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(The FASEB Journal. 2000;14:543-554.)
© 2000 FASEB

Specific phosphorylation of Torpedo 43K rapsyn by endogenous kinase(s) with thiamine triphosphate as the phosphate donor

HOÀNG-OANH NGHIÊM*1, LUCIEN BETTENDORFF{dagger} and JEAN-PIERRE CHANGEUX*

* CNRS UA D-1284, Neurobiologie Moléculaire, Institut Pasteur, 75724 Paris Cedex, France; and
{dagger} Centre de Recherche au Neurobiologie Cellulaire et Moléculaire, University of Liege, Belgium

1Correspondence: CNRS UA D-1284, Neurobiologie Moléculaire, Institut Pasteur, 25–28 rue du Dr. Roux, 75724 Paris Cedex, France. E-mail: honghiem{at}pasteur.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
43K rapsyn is a peripheral protein specifically associated with the nicotinic acetylcholine receptor (nAChR) present in the postsynaptic membrane of the neuromuscular junction and of the electrocyte, and is essential for its clustering. Here, we demonstrate a novel specific phosphorylation of 43K rapsyn by endogenous protein kinase(s) present in Torpedo electrocyte nAChR-rich membranes and identify thiamine triphosphate (TTP) as the phosphate donor. In the presence of Mg2+ and [{gamma}-32P]-TTP, 43K rapsyn is specifically phosphorylated with a 32P-half-maximal incorporation at ~5–25 µM TTP. The presence of TTP in the cytosol and of 43K rapsyn at the cytoplasmic face of the postsynaptic membrane, together with TTP-dependent phosphorylation of 43K rapsyn without added exokinases, suggests that TTP-dependent-43K-rapsyn phosphorylation may occur in vivo. In addition, phosphoamino acid and chemical stability analysis suggests that the residues phosphorylated are predominantly histidines. Inhibition of phosphorylation by Zn2+ suggests a possible control of 43K rapsyn phosphorylation state by its zinc finger domain. Endogenous kinase(s) present in rodent brain membranes can also use [{gamma}-32P]-TTP as a phosphodonor. The use of a phosphodonor (TTP) belonging to the thiamine family but not to the classical (ATP, GTP) purine triphosphate family represents a novel phosphorylation pathway possibly important for synaptic proteins.—Nghiêm, H.-O., Bettendorff, L., Changeux, J.-P. Specific phosphorylation of Torpedo 43K rapsyn by endogenous kinase(s) with thiamine triphosphate as the phosphate donor.


Key Words: nicotinic acetylcholine receptor (nAChR) • cytoskeleton • neuromuscular junction (NMJ) • synapse • phosphohistidine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
THE NEUROMUSCULAR JUNCTION (NMJ) is a sophisticated structure specialized in the transmission of neural signals from the motor nerve to the muscle cell or to the electrocyte, which may be viewed as a simplified muscle cell. 43K rapsyn (reviewed in refs 1 , 2 ) is a membrane-associated peripheral protein (3 , 4) coextensively distributed with the nAChRs at the inner face of the postsynaptic membrane of Torpedo electric organ (5 , 6) and at rodent NMJ (7) . It is necessary for nAChR clustering and formation of functional motor endplates. Mutant mice defective in 43K rapsyn gene die postnatally, display a lack of nAChR clusters and have dysfunctional postsynaptic membranes (8) . In vitro removal of 43K rapsyn renders the nAChRs more mobile within the membrane plane, more susceptible to enzymatic degradation and heat denaturation (reviewed in refs 1 , 2 ), and more accessible to anti-nAChR antibodies (9) .

Phosphorylation is important in cell signaling (reviewed in refs 10 11 12 13 14 ). 43K rapsyn, which contains several putative phosphorylation sites (15) , is partially phosphorylated on serine residues in vivo and phosphorylated in vitro by endogenous protein kinase A (PKA) (16) . However, this phosphorylation is not specific for 43K rapsyn and can occur with other proteins of the postsynaptic membrane (16) . In view of the essential roles of phosphorylation in cell signaling (rfs. in 10–14) and of 43K rapsyn in postsynaptic differentiation (reviewed in refs 1 , 2 , 17 ), we searched for a specific phosphorylation of this synaptic protein.

Thiamine is essential to cell life and may play a role in the central nervous system and in synaptic transmission (18 19 20) . The thiamine pathway includes thiamine and its mono- (TMP), di- (TDP), and triphosphate (TTP) derivatives. TTP, the non-cofactor form of thiamine activates the maxi-chloride channel permeability, possibly via phosphorylation (21) . TTP concentrations are low in most cells (22) , but comparatively high in neuronal (23) and excitable (24 25 26) cells. Thus, TTP is an interesting candidate for a specific phosphorylation of 43K rapsyn.

In this study, we demonstrate that in the presence of radiolabeled TTP, Torpedo 43K rapsyn is the predominant protein phosphorylated by endogenous kinase(s) present in nAChR-rich postsynaptic membrane preparations. Phosphorylation occurs mostly at histidine(s) and at some serine(s). Both TTP- and ATP-dependent phosphorylation of 43K rapsyn are inhibited by TTP and ATP. TTP-dependent kinase(s) thus might share some phosphorylation site(s) with PKA. The possible regulation of 43K rapsyn phosphorylation by endogenous Zn2+ and the modulation of 43K rapsyn functions via its phosphorylation state are discussed. The extension of phosphorylation to rodent brain membranes suggests a more general use of TTP as phosphate donor for synaptic proteins and a possible novel phosphorylation pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Postsynaptic membranes
nAChR-rich postsynaptic membranes (nAChR membranes) were prepared from electric organs excised from freshly killed Torpedo marmorata (T.m.) (Biologie Marine, Arcachon (3 , 16) .

Rodent brain membrane preparations
Brain membrane preparations were performed at 4°C. Mice and rats were anesthetized then killed by decapitation. The brains were dissected and homogenized with a Teflon glass homogenizer in 5 volumes of ice-cold Tris-buffer pH 7.5 containing 10% sucrose (w/w), 1 mM EDTA, 1 mM DTT, and inhibitors of proteases (aprotinin, pefabloc, leupeptin, antipain, pepstatin A, PMSF). The homogenates were centrifuged at 1000 g for 5 min at 4°C. The supernatants were further centrifuged at 30,000 g for 1 h at 4°C. The resulting pellets corresponding to the crude brain membrane fractions were homogenized in ice-cold homogenization buffer without DTT and stored at -80°C.

Phosphate donors, phosphorylation, and quantification
[{gamma}-32P]-ATP (32P-ATP) was from ICN (Irvine, Calif.). [{gamma}-32P]-TTP (32P-TTP) was synthesized (27) . nAChR membranes were phosphorylated with (7–8000Ci/mol) 32P-TTP or 32P-ATP in 50 mM Tris-HCl pH 7.5, 5–15 mM MgCl2, 0.08% CHAPS, inhibitors of proteases at 4–20°C for 60–90 min. Phosphorylation was stopped with sodium dodecyl sulfate (SDS) sample buffer. 32P-phosphorylated membranes were subjected to SDS-PAGE (polyacrylamide gel electrophoresis) designed to separate actin, 43K rapsyn, and {alpha}-nAChR and autoradiographed (Kodak Biomax) and/or 32P-quantified (Molecular Dynamics phosphorImager). Coomassie blue staining was performed when necessary. Where specified, nAChR membranes were treated with 5–20 mM diethylpyrocarbonate (DEPC) (28) in 50 mM Na phosphate buffer pH 6.0 and 7.4 (20 min, 16°C) prior to incubation with 32P-TTP. Common kinase effectors [cAMP; adenosine 3'-5'-cyclic monophosphate, 8-(4-chlorophenylthio)-sodium salt (8-CPT-cAMP); anisomycin; cGMP; calmidazolium; calphostin; cdc2 peptide; genistein; bisindolylmaleimide I (GFX); H7; H89; KN62; KT5720, ML7; protein kinase A inhibitor (PKI); staurosporine; tumor necrosis factor-alpha (TNF-{alpha}); phorbol-12-myristate-13-acetate (TPA)] were tested for their effects on TTP-dependent phosphorylation of 43K rapsyn.

Chemical stability and nature of the phosphate links
For acid treatment, SDS-PAGE gels containing 32P-ATP- or 32P-TTP-treated membranes were cut and incubated with Tris buffer or 16% TCA at 90°C (29 , 30) , washed, and analyzed. Equivalent amounts of 43K rapsyn were ensured by Coomassie blue staining. For base treatment, 32P-labeled membranes were resolved by SDS-PAGE, electroblotted on polyvinylidene difluoride membrane (PVDF). Blots were dried at 55°C to minimize protein loss, wet in methanol, washed with H2O, cut, and incubated in water or 1N KOH at 46°C, and analyzed.

Phosphoamino acid analysis on PVDF-electrotransferred (31) 32P-43K rapsyn
For determination of acid-stable phosphoamino acids, 43K rapsyn was hydrolyzed with 40 µl 5.7N HCl (1 h, 105°C). The supernatant was evaporated and 10 µl H2O was added. Hydrolysates were analyzed by either 1-dimensional (pH 3.5) or 2-dimensional high-voltage electrophoresis on a thin-layer cellulose plate (first electrophoresis, pH 1.9; second electrophoresis, pH 3.5) (32) . For a base-stable analysis, 43K rapsyn was hydrolyzed in 3N KOH (3 h, 105°C), neutralized with 10% HClO4 to pH 7.5 (33) . Supernatants were analyzed by thin-layer chromatography (TLC) on silica gel 60A° plates (ICN) in solvent A (t-butanol:methyl ethyl ketone:acetone:methanol:water:concentrated NH4OH, 10: 20: 20: 5: 40: 5, v :v), which separates phosphohistidine (P-His) from phosphoserine (P-Ser) and phospholysine (P-Lys) (33) . Phosphohistidine and phospholysine were synthesized from polyhistidine and polylysine, respectively (34) . Enzymatic hydrolysis was conducted with 2 µg TPCK-trypsin (Promega, Madison, Wis.) in 40 µl of trypsin buffer (10 mM NaHCO3, 135 mM NaCl, 0.1% SDS, 1 mM CaCl2, pH 8.5) (90 min, 37°C). Two micrograms of TPCK-trypsin was added (2 h, 37°C), followed by 400 µg Pronase (Boehringer Mannheim, Mannheim, Germany) (18 h, 37°C). Supernatants were analyzed by TLC in solvent A. Phosphoamino acids and phosphopeptides were visualized with ninhydrin.

Phosphopeptides
Phosphopeptides were generated by tryptic digestion on PVDF-transferred 32P-43K rapsyn with TPCK-trypsin (o.n.; 37°C in trypsin buffer). Hydrolysates were resolved in 15% SDS-PAGE and autoradiographed for 32P-peptide identification.

Labeling with antibodies or {alpha}-Bungarotoxin (Bgtx)
32P membranes were resolved by SDS-PAGE, electroblotted (35) , treated according to ref 36 , probed with specific anti-43K rapsyn (37) , specific antiphosphoamino acid antibodies (Sigma, St. Louis, Mo.), or 125I-Bgtx (Amersham, Arlington Heights, Ill.), and analyzed.

Immunoprecipitation
32P-membranes were diluted into 1 ml 50 mM Tris-HCl pH 8.8, 0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate, protease inhibitors, 0.15M NaCl, precleared with 50 µl protein, A-agarose beads (Santa Cruz, Santa Cruz, Calif.), and immunoprecipitated with anti-43K rapsyn antipeptide antibodies that specifically recognize 43K rapsyn (37) . Thirty microliters of protein A beads were added (o.n., 4°C). Beads were centrifuged, washed, and analyzed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
The TTP-dependent phosphorylated protein is 43K rapsyn
Equal amounts of nAChR membranes were incubated with [{gamma}-32P]-TTP in the presence of various effectors, separated by SDS-PAGE, and autoradiographed (Fig. 1B ) before Coomassie blue staining (Fig. 1A ). The autoradiogram (Fig. 1B ) showed that most of the phosphorylated protein(s) migrated at ~43 kDa (Fig. 1B , arrow). The phosphorylation occurs without externally added kinases, is enhanced by Mg2+ (5 mM, Fig. 1B , lanes 3, 6), partially inhibited by DTT (Fig. 1B , lane 1), and inhibited by Zn2+ (Fig. 1B , lane 2) and TTP in a dose-dependent manner [Fig. 1B : 24 µM (lane 8 vs. 4, 9); 240 µM (lane 5 vs. 3, 6; lane 7 vs. 4, 9)].



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  		Figure 1. Phosphorylation of a 43 kDa protein with TTP as the phosphate donor by endogenous kinase(s) present in the nAChR-rich postsynaptic membrane. A) Coomassie blue staining of the panel B autoradiogram. B) SDS-PAGE-autoradiogram of electrocyte postsynaptic membranes phosphorylated with 8 µM [{gamma}-32P]-TTP in the presence of various effectors reveals one major radioactive band at ~43 kDa (arrow). Phosphorylation is inhibited by cold TTP in a dose-dependent manner [24 µM (lane 8/control lanes 4, 9) and 240 µM (lane 7/control lanes 4,9 and lane 5/control lanes 3,6)]. Molecular mass markers: far right. C) A sister gel of panel A was blotted and split into two parts. Lanes 1–3 were incubated with 125I-Bgtx and autoradiographed. In lane 2, with phosphorylation prevented, the radioactive band observed with 125I-Bgtx is {alpha}-nAChR (arrowhead). In lanes 1 and 3, with the 32P membranes further incubated with 125I-Bgtx, two radioactive bands are observed. Note that in lane 1, with phosphorylation partially prevented, the upper radioactive band corresponding to the 32P-43 kDa band (arrow) is fainter than that observed in lane 3. This demonstrates that the TTP-dependent phosphorylated 43 kDa protein is not {alpha}-nAChR.

A sister gel of 1A was blotted, and the blotted 32P-labeled membrane corresponding to lanes 1 to 3 and to various degrees of membrane phosphorylation were further incubated with 125I-Bgtx (a toxin specific for {alpha}-nAChR), then autoradiographed (Fig. 1C ). With phosphorylated membranes, two radioactive bands were observed (Fig. 1C , lanes 1, 3). In lane 2, with phosphorylation prevented, only one radioactive band corresponding to the 125I-Bgtx-labeled band (arrowhead) and distinct from the 32P-labeled 43 kDa band (arrow) was observed. This demonstrates that {alpha}-nAChR is not phosphorylated by 32P-TTP.

The 32P-labeled band was recognized by anti-43K rapsyn antibodies (immunoblot, data not shown). To ascertain that the 32P-phosphorylated protein is 43K rapsyn, immunoprecipitation of 32P-labeled membranes was conducted with three specific anti-43K rapsyn antipeptide antibodies (37) . Figure 2A shows that the 32P-labeled protein was specifically immunoprecipitated by anti-43K rapsyn antibodies. One anti-43K rapsyn antibody (Fig. 2A , lane 1) used in a semiquantitative analysis (Fig. 2B ) showed that the radioactivity immunoprecipitated is directly correlated to the amount of anti-43K rapsyn used (Fig. 2B , lanes 3–5). Supernatants of immunoprecipitation showed the opposite situation (Fig. 2C ). The specificity of the immunoprecipitation was verified with preimmuneserum and preabsorbed antibodies (Fig. 2B , lanes 1, 2). This demonstrates that 43K rapsyn is the TTP-dependent phosphorylated protein.



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  		Figure 2. The TTP-dependent 32P-phosphorylated 43 kDa protein is 43K rapsyn. A) Postsynaptic membranes phosphorylated in the presence of 32P-TTP were solubilized and immunoprecipitated with three specific anti-43K rapsyn antipeptide antibodies (lanes 1–3). No radioactivity was precipitated with preimmuneserum (lane 4). B) Immunoprecipitation with increasing volumes of anti-43K rapsyn shows that the immunoprecipitated radioactivity is proportional to the amount of antibodies used (lanes 3–5). The specificity of the immunoprecipitation is demonstrated with preimmuneserum and preabsorbed anti-43K rapsyn antibodies (lanes 1, 2). C) The 32P-radioactivity remaining in supernatants of immunoprecipitation decreases proportionally to the amounts of antibody added, indicating that most if not all radioactive phosphate is on 43K rapsyn.

The TTP-dependent-phosphorylation is driven by endogenous kinase(s) present in the nAChR-rich membranes
The phosphorylation of 43K rapsyn that occurs at 4–22°C without externally added kinases is Mg2+ (5 mM, Fig. 1B , lanes 3, 6), pH, and time dependent (Fig. 3 ). It requires TTP, is dose dependent and saturable (KD ~5–10 µM TTP, Fig. 3C ; with one membrane preparation, KD ~25 µM TTP), and presents characteristics of an enzymatic reaction. Thus, the TTP-dependent phosphorylation of 43K rapsyn is driven by endogenous kinase(s) copurified with the postsynaptic membrane. The IC50 for TTP, ATP, GTP are around 40 µM, 500 µM, and 1000 µM, respectively (Fig. 3A ). CTP inhibits poorly. The TTP-dependent kinase or TTP-43K-kinase activity is favored by light alkaline pH (Fig. 3B ) and partially inhibited by DTT (30–40% inhibition/10 mM; Fig. 1B , lane 1). Zn2+ inhibits the TTP-dependent phosphorylation of 43K rapsyn in a Mg2+ independent manner [~70% inhibition/0.5–3 mM Zn2+/8 µM 32P-TTP and (Fig. 1B , lane 2)].



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  		Figure 3. Characteristics of the endogenous TTP-dependent kinase(s) that catalyze phosphorylation of 43K rapsyn. A) Inhibition of TTP-dependent phosphorylation of 43K rapsyn by TTP, ATP, and GTP triphosphates. B) The TTP-kinase activity is optimum at light alkaline pH. C) 43K-rapsyn phosphorylation is dose dependent and saturable (KD~5–10 µM TTP). D) Kinetics of TTP-dependent phosphorylation of 43K rapsyn.

TTP-43K-kinase is not PKA
Although 43K rapsyn is, within the detection sensitivity, the only protein phosphorylated in the presence of 32P-TTP (Figs. 1 and 4 , lane 1), additional proteins, including nAChR subunits, are phosphorylated with 32P-ATP (Fig. 4 , lane 2), in agreement with our former results (16) . 43K rapsyn phosphorylation is saturated with ~25–50 µM 32P-TTP (Fig. 3C ) but is not saturated with 200 µM 32P-ATP. 32P-ATP- and 32P-TTP-dependent 43K rapsyn phosphorylation are specifically inhibited by both TTP and ATP, suggesting the presence of common phosphorylation sites between PKA and TTP-kinase. However, analysis conducted with PKA effectors showed that they are different. PKI inhibited PKA (60±13% inhibition) but not TTP-kinase (6±1% inhibition). Exogenous PKA catalytic subunit increased the ATP-dependent phosphorylation (603±14% 32P vs. 100±23% in control) (ref 16 ; this study) while inhibiting that driven by TTP (41±6% vs. 100±2% in control).



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  		Figure 4. The TTP-dependent kinase that specifically phosphorylates 43K rapsyn is different from ATP-dependent kinases. Autoradiogram of postsynaptic membranes phosphorylated with 32P-TTP or 32P-ATP shows that with 32P-TTP, only 43K rapsyn (arrow) is phosphorylated (lane 1) whereas with 32P-ATP (lane 2) many proteins, including nAChR subunits and 43K rapsyn, are phosphorylated. This suggests the involvement of different kinases depending on the nature of the phosphodonor.

TTP-43K-kinase, a novel kinase?
Putative phosphorylation sites (15) for PKA [Ser-406 (38) ] and tyrosine kinase [Tyr-98, Tyr-189, Tyr-325 (39) ] are present on Torpedo 43K rapsyn. Searches on Prosite (40) and PhosphoBase (41) showed putative sites for CaMKI, CKI, CKII, PKA, PKC, and PKG protein kinases. Of 18 common kinase effectors, only staurosporine caused a slight inhibition (33±3% inhibition/200 nM). Numerous activators or inhibitors of PKA, PKC (TPA, calphostin, GFX), MAP kinase, Protein kinase G, CaM kinase II, JNK2 kinase, cdc2 kinase, MLCK, SAP kinase, and TyrPK (data not shown) did not drastically alter the activity of TTP-kinase, which is likely of a novel type.

Nature of the amino acids phosphorylated with TTP
A 2-dimensional high-voltage electrophoresis of acid hydrolysates of 32P-ATP-dependent, phosphorylated 43K rapsyn (ATP-32P-43K rapsyn) has shown that phosphorylation by PKA occurs predominantly on serine(s) (16) . Similar analysis on TTP-dependent 32P-phosphorylated 43K rapsyn (TTP-32P-43K rapsyn) showed different results, with a faint radioactive signal at serine and a strong one at inorganic phosphate (Pi). No detectable radioactivity was observed at the phosphothreonine (P-Thr) or phosphotyrosine (P-Tyr) region (data not shown). Similar results conducted by immunoblot analysis on TTP-dependent 32P-labeled nAChR-rich membranes with specific antiphosphoamino acid antibodies were obtained (data not shown). Anti-P-Tyr strongly stained several nonradioactive bands, but not 43K rapsyn. This suggests the presence of in situ Tyr-phosphorylated proteins and nAChR-associated protein tyrosine kinases (43) , and also suggests that Tyr is not phosphorylated in 43K rapsyn [but see (44) ]. No staining of 32P-43K rapsyn was observed with anti-P-Thr. Anti-P-Ser faintly stained 43K rapsyn both in control [this is consistent with the presence of in situ P-Ser in 43K rapsyn (16) ] and in TTP-32P- membranes. A stronger staining of 32P-43K rapsyn suggests some phosphorylation on serine driven by TTP and is consistent with the reciprocal inhibition of ATP- and TTP-dependent phosphorylation of 43K rapsyn by TTP and ATP, respectively.

To gain insight into the unexpected high 32Pi content in TTP-32P-43K rapsyn hydrolysate, ATP- and TTP-32P-43K rapsyn were simultaneously hydrolyzed with HCl and analyzed by 1-dimensional electrophoresis. Similar ninhydrin-stained phosphopeptide patterns but different autoradiograms were obtained (Fig. 5 ). ATP-32P-43K rapsyn hydrolysate (lane 1) led to high radioactivity at P-Ser (arrowhead) and low radioactivity at Pi. TTP-32P-43K rapsyn hydrolysate (lane 3) showed very faint radioactivity at P-Ser (arrowhead), and high radioactivity at Pi. The radioactivity in the P-Thr or P-Tyr regions appeared mainly as a trailing background. This confirms serine phosphorylation with ATP and suggests that phosphorylation with TTP occurs predominantly on residues other than serine; furthermore, TTP driven phospholinkages are mainly acid labile.



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  		Figure 5. Phosphoamino acid analysis of phosphorylated 43K rapsyn. 43K rapsyn phosphorylated by 32P-ATP or 32P-TTP were separated by SDS-PAGE, blotted onto PVDF, hydrolyzed in 5.7N HCl (1 h, 105°C), and analyzed for phosphoamino acids by 1-dimensional high voltage electrophoresis on thin-layer cellulose in pH 3.5 solvent. Nonradioactive P-Ser, P-Thr and P-Tyr (lane 2) were run as standards. The ATP-dependent 32P-43K rapsyn hydrolysate (lane 1) shows several main radioactive spots stained by ninhydrin (dotted lines) at phosphopeptide regions and at P-Ser level. This is consistent with serine phosphorylation reported in ref 16 . The TTP-dependent 32P-43K rapsyn hydrolysate leads to a similar ninhydrin-stained pattern (lane 3, dotted lines), but a quite different radioactivity pattern with little radioactivity at phosphopeptide regions, a very faint radioactivity at P-Ser level, and most of the radioactivity at the inorganic phosphate (Pi) region (lane 3). These results show that ATP and TTP drive different phosphorylations on 43K rapsyn.

A pH stability analysis was further performed on ATP- and TTP-32P-43K rapsyn. SDS-PAGE gels containing both phosphoproteins were treated with TCA at 90°C and 32P-quantified (Table 1) . TTP-dependent phosphorylated 43K rapsyn is acid sensitive and the 32P-phosphate loss is a function of time in TCA (50±4 and 16±1% 32P after 5 and 10 min vs. 100±13% for control). In contrast, ATP-32P-43K rapsyn is less sensitive (79±5 and 49±9% 32P after 5 and 10 min vs. 100±8% for control). A similar test conducted at alkaline pH (Table 1) showed a remarkable stability of the TTP-dependent phospholinkages (72±4% 32P after 2 h in 1N KOH at 46°C vs. 100±5% for control) in contrast with the ATP-driven phospholinkages (18±2 vs. 100±6%32P in control). Thus the phosphate links elicited by ATP are acid stable and alkaline labile, a signature of O-linked phosphoamino acids phosphoserine and phosphothreonine (45) . Serine is indeed phosphorylated with ATP (16 ; foregoing text). Conversely the phosphoryl linkages introduced by TTP are acid labile and alkaline stable, a characteristic of N-phosphate linkages at phosphohistidine or phospholysine (45) .


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  		Table 1. Determination of the predominant phosphate links in ATP- and TTP-dependent phosphorylated 43K rapsyn by chemical stability and phosphoamino acid (Paa) analysisa

TTP causes phosphorylation predominantly on histidine residues.
To identify the N-phosphoamino acids in TTP-phosphorylated 43K rapsyn, a TLC of 43K rapsyn hydrolysates was performed in solvent A. Nucleoside diphosphate kinase (NDPK) (46) which autophosphorylates histidine (47) was used as control (Fig. 6A , B , lanes 4). All hydrolysates (Figs. 6A , B , lanes 2–4) displayed radioactive material migrating similarly to phosphohistidine, the highest intensity being observed with the enzymatic hydrolysate from TTP-32P-43K rapsyn (Fig. 6A , lane 2). The low radioactivity at "P-His" in both TTP-32P-43K rapsyn (Fig. 6A , lane 3; Fig. 6B , lanes 2, 3) and ATP-32P-NDPK alkaline hydrolysates (Figs. 6A , B , lane 4), probably derived from P-His partial destruction during hydrolysis. Added phosphohistidine (internal standard) comigrated with the radioactive spots (Fig. 6B , lanes 2–4). These results favor phosphorylation on histidine with TTP.



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  		Figure 6. TLC analysis of TTP-32P-43K rapsyn. Alkaline hydrolysates (3N KOH, 1 h, 105°C) of TTP-32P-43K rapsyn and ATP-32P-NDPK, and trypsin/Pronase digest of TTP-32P-43K rapsyn were resolved by TLC in solvent A, stained with ninhydrin (dotted lines) and autoradiographed. External standards were P-Ser (lanes 1), P-His (lanes 5). A) Trypsin/Pronase digest (lane 2), alkaline hydrolysates of TTP-32P-43K rapsyn (lane 3) and ATP-32P-NDPK (lane 4) all show radioactivity at P-His level. B) P-His (dotted circle) added to alkaline hydrolysates of TTP-32P-43K rapsyn (lanes 2, 3) or of ATP-32P-NDPK (lane 4) comigrates with the radioactive spot. This strongly suggests histidine phosphorylation driven by TTP in 43K rapsyn.

To assess the importance of histidine(s), nAChR membranes were pretreated with DEPC, then incubated with 32P-TTP [DEPC modifies histidines, thus preventing their subsequent phosphorylation (28) ]. 43K rapsyn phosphorylation was effectively decreased in DEPC membranes (20±2% vs. 100±19% 32P in mock membranes).

Partial tryptic digestion conducted on ATP- and TTP-32P-43K rapsyn, followed by 15% acrylamide SDS-PAGE, showed one major radioactive band at ~6.5–15 kDa for ATP- and several radioactive bands from ~6.5 to 35 kDa for TTP-phosphorylated 43K rapsyn (data not shown). Again, this indicates different phosphorylation sites depending on the nature of the phosphodonor. ATP probably leads to phosphorylation mainly on one serine residue; with TTP, one or several histidine residues might be mainly phosphorylated.

TTP is not a phosphodonor for NDPK
NDPK is a highly conserved enzyme that plays a key role in growth and metastasis control (47) . As the enzyme autophosphorylates histidine and presents a broad specificity, phosphorylation was assayed with TTP. NDPK was strongly phosphorylated with 32P-ATP but not with 32P-TTP (data not shown).

TTP, a phosphate donor in the central nervous system (CNS)
Mouse and rat brain membranes incubated with 32P-TTP or 32P-ATP were phosphorylated. However, as with Torpedo postsynaptic membranes (Fig. 7A , B , lanes 1), ATP phosphorylated many proteins in mouse brain membranes (Fig. 7A , lane 2) whereas TTP phosphorylated very few. Two major 32P-labeled bands were observed at ~43–46 kDa (Fig. 7B , lane 2). Phosphorylation was partially inhibited by ATP (Fig. 7A , lane 3) or TTP (Fig. 7B , lane 3). Thus, in vitro, TTP is also a phosphodonor for proteins in the CNS.



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  		Figure 7. TTP is a phosphodonor for brain membrane proteins. Rodent crude brain membrane extracts incubated with 32P-ATP (A) and 32P-TTP (B) were analyzed by SDS-PAGE and autoradiography (molecular mass markers: far left). Torpedo postsynaptic membranes were used as controls. A) Torpedo ATP-32P membranes (lane 1). ATP phosphorylates many proteins in brain membrane extracts (lane 2) and phosphorylation is inhibited by cold ATP (lane 3). B) Brain membranes incubated with 32P-TTP offer a much simpler radioactive pattern, with two major 32P-bands around 46 kDa (lane 2). Phosphorylations are partly inhibited by cold TTP (lane 3). Lane 1: control Torpedo TTP-32P membranes were incubated with low concentrations of 32P-TTP to match the weak brain membrane signals.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Endogenous PKA associated with nAChR-rich membrane preparations phosphorylate 43K rapsyn and other proteins (16) . The essential role of 43K rapsyn in nAChR clustering and postsynaptic structure formation prompted us to search for a specific phosphorylation of 43K rapsyn. By immunoprecipitation and Western blot analysis, we have identified this by using TTP as phosphodonor.

The TTP-dependent kinase(s), (a) novel kinase(s)?
Specific phosphorylation of 43K rapsyn with TTP as phosphodonor occurs at 4–30°C, temperatures compatible with that of the sea water surrounding the Torpedo. 43K rapsyn is localized at the postsynaptic membrane inner face (5 , 6) , hence topologically accessible to the high cytosolic TTP content (~4–30 nmol/g wet tissue; 24, 26). Thus, conditions necessary for a successful endogenous phosphorylation of 43K rapsyn are met, supporting the notion that phosphorylation of 43K rapsyn with TTP as phosphoryl donor occurs in vivo in Torpedo electrocytes.

The phosphorylation is Mg2+ and TTP dependent, with characteristics of an enzymatic reaction driven by endogenous kinase(s) present in the nAChR-rich postsynaptic membrane and specific for TTP, although with some affinity for ATP. They were named ‘TTP-dependent-43K rapsyn kinase(s) or TTP-kinase(s)’.

These TTP-kinase(s) seem to be of a novel type, different from PKA, PKC, or common kinases. Their affinity is not drastically affected by inhibitors of PKA, activators (TPA) or inhibitors (calphostin, GFX) of PKC, or effectors of other common kinases. The question of their classification in a new eukaryotic protein kinase family or as members of known kinase families will be solved with their identification, purification, characterization, and sequencing. However, the possibility of a TTP-dependent autophosphorylation of 43K rapsyn must also be considered.

Histidine phosphorylation of 43K rapsyn
Phosphoamino acid and antibody analysis suggests that besides some minor phosphorylation on serine residues, histidines are predominantly phosphorylated. The inhibition of TTP-dependent 43K phosphorylation by both ATP and TTP suggests that TTP-kinase might share some common phosphorylation sites with PKA. However, since most of the detectable phosphoryl groups introduced by TTP are on histidine(s) and those by ATP on serine (16) , the inhibition through shared serine site(s) should be only partial. The strong inhibition of phosphorylation by high concentrations of heterologous triphosphate ATP may reside on the ability of TTP-kinase to recognize and link either ATP or TTP, although with different affinities. In addition, a modification of the histidine(s) microenvironment brought about by phosphorylation of serine(s) might occur and result in a decrease of histidine phosphorylation by TTP-kinase(s) (Ser-406, a strong PKA consensus site, is close to His-384 and His-387).

Zn2+ modulates the activity of many proteins and may play a role in synaptic transmission (48) ; we have shown that TTP-kinase activity is prevented by 500 µM Zn2+. At its carboxyl terminus, ahead of Ser-406, 43K rapsyn displays two zinc finger motifs that could be important for nAChR clustering (42 , 49 , 50) . In addition, two conserved histidines, His-384 and His-387, are present in the zinc finger motifs. In vitro, 43K rapsyn binds Zn2+ probably through the two histidines (42) , which consequently might become less available for an eventual phosphorylation. Binding of Zn2+ might also elicit conformational changes inducing a decrease of 43K rapsyn accessibility for histidine phosphorylation by TTP-kinase. If Zn2+ binds to 43K rapsyn in vivo, the zinc finger domain might play a role in regulation of the protein phosphorylation state. An intrinsic sensitivity of TTP-kinase to Zn2+ should account only partially at these Zn2+ concentrations.

Tryptic digestions suggest that ATP probably leads to the phosphorylation of one main serine (possibly Ser 406, a strongly conserved PKA consensus site) whereas TTP may drive phosphorylation on one or several histidines. 43K rapsyn possesses 13 histidines that are potential candidates. Ten of these residues are conserved among chick (51) , human (52) , mouse (53) , Torpedo (54) , Xenopus (55) . Some of the conserved histidines also have their neighboring sequence conserved: e.g., His-154; His-239; His-256; His-384 and His-387 of the tandem zinc fingers. Highly homologous, although not totally conserved, neighboring sequences of His-53, His-329, His-348, and His-353 are located in regions possibly important for 43K rapsyn functions. His-53 is present in a domain involved in 43K rapsyn self-association (56) . Mutations of His-384 and His-387 reduce 43K rapsyn ability to form clusters (42) . His-348 and His-353 are located between these two important regions of 43K rapsyn. The neighboring sequence RYAH of His-154 is conserved in K. aerogenes (57) , N. meningitidis (58) , and E. coli (59) and has been identified as a phosphorylation site essential for polyphosphate kinase activity in prokaryotes (59) . The phosphate in phosphohistidine is of a high energy state and is often further transferred to an acceptor residue (on the same or another molecule), an important step in the two-component signaling mechanisms in cell regulation (60 , 61) . It will be of interest to identify the histidine(s) phosphorylated by TTP and determine by mutational analysis whether a similar role of histidine phosphorylation can be related to 43K rapsyn phosphorylating and clustering functions in the postsynaptic domain.

TTP-dependent phosphorylation of 43K rapsyn, TTP-kinase(s), and nAChR clustering.
43K rapsyn is present as cytosolic and membrane-attached pools in a ratio depending on tissue maturation (37) . The question of a relationship between 43K rapsyn phosphorylation and its cellular compartmentalization is raised.

nAChR phosphorylation has been reported in several instances (62 63 64 65) . 43K rapsyn regulates tyrosine phosphorylation of several postsynaptic membrane proteins, including the nAChR ß-subunit (44) . nAChR tyrosine phosphorylation regulates the rapid rate of receptor desensitization and may play a role in nerve-induced nAChR clustering (65 66 67) . Two protein tyrosine kinases associated with the nAChR have been cloned in Torpedo electrocyte (43) . The TTP-kinase(s) that drive specific phosphorylation(s) of 43K rapsyn predominantly on histidine(s) are also present in nAChR-rich postsynaptic membrane. Their purification (see above) will allow further analysis of their possible involvement in the cascade responsible for nAChR phosphorylation and clustering.

Of 18 common protein kinase effectors tested, only staurosporine, a potent but nonspecific protein kinase inhibitor (68) , causes some inhibition. Staurosporine also inhibits agrin-induced nAChR phosphorylation and aggregation (69) . This raises the question of an eventual connection between these events and 43K rapsyn phosphorylation via TTP. Agrin plays an important role in NMJ differentiation (70 71 72) . Cotransfected 43K rapsyn causes clustering of dystroglycan, the agrin binding component of the dystrophin glycoprotein complex (73) . It also induces clustering and activation of MuSK, a synapse-associated muscle specific kinase (74 75) and component of the agrin-MuSK-MASC signaling complex responsible for nAChR clustering and postsynaptic differentiation. It will be interesting to study the influence of TTP-dependent phosphorylation on the involvement of 43K rapsyn in the agrin-dystroglycan-MuSK-MASC cascade.

Phosphorylation of 43K rapsyn through TTP also suggests the possibility of an interplay between 43K rapsyn and the thiamine pathway in excitable cells. Increased nervous activity leads to dephosphorylation from TTP and TDP to TMP and thiamine (18 , 76) , and deafferentation of the cerebellum decreases turnover of thiamine phosphate derivatives (77) .

Extension of TTP-dependent phosphorylation to other eukaryotic systems. TTP, a phosphodonor for mammalian synaptic proteins?
Occurrence of the TTP-dependent phosphorylation of 43K rapsyn at the vertebrate NMJ remains to be defined as well as its potential role in protein–protein interactions, nAChR aggregation, and stabilization at the NMJ.

Although TTP is not a phosphodonor for NDPK histidine [despite NDPK’s broad specificity (47) ], TTP can cause phosphorylation of proteins present in rodent central nervous membranes. TTP thus represents a valuable tool for defining a possibly novel phosphorylation pathway specific for synaptic proteins.

43K rapsyn causes clustering of cotransfected GABAA receptors (78) and is present in chick ciliary ganglion neurons (51) . Analysis of a possible involvement of TTP as a phosphodonor in the phosphorylation of brain receptors, chick ciliary ganglion 43K rapsyn and putative brain 43K rapsyn homologues should permit a better understanding of the molecular processes underlying synaptic functions.

The novel and specific TTP-dependent phosphorylation of 43K rapsyn highlights the possible importance of TTP-dependent phosphorylation in the modulation of synaptic organization. It also opens up a new phosphorylation pathway for synaptic proteins that differs from the more classical purine triphosphate pathway.

Histidine phosphorylation in eukaryotes
In eukaryotes, phosphorylation has been estimated to occur predominantly on serine residues (~90%), ~9.9% on threonine residues and only ~0.1% on tyrosine residues despite its key role in cell modulation (32). In prokaryotes phosphorylation on histidine (~6%) has been documented and often related to regulation processes (61) . Fewer cases are reported in eukaryotes (79) . Our findings add a new example of histidine phosphorylation on a synaptic protein in eukaryotic cells, thus broadening the importance of histidine in eukaryotic phosphorylation.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
We show for the first time the phosphorylation of proteins by TTP, a triphosphate component distinct from ATP or GTP. The protein kinase(s) seems to be of a novel type. The amino acid phosphorylated also is not common in eukaryotes, since it is mainly histidine. We show that the protein target in this phosphorylation is 43K rapsyn, which is specifically present in postsynaptic membranes and essential for the synapse to function properly. This new type of TTP-dependent phosphorylation is not restricted to 43K rapsyn, but is also observed with mouse and rat brain membranes. This favors a more general use of TTP as a phosphate donor in a novel phosphorylation pathway. It also opens up a new topic in the phosphorylation domain.


   ACKNOWLEDGMENTS
 
We thank Pr. T. Grisar for his continual support, Drs. P. J. Corringer, Y. Paas, and L. Marubio for helpful discussions and reading of the manuscript, Drs. O. Acuto and V. Di Bartolo for help and access to their high-voltage electrophoresis device, and Dr. M. Véron and collaborators for a generous gift of NDPK. This research is supported by grants from the Collège de France, the Association Française contre la myopathie, a Biotech contract from the Commission of the European Communities to J.P.C., and grants (1.5.083.98) from the National Funds for Scientific Research, Belgium (FNRS) and the Fondation Léon Fredericq to L.B. L.B. is research associate at the FNRS.


   FOOTNOTES
 
Received for publication March 14, 1999. Revised for publication November 1, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
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