli activate several protein kinases to phosphorylate the Ser 603 residue. Thus, to investigate the existence of a novel (or another) protein kinase that phosphorylates Ser 603 on synapsin I in neuronal cells, we tested several agonists that induce the phosphorylation of Ser 603 but not the autophosphorylation of CaMKII.
Preinflammatory factors are reported to induce or enhance the release of neurotransmitters through the activation of cell surface receptors (13)(14)(15), probably in part via a small GTPbinding protein-involved mechanism (16). Among these agonists, bradykinin (BK) is recognized as a potent agonist of the release of catecholamine from PC12 cells in an intracellular Ca 2ϩ -dependent manner (13,16). In some of these, BK stimulation seems to be involved in small G protein-dependent pathway(s) in PC12 cells (17). Recently, it has been accepted that BK stimulates a small G protein Cdc42/p21-activated kinase (PAK)-involved pathway in several cell systems (18 -21).
In this study, we found that BK evokes the phosphorylation of synapsin I at Ser 603 but not the autophosphorylation of CaMKII in PC12 cells and that bovine brain homogenate and PC12 cell lysate contain fractions that have Ca 2ϩ /calmodulinindependent Ser 603 kinase activities.

MATERIALS AND METHODS
Materials and Chemicals-Synapsin I was purified from bovine brain as described by Schiebler et al. (22). GST-Cdc42 was prepared according to the method of Chuang et al. (23). His-tagged PAK2 was prepared by GeneStorm Expression-Ready clone of human PAK2 (Invitrogen). In brief, COS7 cells were transfected with pcDNA3.1/PAK2 using FuGENE 6. At 48 h post-transfection, the cells were harvested and lysed. His-tagged PAK2 was purified through a Ni column (Qiagen) and dialyzed against 20 mM Tris-HCl (pH 7.5). Chromatography columns, SP-, DEAE-, phenyl-and heparin-Toyopearl, and HA1000 (hydroxyapatite column), and MonoQ were purchased from Tosoh (Tokuyama, Japan). Anti-PAK1, anti-PAK3, and anti-Rho-kinase antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-synapsin I antibody was from Calbiochem (San Diego, CA). KN62 was from Seikagaku Kogyo (Tokyo, Japan). Other materials and chemicals were purchased from commercial sources.
Preparation of Phosphorylation Site-specific Antibodies against Phosphorylated Synapsin I-Monoclonal antibody against Ser 603 (site 3)phosphorylated synapsin I was raised in mice immunized with keyhole limpet hemocyanine (klh)-conjugated phosphopeptide GPIRQApSQAG-PGPC-klh, as previously described (24). In brief, Ribi adjuvant emulsion containing 25 g of the phosphopeptide preparation was injected into BALB/c mice. Three days after the final booster, the spleen was removed and the spleen cells were fused with mouse P3Ui myeloma cells. After screening for useful hybridomas by enzyme-linked immunosorbent assay (ELISA), the antibody (pS603-Syn I-Ab) obtained was subjected to immunoblot analysis with synapsin I phosphorylated by CaMKII and with the extract of PC12 cells stimulated by 50 mM KCl (Fig. 1, A and B). The immunoactive band was completely quenched with 10 g/ml Ser 603 -phosphorylated peptide (Fig. 1A), and the cell extract showed almost a single immunoactive band (Fig. 1B). Polyclonal antibodies (pS566-Syn I-Ab and pS603-Syn I-Ab) against synapsin I phosphorylated either at site 2 or site 3 were raised in rabbits immunized with klh-conjugated phosphopeptides ATRQApSISGPAPC-klh (site 2) and GPIRQApSQAGPGPC-klh (site 3) and characterized as described previously (25). Other polyclonal antibodies were also raised in rabbits using phosphopeptides YLRRRLpSDSNFMAC-khl (site 1) for pS9-Syn I-Ab, ASPAAPpSPGSSGGC-klh (site 5) for pS67-Syn I-Ab and CAARPPApSPSPQRQC-klh (site 6) for pS551-Syn I-Ab and characterized as described above. The polyclonal antibody against Thr 286/287phosphorylated CaMKII was prepared and characterized as described previously (26).
Culture of PC12 Cells-PC12 cells were plated and grown at a density of 9 ϫ 10 5 cells/60 mm dish in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and 10% horse serum at 37°C for 2 days. After a 12-h serum starvation, the cells were used in the experiments.
Transient Transfection of PC12 Cells-We made use of a HA-tagged rat ␣PAK-encoded plasmid pXJ40HA from Dr. Manser, in which Lys 298 is mutated to Ala in order to make a dominant-negative form and Thr 422 is replaced with Glu to produce a constitutively active form (27). The transfection of both was conducted according to the method of Bokoch (28). Briefly, PC12 cells (1 ϫ 10 5 ) were plated on polyimine-coated cover glass in 35-mm diameter dishes 24 h before transfection, washed with PBS, and then treated in 0.6 ml of Dulbecco's modified Eagle's medium containing 25 l of Superfect transfection reagent (Qiagen) and 5 g of the plasmid for 2 h at 37°C. The resulting cells were washed with PBS and then further incubated in regular growth medium for 24 h at 37°C. The medium was replaced with Dulbecco's modified Eagle's medium without serum for serum starvation overnight. The cells were subjected to the stimulation with BK or KCl and processed for immunocytochemistry.
Immunofluorescent Staining-PC12 cells were fixed in 4% paraformaldehyde containing 4% sucrose and 4 mM EGTA for 30 min at room temperature. After fixation, the cells were washed three times with PBS and then permeabilized with 0.1% (v/v) Triton X-100 in PBS. After a wash with PBS, the cells were blocked with 5% bovine serum albumin in PBS for 1 h at room temperature and washed once with PBS. The cells were incubated with a 1:200 dilution of monoclonal anti-HA antibody (Santa Cruz Biotechnology) and 1:2000 dilution of polyclonal pS603-Syn I-Ab in PBS containing 5% bovine serum albumin overnight at 4°C. They were washed three times with PBS for 5 min and then incubated with 7.5 g/ml fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) and 0.75 g/ml tetramethylrhodamine isothiocyanate-conjugated donkey anti rabbit IgG (Jackson ImmunoResearch Laboratories) for 1 h.
Immunological Detection of Phosphorylation of Synapsin I at Ser 603 -PC12 cells were washed with HEPES-buffered saline (HBS) which contained 150 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , 20 mM glucose, and 20 mM HEPES (pH 7.2) and incubated for 15 min at 37°C. After preincubation with or without KN62, the cells were stimulated by 1 M BK or 50 mM KCl at 37°C for a specified period. The reaction was terminated by adding 5% trichloracetic acid, and the cells were then scraped and sedimented. The trichloroacetic acid cell pellet was dissolved in 200 l of Laemmli sample buffer containing 8 M urea and a protease inhibitor mixture and then neutralized. 10 l of the sample was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on a 5-20% polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA.). After blocking with skimmed milk, the membrane was treated with the primary antibody (polyclonal (1:500) or monoclonal (1:100) pS603-Syn I-Ab) followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig antibody (Amersham Biosciences) using an ECL system (Amersham Bisocsciences).
Assay of Protein Kinase Activity-CaMKII was prepared as previ- ously described (29), and the enzyme reaction was started by adding CaMKII to a 100-l reaction mixture containing 20 g of synapsin I, 0.1 mM CaCl 2 , 3 M calmodulin, and 0.1 mM [␥-32 P]ATP or cold ATP for 10 -30 min at 30°C. The resulting mixture was subjected to autoradiography or immunoblotting analysis using pS603-Syn I-Ab. Phosphorylated synapsin I was used as the standard. The assays for other protein kinases, PKA, Rho-kinase, and PAKs were performed as described previously (30).
Determination of the Ser 603 (Site 3) Kinase Activity by ELISA-Ser 603 (site 3) kinase activity was determined by ELISA using pS603-Syn I-Ab and synapsin I. Each fraction (10 l) obtained by column chromatography was added to the well of a synapsin I (1 g/well)-adsorbed 96-well immunoplate containing 100 l of reaction mixture (25 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1 mM ATP, 0.1 mM DTT, and 0.1 mg/ml bovine serum albumin) and incubated for 30 min at 30°C. The reaction was terminated by adding 100 l of 20% H 3 PO 4 , followed by washing with washing buffer (0.1% Triton X-100 in PBS). Phosphorylation of synapsin I was determined using monoclonal pS603-Syn I-Ab (1:100) followed by anti-mouse Ig antibody conjugated with HRP (1:1000).
Immunoprecipitation of PAK1 and PAK3 from Bovine Brain Homogenate and PC12 Cell Lysate-To prepare the immunoprecipitants of PAK1 and PAK3 from a bovine brain homogenate and PC12 cell lysate, anti-PAK1 and anti-PAK3 antibodies were prebound to protein G-Sepharose beads (Amersham Biosciences) by incubation for 1 h at room temperature and then washed in lysis buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1% Nonidet P-40, 0.1 mM DTT, 0.1 M okadaic acid, 1 mM Na 3 VO 4 , 0.1 mM Na 2 MoO 4 , 10 g/ml leupeptin, 10 g/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride). 300 l of bovine brain homogenate (ϳ30 g of protein) or PC12 cell lysate (2 ϫ 10 6 cells) was incubated with 20 l of a 1:1 slurry of anti-PAK antibodybeads for 1 h at 4°C, and the beads were washed three times with the lysis buffer and twice with 25 mM Tris-HCl, pH 7.5, and 0.1 mM DTT. The bead pellet was used to assay the synapsin I phosphorylation activity. The protein kinase activity for each phosphorylation site of synapsin I was assessed by immunoblot analysis using pS9-Syn I-Ab, pS67-Syn I-Ab, pS551-Syn I-Ab, pS553-Syn I-Ab, and pS603-Syn I-Ab.
Purification of PAK1 (p68) and PAK3 (p65) from Bovine Brain-Bovine brain gray matter (300 g) was homogenized in 1000 ml of homogenizing buffer (25 mM Tris-HCl (pH 7.5), 5 mM EGTA, 1 mM EDTA, 1 mM DTT, 10 g/ml leupeptin, 10 g/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride). The 100,000 ϫ g supernatant of the homogenate was applied to a SP-Toyopearl column (50 ml) equilibrated with buffer A (25 mM Tris-HCl, pH 7.5, 1 mM DTT) and the flow-through fraction was applied to a DEAE-Toyopearl column (250 ml). After a wash with buffer A, the proteins were eluted with a linear gradient to 0.3 M NaCl in buffer A. Ser 603 kinase activity was detected as four major peaks in the chromatogram. Peak I eluted with 90 mM NaCl, peak II with 120 mM NaCl, peak III with 175 mM NaCl, and peak IV with 200 mM NaCl. Each combined fraction of peaks II and III were made in 1 M ammonium sulfate and then applied to a phenyl-Toyopearl column (50 ml) equilibrated with buffer A containing 1 M ammonium sulfate. The kinase activity was eluted with a linear gradient to 10% of dioxan in buffer A and dialyzed against buffer A. The dialysate of combined fractions containing protein kinase activity, which originated from peak II obtained by DEAE column chromatography, was further applied to a heparin-Toyopearl column (10 ml) equilibrated with buffer A and eluted with a linear gradient to 0.7 M NaCl in buffer A. The resulting fractions were applied to a hydroxyapatite column (HA1000) (1 ml) equilibrated with buffer B (10 mM potassium phosphate, pH 7.0). Kinase activity was eluted with a linear gradient to 0.4 M potassium phosphate. An aliquot of each kinase-containing fraction was resolved by SDS-PAGE in a 5-20% (w/v) acrylamide gel, and proteins were visualized with Coomassie Brilliant Blue R-250. For the purification of peak III on DEAEcolumn chromatography, the fractions containing Ser 603 kinase activity were subjected to phenyl-column chromatography. The protein kinasecontaining fractions were dialyzed and further applied to a Source 15 Q (Amersham Biosciences) column (8 ml) equilibrated with buffer A and eluted with a linear gradient to 0.5 M NaCl in buffer A. The fractions containing protein kinase activity were subjected to heparin-Toyopearl column chromatography (10 ml) followed by hydroxyapatite column chromatography (HA1000) (1 ml) as for peak II.
Internal Peptide Microsequencing of p68 and p65-Internal peptide microsequencing of p68 and p65 was carried out by the method of Hellman et al. (31). The purified p68 and p65 were subjected to SDS-PAGE in 5-20% (w/v) acrylamide and the protein bands were cut out. The tryptic fragments were purified employing the SMART system using a reversed-phase column (RPC C2/C18 SC 2.1/10) (Amersham Biosciences), and the sequence was determined with a PPSQ-23 system (Shimadzu, Kyoto, Japan).

Phosphorylation of Synapsin I at Ser 603 (site 3) in PC12
Cells-Of the several agonists tested, five evoked the phosphorylation of synapsin I at the Ser 603 residue as determined with pS603-Syn 1-Ab (Fig. 1C). The Ser 603 residue was phosphorylated rapidly (within 30 s) in response to KCl, nicotine, glutamate, and BK, whereas nerve growth factor-evoked phosphorylation required a longer treatment (over 1 h). Bradykininevoked phosphorylation of Ser 603 was accompanied by little or no autophosphorylation of CaMKII. Because the phosphorylation of Ser 603 is generally considered to be CaMKII-dependent, we further investigated the BK-evoked phosphorylation at Ser 603 in PC12 cells (Fig. 2). Both high KCl (60 mM) and 1 M BK evoked a transient phosphorylation, and the extent of the KCl-evoked phosphorylation was 2-to 2.5-fold higher. The KClevoked, but not BK-evoked phosphorylation, was suppressed by 3 M KN62, a CaMKII inhibitor, whose IC 50 was around 1.5 M (Fig. 3). These findings led us to speculate that there is another pathway that might be activated by BK for the phosphorylation of Ser 603 in PC12 cells besides the CaMKII-involved one.
Ca 2ϩ -independent Phosphorylation of Synapsin I at Ser 603 in a Bovine Brain Homogenate-We tried to detect and isolate the Ser 603 kinase activity under Ca 2ϩ -free conditions in 100,000 ϫ g supernatant fractions of a bovine brain homogenate by sequential column chromatography using an ELISA system consisting of a pS603-Syn I-Ab and synapsin I-adsorbed 96-well plate (Fig. 4). The first DEAE-Toyopearl column chromatography yielded four major peaks of Ser 603 kinase activity; eluted with 0.09 M NaCl (peak I), 0.12 M NaCl (peak II), 0.175 M NaCl (peak III), and 0.2 M NaCl (peak IV), respectively (Fig. 4A). The peak I fractions reacted with anti-Rho-kinase antibody (data not shown). CaMKII was detected with anti-CaMKII antibody in fractions 10 -22 (data not shown). Peak II and peak III fractions were further purified through a phenyl-Toyopearl column, a Source15Q column only for peak III, a heparin-Toyopearl column, and finally a hydroxyapatite column as described under "Materials and Methods." The elution pattern of peak II and III fractions from the hydroxyapatite column is shown in Fig. 4 Fig. 4 (D and E). The final eluates consisted of proteins of molecular mass 68,000 Da (for peak II) and 65,000 Da (for peak III), termed p68 and p65, respectively. The p68 and p65 obtained by SDS-PAGE were subjected to peptide microsequencing. The trypsin-digested peptide of p68 was sequenced as six internal peptides as follows: EKERPEISLPSDFEHTIHVGFDA, SVYTR, DIKSD-NILLGMDG, LTDFGFCAQITPEQSKR, TMVGTPYWMAPE-VVTR, and ELLQHQFLK; and that of p65 as four internal peptides: ERPEISLP, LLQTSNITKLEQK, YMSFTSGDKSAH-GYIAA, and STMVGTPYWMAPEVV. The amino acid sequences of p68 peptides fully matched the p21-activated protein kinase isoform PAK1 as compared with the Swiss-Prot protein sequence data base entries, and the sequences of p65 matched PAK3 (Fig. 5).

(B and C) and the SDS-PAGE staining with Coomassie Brilliant Blue R-250 is shown in
Immunoprecipitation of PAK1 and PAK3 in Bovine Brain Homogenate-As p68 and p65 were identified in the amino acid sequence of PAK1 and PAK3, respectively, we examined whether the immunoprecipitants from a bovine brain homogenate obtained using anti-PAK1 and PAK3 antibodies could phosphorylate synapsin I at the Ser 603 residue. The immunoprecipitants of PAK1 and PAK3 incorporated 32 P into synapsin I (both subtypes a and b) in a GST-Cdc42/GTP␥S-dependent manner, and the extent of GST-Cdc42/GTP␥S activation was 10 -30 times higher (Fig. 6A). The phosphorylation site was identified as Ser 603 using the pS603-Syn 1-Ab. Moreover, we tested the phosphorylation of other sites on synapsin I with the immunoprecipitants using phosphorylation site-specific antibodies (Fig. 6B). The synapsin I preparation, which was treated with the immunoprecipitants of PAK1 and PAK3, reacted with pS9-Syn I-Ab to a similar extent to pS603-Syn I-Ab, whereas the intensity of the immunoreaction with pS551-Syn I-Ab was lower. Immunoreactivity for Ser 67 and Ser 553 was scarcely detected. To avoid phosphorylation by contaminants in these immunoprecipitants and also to check the activity of other PAK subtypes, we examined whether His-tagged recombinant PAK2 can phosphorylate synapsin I at Ser 603 . The preparation of recombinant PAK2 required GST-Cdc42/GTP␥S to phosphorylate the Ser 603 residue (Fig. 6C). Recombinant PAK2 could also phosphorylate the Ser 9 and Ser 551 residues in a GST-Cdc42/ GTPrS-dependent manner (Fig. 6D).
Detection of PAK1 and PAK3 Activity in PC12 Cell Lysate-Like the case of the bovine brain homogenate, we detected Ca 2ϩ -independent Ser 603 kinase activity in a PC12 cells lysate by MonoQ column chromatography (Fig. 7A). Three major peaks of activity were obtained; the first peak was eluted with 0.25 M NaCl (fractions 16 -20), whereas the second and third peaks were eluted with 0.35 and 0.45 M NaCl, respectively. CaMKII was detected in fractions 12-18 using anti-CaMKII antibody (data not shown). The immunoprecipitants of PAK1 and PAK3 from the PC12 cell lysate also phosphorylated the Ser 603 residue in a Cdc42/GTP␥S-dependent manner (Fig. 7B).
Modulation of Ser 603 Phosphorylation by Transient Expression of PAK Mutants-Finally, to confirm the phosphorylation of synapsin I at Ser 603 by PAK in neuronal cells, we used PC12 cells transfected with a plasmid encoding a constitutively ac-  4. Chromatography and purification of Ser 603 kinase activity in a bovine brain homogenate. A, DEAE-Toyopearl column chromatography of the 100,000 ϫ g supernatant of bovine brain homogenate. The flow-through fractions of the 100,000 ϫ g bovine brain supernatant obtained by SP-Toyopearl column chromatography were subjected to DEAE-Toyopearl column chromatography, washed, and eluted with a linear gradient of NaCl. Ser 603 kinase activity was detected by ELISA as described under "Materials and Methods." The bar indicates the CaMKII-containing fractions (10 -22). Chromatograms of peaks II and III after hydroxyapatite column chromatography are shown in B and C, respectively. Coomassie Brilliant Blue (R-250) staining of the kinase peak fractions obtained at each chromatography step from peak II and peak III is shown in D and E, respectively. D, DEAE-Toyopearl; P, phenyl-Toyopearl; Q, Source 15Q; H, heparin-Toyopearl; HA, hydroxyapatite column chromatography.
tive and dominant-negative HA-tagged rat ␣PAK (identical to PAK1). In PC12 cells transfection efficiency was low, so we thus investigated the phosphorylation in an immunocytochemical analysis with pS603-Syn I-Ab. The culture, which con-tained wild-type and mutant cells, has the advantage of detecting the phosphorylation in both cells in the same time and the field of microscope. The expression of constitutively active ␣PAK evoked an immunostaining signal for synapsin I phosphorylation even in resting HA-positive cells and enhanced the immunostaining signal upon stimulation with BK and KCl (Fig. 8). The BK-evoked immunostaining signal was observed in a diffusing pattern in wild-type PC12 cells. In contrast, the expression of dominant-negative ␣PAK dramatically reduced the immunostaining signal in HA-positive cells on BK stimulation (Fig. 9). However, the KCl-evoked immunostaining signal in HA-positive cells was not reduced by expression of the dominant negative form. The results were confirmed in three other separate experiments. DISCUSSION We demonstrated in this report that our system, i.e. the protein kinase-probing system facilitates the detection of the phosphorylation site-specific protein kinase of a target molecule in tissue and cell homogenate, and that it is particularly effective for proteins phosphorylated at multiple sites. The cardinal point of this system is to use a phosphorylation sitespecific antibody and whole target protein-coated plates for immunoreactions. In the case of a target protein phosphorylated at multiple sites, the 32 P incorporation system cannot easily detect any sharp peaks or fractions of protein kinase on column chromatography, probably yielding broad and undistinguishable peaks. In this study, we could detect and identify PAK1 and PAK3 as Ca 2ϩ /calmodulin-independent Ser 603 kinases in a bovine brain homogenate. Fortunately, our protein kinase probing system detected these activities in the column chromatography fractions even in the absence of any other activators such as Cdc42/GTP␥S, or Rho/GTP␥S (Figs. 4 and 7), which may be capable of detecting the basal activities of Rhokinase and PAKs, whereas the PAK1 and PAK3 immunopre- 5. Internal peptide microsequencing of p68 and p65. The amino acid sequence of six tryptic peptides from p68 and four from p65 was determined. The amino acid sequences are shown with the singleletter abbreviations. Identified amino acids are boxed. These sequences were compared with those of the bovine p21-activated protein kinase isoforms PAK1 and PAK3.
FIG. 6. Synapsin I phosphorylation activity of immunoprecipitants from a bovine brain homogenate obtained with anti-PAK1 and PAK3 antibodies. A, synapsin I was incubated with each immunoprecipitant or CaMKII in a kinase assay mixture containing [␥-32 P]ATP or cold ATP in the presence or absence of 0.1 mg/ml GST-Cdc42/GTP␥S. After a 30-min incubation, the resulting mixture was subjected to autoradiography and immunoblotting analysis using pS603-Syn I-Ab. B, protein kinase activity of immunoprecipitants for other sites. Synapsin I was incubated with the PAK-immunoprecipitants in the presence of GST-Cdc42/GTP␥S, and the reaction was stopped by adding SDS-PAGE buffer. Synapsin I was subjected to immunoblotting analyses using various phosphorylation site-specific antibodies. Note that phosphorylation at Ser 9 , Ser 551 , and Ser 603 was detected in the immunoprecipitants. C, Ser 603 kinase activity of recombinant His-tagged PAK2. The recombinant His-tagged PAK2 (0.15 mg/ ml) was incubated in the presence (ϩ) or absence (Ϫ) of 0.1 mg/ml GST-Cdc42/GTP␥S, and the phosphorylation was assessed using pS603-Syn I-Ab. D, phosphorylation of various sites on synapsin I by His-tagged PAK2, as determined in B.

FIG. 7. Ser 603 kinase activity in PC12 cells.
A, MonoQ-column chromatography of the PC12 cell lysate. The 100,000 ϫ g supernatant of the cells lysate was subjected to MonoQ-column chromatography and eluted with a linear gradient of NaCl. Kinase activity was detected by ELISA using pS603-Syn I-Ab. The bar indicates the fractions containing CaMKII (12)(13)(14)(15)(16)(17)(18). B, Ser 603 kinase activity of the immunoprecipitants obtained from PC12 cell lysate with anti-PAK1 and PAK3 antibodies. Synapsin I was incubated with the immunoprecipitants in the presence or absence of 0.1 g/ml GST-Cdc42/GTP␥S, or with CaMKII/ calmodulin-Ca 2ϩ , and assessed as described under "Materials and Methods." cipitants from a brain homogenate required Cdc42-GTP␥S in immunoblot analysis (Fig. 6). This might be due to pS603-Syn I-Ab possibly being highly specific and able to detect a native phosphorylated form of synapsin I (in ELISA) preferentially to the denatured form (in immunoblotting system). Our protein kinase probing system may be useful for detecting new protein kinase(s) in many samples in a short time.
In this study, we showed that the bovine brain homogenate contained four major Ser 603 kinases other than CaMKII (Fig.  4). CaMKII was detected by anti-CaMKII antibody in the fractions 10 -22 in DEAE-column chromatography, and Rho-kinase was detected in peak I by anti-Rho-kinase antibody. Moreover, peak I was also identified as Rho-kinase by tryptic peptide analysis, and the Rho-kinase preparation from bovine brain incorporated 32 P into the Ser 603 residue of synapsin I in vitro. 2 Although we could identify neither peak VI nor the shoulder between peak I and II (Fig. 4), we showed that bovine brain contains at least four protein kinases for synapsin I at Ser 603 (site 3): CaMKII, Rho-kinase, PAK1 and PAK3. These findings are surprising, because the Ser 603 site has been believed to be specific for CaMKII (1,4,5,8). There is a growing body of evidence that PAKs can phosphorylate certain proteins possessing an appropriate amino acid sequence. King et al. (32,33) have reported that PAK3 phosphorylates Raf-1 at Ser 338 , whose flanking domain contains the amino acids sequence 333 RXXRXS 338 , and such positively charged arginines at 333 (Ϫ5 N-terminal-side of Ser 338 ; P-5) and 336 (P-2) are essential for phosphorylation. Similar findings concerning the consensus sequence for PAK-phosphorylation are reported for several kinds of proteins (34 -36), proposing that PAK requires a basic amino acid sequence, such as arginine and lysine, at Ϫ2 or more N-terminal-side positions (up to Ϫ5) from the phosphorylation site. The Ser 603 flanking domain RXXS of synapsin I is compatible with the consensus motif for PAK-phosphorylation. Thus, these findings suggest that synapsin I is a good substrate for PAKs at least in the in vitro system.
Many laboratories have reported that PAK activity is regulated by various external stimuli through the activation of cell surface receptors, including G-protein-coupled receptors, growth factor receptors (37), and proinflammatory cytokine receptors (15), and that its activation requires the GTP-activated form of Cdc42/Rac1, a Rho family member (38,39). The activation of such protein-involved pathways may promote the activation of peripheral membrane associated with filopodia and lamellipodia formation through the reorganization of Factin-containing microfilaments and/or intermediate filaments (40,41). The role of Cdc42/Rac1-PAKs in regulating cytoskeleton-based membrane activities has been defined extensively in muscle and non-muscle cell systems, and in part in neuronal cells (28). However, little is known about the precise molecular mechanism of Cdc42/Rac1-PAKs in neuronal functions other than actin-involved events such as synaptic vesicle trafficking or nerve-end membrane events. This may be due to the lack of identification of an effector of PAKs in neurons or brain. In this study, we demonstrated that the immunoprecipitants from bovine brain obtained with anti-PAK1 and PAK3 antibodies, and the recombinant PAK2 preparation phosphorylated synapsin I at Ser 603 in a Cdc42/GTP␥S-dependent manner (Fig. 6). Furthermore, we confirmed that an ␣PAK preparation (provided by Dr. Inagaki) phosphorylates the Ser 603 residue in vitro. 3 In neuronal PC12 cells, BK evoked the phosphorylation of synapsin I at Ser 603 , which was more transient and resistant to KN62 treatment (Figs. 2 and 3), whereas the KCl-evoked phosphorylation was sensitive to KN62 treatment. In immunocytochemical analysis, we demonstrated that the expression of dominant negative ␣PAK reduced the BK-evoked phosphorylation of Ser 603 in the mutant cells, but not the KCl-evoked one (Fig. 9). However, it should be evaluated which kinase the dominantnegative ␣PAK suppresses preferentially, PAK1 or PAK3, in BK-stimulated cells. Moreover, the expression of constitutively active ␣PAK elicited the phosphorylation even in the resting mutant cells (Fig. 8). Taken together with other reports, the present findings allow us to speculate that BK evokes the activation of the Cdc-42/PAK pathway and thereby the phosphorylation of Ser 603 on synapsin I. This is the first proposal that PAKs phosphorylate a physiologically significant site on synapsin I in neuronal cells.
PAKs were first detected while screening for binding targets of Rac and Cdc-42 GTPase in the rat brain (39). Thereafter, PAK1 and PAK3 were detected in large amounts in certain 2 K. Sakurada 8. Augmentation by expression of constitutively active ␣PAK of bradykinin-evoked phosphorylation of Ser 603 on synapsin I in PC12 cells. PC12 cells were transfected with HA-tagged constitutively active rat ␣PAK (T422E)-encoded plasmid as described under "Materials and Methods." The transfection efficiency of PC12 cells was low, so the culture contained wild-type and mutant cells. The resulting cells were stimulated for 30 sec with 1 M BK or 50 mM KCl, and the reaction was terminated with 4% paraformaldehyde containing 4% sucrose and 4 mM EGTA applied for 30 min at room temperature. After permeabilization with 0.1% (v/v) Triton X-100, the cells were blocked with 5% bovine serum albumin and then processed to immunocytochemical double staining with monoclonal anti-HA antibody and fluorescein isothiocyanate-conjugated goat anti-mouse IgG and with polyclonal pS603-Syn I-Ab and tetramethylrhodamine isothiocyanateconjugated donkey anti-rabbit IgG. Note that the immunostaining signal for Ser 603 phosphorylation is observed in the resting HA-positive cell, and that upon stimulation with BK the immunostaining signal is more intensive in HA-positive than that HA-negative cells. Arrows indicate transfected cells. portions of the cortex, whereas PAK2 seemed to be ubiquitously expressed in all tissues (28). At a subcellular level, PAKs may localize in actin-containing structures and regulate several cellular events such as the formation of filopodia and actin spikes, and the actin-containing meshwork in the cell periphery (42). It is known that microinjection of Cdc42He into Swiss 3T3 cells promotes the formation of peripheral actin microspikes and filopodia (21). Some of these events may be accompanied by the phosphorylation of actin-associated proteins (34 -36), suggesting that PAKs co-localize with F-actin to phosphorylate actin binding proteins. As non-phosphorylated synapsin I binds to actin-containing structures and anchors synaptic vesicles (43,44), it is possible that at neuronal sites, Cdc42/PAK coexists with synapsin I through an association with F-actin and facilitates the phosphorylation of synapse I in response to neuronal stimulation, because synapsin I is characterized as an actin binding protein.
It has been reported that BK promotes a transient increase of intracellular Ca 2ϩ via the activation of the phosphatidylinositol signaling pathway and, consequently, induced the autophosphorylation of CaMKII in PC12 cells (45,46). These results differ from our findings, but at the present time we do not have data to explain the discrepancy, except for the difference in experimental conditions. However, we have obtained similar results using another neuronal cell line; i.e. BK evoked the phosphorylation of synapsin I at Ser 603 without autophosphorylation of CaMKII. 4 Thus, further study is required to solve this issue.
In conclusion, we propose the possibility of at least two pathways for the phosphorylation of synapsin I at Ser 603 in the brain or neuronal cells; one involves the CaMKII and the other Cdc42/PAK.