John Scott, Vollum Institute) was treated to remove bound CAMP, as described previously for bovine RIa (29), and used immediately for the titration of C subunits ...
” H E JOURNALOF BIOLOXCAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 269, No. 38, Issue of September 23, pp. 23722-23730, 1994 Printed in U.S.A.
Differential Phosphorylation of Neuronal Substrates by Catalytic Subunits of ApZysia CAMP-dependent Protein Kinase with Alternative N Termini* (Received forpublication, January 21, 1994, and in revised form, June 14, 1994)
Rekha G. Panchal, Stephen Cheley, and Hagan Bayley From the Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545
CAMP-dependent protein kinase (PKA) is an impor- mitters suchas serotonin results in the temporary elevation of of PKA tant participant in neuronal modulation: the ability of intracellular CAMP(3,4) and the consequent activation facilineurons to change their properties in response to exter- (4,5). Activated PKA participates in both the short term by mild stimulation nal stimuli. In Aplysia mechanosensory neurons, PKA tation of minutes to hours that is mediated plays roles in both short and long term presynaptic fa-of sensory cells and thelong term facilitation lasting days ariscilitation, which is asimplemodelforlearningand ing from strong stimulation. Bothshort and long term facilitamemory. PKA in Aplysia is a collection of structurally tion are evoked by a common extracellular signal, serotonin, and functionally diverse regulatory and catalytic (C) and they share features such as the increasedexcitability of the subunits. We have argued thatthis diversity may in part affected sensory neurons(6) and enhanced transmitter release account for the ability of the enzyme to take part in (7). Nevertheless, the two processes can be separated. Long neuronal events that are spatially and temporally sepa- term facilitation requires the synthesisof new proteins (8)in rated. Here, we add credence to this hypothesis by show- response t o which PKA activity persists, despite the returnof ing that C subunits of Aplysia PKA with alternative N intracellular CAMPto the basal concentration(9). The persisttermini target different substrates in subcellular frac- ent kinase activity is mediated by ubiquitin-dependent breaktions from Aplysia neurons, despite their similaractions down of PKA regulatory subunits (10). Concurrently, synaptic on synthetic peptide substrates. Purified recombinant growth is activated leading to changes in the number and patC,.,NlAl, which has an N terminus that is homologous tern of synaptic contacts (2,11, 12). These events are initiated to the myristylated sequence described in mammals, by the PKA-mediated phosphorylation of transcription factors such as CREB (CAMP response element-binding protein) locatalyzes the formation of two phosphoproteins of 24 which has a cated in the nucleus of sensory cells (5, 13, 14). By contrast, and 8 kDa more rapidly than Cm,,N2A1, distinct N terminus weaklyrelated to that of the yeast short term changes involve the rapid phosphorylation of ion TPKl gene product. The 24-kDa phosphoprotein, but not channels orassociated proteins in the presynaptic terminals of the 8-kDa species, is detected in taxol-stabilized micro- sensory neurons (15-19). tubules, suggesting that it is associated with the cyHow can a single enzyme, PKA, participate in thecontrol of toskeleton. CmL,N2A1, in contrast, generates a 55-kDa events that are spatially and temporally separated? Possible phosphoprotein that is not observed with C,,,NlAl. solutions include: selective neural inputs to the sensory cell The 5s-kDa species is found in the detergent superna- body and its terminals (20,211, bidirectionalsignaling between tant of the cytoskeleton fraction. Differential targeting the nucleus and terminals through fast axonal transport(22, of substrates by C subunits of PKA may therefore con- 23), localized elevations of CAMP in response to cell-wide stimutribute to the ability of this kinase to play multiple roles lation (41, and functional diversity inPKA (24). It is the latter in neuronal modulation. on whichwe have focused, although it seems likely that various mechanisms cooperate in the control of facilitation. PKA in Aplysia is structurally diverse. At least sixforms of In sensory neurons of the marine mollusk Aplysia, CAMPregulatory (R) (24-26) and four forms of catalytic (C) subunit dependent protein kinase (PKA)’ plays several roles in the (27, 28) have been identified. We are attempting todefine the presynaptic facilitation that contributes to behavioral sensitiextent towhich the structural diversity is translated funcinto zation, a simple form of learning and memory (I,2). Stimulational diversity in the form of substrate specificity (291, regution of Aplysia sensory neurons with facilitatory neurotranslation (24, 25), subcellular location (24, 251, and subcellular translocation. In this paper, we report an investigation of the * This work was supported by National Institutes of Health Grant NS26760. The costs of publication of this article were defrayed in part neuronal substratesof two forms of the C subunit with differby the payment of page charges. This article must therefore be hereby ent N termini (28). marked “aduertisement” in accordancewith 18 U.S.C.Section 1734 The four formsof C subunit areencoded by a singlegene and solely to indicate this fact. The abbreviations used are: PKA, CAMP-dependent proteinkinase; arise through alternative RNA splicing and alternative proC subunit, catalytic subunitof PKA, C,,,NlAl, the N l A l splice form moter use, which generate two internal amino acid cassettes combinaof the C subunit from Aplysia; C,,,NSAl, the N2A1 splice form of the (A1 and A 2 ) and two N termini (N1 and N2) that are C subunit from Aplysia; Ht 31, a human thyroid PKAanchoring protein; torially expressed(28). Earlier studies have shown that A1 the MOPS, 3-(N-morpholino)propanesulfonicacid; N1 and N2, alternative cassette andA2 cassette formsof the C subunit have different N termini of the C subunit of Aplysia PKA, PIPES, piperazine-N,N“ bis(2-ethanesulfonic acid); P, pellet fraction; PKIP, PKA inhibitor pep- specificities toward peptide substrates in vitro and differ in tide; PMSF, phenylmethanesulfonyl fluoride;R subunit, regulatory sub- afinity for acommon R subunit (29). These propertiesof C may unit of PKA, RI, type I regulatorysubunit of PKA, RII,type I1 contribute t o the fine-tuning of neuronal responses (29). For regulatory subunit of PKA; type N4 RI subunit from Aplysia example, holoenzymes with differentaffinities for CAMP(a conPKA; S , supernatant fraction; D m ,dithiothreitol; BSA, bovine serum albumin; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyllglycine; sequence of the differing affinities for a common R (29)) and different cellularlocations with respect to key substrates (241, PAGE, polyacrylamide gel electrophoresis.
23722
Neuronal Substrates of Aplysia PKA could modulate one set of ion channels at a low intracellular CAMPconcentration and another setat a higher CAMPlevel, a phenomenon that has been observed in Aplysia sensory neurons (18). Separate functions for the alternative N termini have not been reported. The N1 terminus of the Aplysia C subunit is homologous to the classical myristylated mammalian N terminus, whereasN2 weakly resemblesthe N terminus of the yeast TPKl geneproduct and theN terminus of a C subunit from the nematode Caenorhabditis elegans(28).The crystal structureof the mouse C a subunit (30) reveals that the N terminus is attached at a point distant from the active site andis therefore unlikely t o be involved in the direct modulation of catalytic activity. One hypothesis is that the alternative N termini, N1 and N2, are involved in substrate targeting, inwhich a C subunit released from holoenzyme by the action of CAMPbinds a t a cellular location in the vicinity of key substrates or to the substrates themselves. Such an interaction could orient the active site and thus enhance the phosphorylation of target substrates on selected residues (28).To test this idea,we have now used recombinant Aplysia C subunits with the alternative N termini t o phosphorylate substrates inAplysia neural extracts. N1 and N2 forms of C, with a common internal cassette (Al), indeed phosphorylate neuronal substrates at different rates.
23723
droxymyristicacid,inmethionineand serum-free Grace’s medium. Without changing the medium, the cells were then labeledfor 4 h with a 35S-protein labeling mix (DuPont NEN, NEG-072: 1175 Ci/mmol, 1 mCi/ml of medium). The cells treated with 2-hydroxymyristic acid had an unhealthy appearance. After lysis in 20 mM Tris.HC1, pH 7.4, 2 mM MgCl,, 1m DTT, 1mMATP, 0.5 mM PMSF, and 0.1% Nonidet P-40 (500 pl), supernatants from a 16,000 x g centrifugation were mixed with PKIP (PKA inhibitor peptide)-AfE-Gel 10 (50 pl), which retains the C 1h at 4 “C, the PKIP-Affi-Gel was washed subunit of P K ~ ( 2 9 , 3 5 )After . with 15 vol of buffer containing 20 mM Tris-HC1, pH 7.4, 2 mM MgCl,, 200 mM NaCl, 1 m DTT, 0.5 mM PMSF, and 0.1% Nonidet P-40. The C subunit was then eluted with electrophoresis sample buffer, subjected to SDS-PAGE (36) in a 38-cm-long 10% gel for 24 h a t 200 V, and visualized by autoradiography. I n Vitro Dunslation of Aplysia C Subunits The construction of a pT7 expression vector containing the C, ANlAl cDNA insert has been described (29). A similar N2A1 construct was made by cutting the CWL,N2A1 insert from Bluescript KS(-) (see above) with NdeI and HindIII and ligating it into thepT7 vector that had been prepared with the same enzymes. The pT7 constructs were linearized with Hind I11 and transcribed in uitro with T7RNA polymerase, in the presence of 1 m m7Gt5‘)ppp(5’)G. The integrity of the transcripts was verified by electrophoresis in a denaturing formaldehyde-agarose gel. A nuclease-treated rabbit reticulocyte lysate was preBSA or BSA treated for15 min a t room temperature with fatty acid-free complexed with 2-hydroxymyristic acid. The RNAs were then translated for 1h at 30 “C, in thepresence of [35Slmethionine(1175 Ci/mmol, 1 pCi/pl translation mix).
EXPERIMENTAL PROCEDURES Determination of kc,, a n d K,,, Values for Kemptide Construction of a Baculouirus Containing a CML.,N2A1 cDNA kc,, and K, values for the synthetic peptide substrate Kemptide The C,,.,NBAl cDNA was assembled in BluescriptKS(-) by three- (LRRASLG) were determinedas described previously 129,37). Reaction way ligation of the following DNAs. 1)The 5”coding region of an N2A1 mixtures contained 50 mM MOPS, pH 6.8, 0.1% (wh) Nonidet P-40, 15 cDNA, encompassing the N2 codons, generated by polymerase chain mM MgCl,, 2.5 mM 3-isobutyl-l-methylxanthine, 100 pg/ml BSA, 1.56 to reaction from the plasmidAC-A2 (28) by using the 5’ primer HB217 5’ 200 p Kemptide, and 100 p~ ATP containing [Y-~~PIATP at 550 dpml GCGCAAGCWGGATCCATATGGCTGATATTATTCACAAG 3‘, which pmol. contains 5’ HindIII, BamHI, and NdeI restriction sites and the first seven codons of N2 and the 3’ primer HB218 5’ GCGCGAATTCTTTTitration of C Subunits with CAMP-free R Subunits GAAGAACTCG 3’, which is complementary to the N2A1 coding se(24) was Qpe I Subunit-Apurified Aplysia type I R subunit(R,,,) 5’ end of the quence and encompassesa n EcoRI restriction site near the 50 mM MOPS, pH6.8, subjected t o 2-fold serial dilutions in assay buffer: exon 2 sequence. The polymerase chain reaction product was digested 0.1% ( w h ) Nonidet P-40, 15 mM MgCl,, 100 pg/ml BSA. After the addiwith HindIII and EcoRI for ligation. 2) The A1 coding region excised or C,,,NPAl, to 100 PM) tion, t o each tube, of C subunit (C,,,NlAl from the plasmid pT7-Cm,,A1 (29) by using the EcoRI site and a and [Y-~~PIATP (550 dpdpmol) in assay buffer, the mixtures (40 p1) BamHI site present in pT7 the plasmid downstreamof the cDNAinsert. were incubated for 20 min at 30“C in the absence of substrate. Phos3) Bluescript KS(-) linearized withHind111 and BamHI.After assembly, phorylation was then initiatedby adding Kemptide (100 p ~ )followed , the integrity of the N2A1 insert wasconfirmed by DNA sequence anal- by incubation for a n additional 20 min at 30 “C. Phosphorylated Leuysis. To obtain a recombinant baculovirus, the N2A1 cDNA was excised Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)wasdetermined as described from Bluescript KS(-) with BamHI and inserted after the polyhedrin (29, 37). vector pVL941 (31), promoter at the uniqueBanHI site in the transfer Qpe II Subunit-Recombinant murine RIIa (gift of Daniel Carr and yielding pVL-C,,,NBAl. Recombinant virus derived by cotransfection John Scott, Vollum Institute) was treated to remove bound CAMP, as of Sf9 cells withAcMNPV (baculovirus) DNA and pVL-C,,,NBAl was described previously for bovine RIa (29), and used immediately for the produced by three rounds of limiting dilution (29, 32). titration of C subunits as described above for RAPL.N4. Expression a n d Purification of Aplysia C Subunits Tissue Fractionation C,,.,NlAl and C,,,N2Al were expressed in insect Sf9 cells and Tissue fractions were stored at -70 “C or used directly for in uitro then purified, as described earlier for other Aplysia C subunits (29). The phosphorylation reactions, with similar results. The abdominal, pleupurified kinases were storeda t -70 “C in 20mM TrisSHC1, pH 7.4, 1mM ral-pedal, cerebral, andbuccal ganglia wereremoved from Aplysia caliDTT, 1 mM EDTA with glycerol added to 10% (v/v). Protein concentra- fornica (200-250 g, Marinus) and the connective sheaths were microtions were determined with a solid-phase colloidal gold binding assay dissected away toyield neuronal components (cell bodies and neuropil) (33). (24, 38). An unfractionated extract of neural tissue was obtained by homogenizingneuronalcomponentsinbuffer containing10mu Solubilization of 2-Hydroxymyristic Acid Tris.HC1, pH 7.4, 1 mM MgCl,, 1 mM EDTA, 1 mM D m , 5 mM NaF, 100 2-Hydroxymyristic acid (Sigma, H6771) was added t o Sf9 cells in p sodium vanadate,1mM PMSF, and 10 p~ leupeptin. The preparation culture or to rabbit reticulocyte lysate as serum albumin(BSA) of subcellular fractionsfrom Aplysia nervous tissue hasbeen described a bovine complex. The fatty acid was first coated onto Celite at 1 mmoVg (34). (24). In brief, the membrane pellet fraction (P) was a 150,000 x g pellet Coated Celite (50 mg) was mixed with fatty acid-freeBSA (Boehringer solubilized in 50 mM Tris.HC1, pH 7.4, 1 mM MgCl,, 1 mM EDTA, 1 mM Mannheim, 100375: 250 mg in 1 ml of water) and allowed to stand for DTT, 5 mM NaF, 100 p sodium vanadate containing1% (w/v) Nonidet 1 h at room temperature, afterwhich the Celite wasremoved by filtraP-40. The corresponding membrane supernatant fraction (S) was in 10 tion through a 0.22-pm polyvinyldifluoride filter. The fatty acid-BSA mM Tris.HC1, pH 7.4, 1 mM MgCl,, 1 mM EDTA, 1 mM DTT, 0.25 M complex was added to cells or reticulocyte lysate a t 1-5 mg of B S N ml, sucrose, 5 mM NaF, 100 p~ sodium vanadate, 1 mM PMSF, and 10 p~ i.e. a maximum final concentrationof 0.2-1 m 2-hydroxymyristic acid. leupeptin. Membrane fractions of ovotestis and buccal muscle were prepared in the same way, from the same wet weights of tissue. The 35S-Labeled AplysiaC Subunits from Sf9 Cells detergent(NonidetP-40)-extractedcytoskeletonfraction(P)wasa sf9 cells (1 x lo6)in “normal” medium (1ml, Ref. 29) were infected 16,000 x g pellet suspended in0.1 M PIPES, pH 6.9,1mM MgSO,, 2 mM with AcMNPV-C,,,NlAl or AcMNPV-CM,,N2A1 at a multiplicity of EGTA, 1.0 M glycerol, 5 mM NaF, 100 p~ sodium vanadate,1 mM PMSF, 10 plaque forming unitskell. At 68 h post-infection, the cells were in- and 10 p leupeptin. The corresponding cytoskeleton supernatant(S) cubated for 1 h with fatty acid-free BSA or BSA complexed with 2 - h ~ - was in the same buffer containing 0.1% (w/v) Nonidet P-40. Taxol-
23724
Substrates Neuronal
stabilized microtubules (P) were suspended in 0.1 M PIPES, pH 6.6, 1 mM EGTA, 1 mM MgSO,, 10 p~ leupeptin, and 1 mM PMSF. The corresponding microtubule supernatant (S)contained the same buffer with 1 mM GTP and 20 PM taxol.
PKA
of Aplysia
C
B
A
1
Sf 9
-
+ 2
IVT
1
In Vitro Phosphorylation
2 s
-e*
-1
+ ---
Sf9
+ HMA 2
For phosphorylation of substrates in neuronal extracts C,,,NlAl “ and C,,,,NBAl concentrations were normalizedfor Kemptide phosphorylating activity, which was determined by using saturating substrate and CA,,~,N2A1are (200 PM). Because the k,, values for C,,,,NlAl :.a similar, the protein concentrations of C subunits used for phosphorylN1 N2 N1 ation were also similar (see “Results”). Phosphorylation reactions were FIG.1. CmLANIA1expressed in Sf9 cells or reticulocyte lysate carried out in volume a of 30 pl containing 50 mM MOPS, pH 6.8.15mM MgCI,, 2.5 mM 3-isobutyl-l-methylxanthine,0.1% (w/v) Nonidet P-40, is N-myristylated. A, Sf9 cells infected with AcMNPV-C,plr,NIA1 100 phi sodium vanadate, 5 mM NaF, 3.3 pmol[y-s2PlATP (3000 Ci/ were pulse-labeled with %-amino acids either during treatment w t h mmol) with purified recombinant C subunit (0.86 ng, C,,,,NlA1; 0.92 fatty acid-free BSA (lane 1 ) or BSA complexed with 2-hydroxymyristic or with no added enzyme. Reactions were initiatedby acid ( E ” : 1 mg BSNml) (lane 2). C subunits were enrichedfrom cell ng, C,,,,N2Al), adding membrane, cytoskeleton,or microtubule fractions or the corre- extracts by using PKIP-M&Gel. Samples weresubjected to prolonged of sponding supernatants (5.0 pgof protein) and incubated for 5 min a t SDS-PAGE and visualized by autoradiography. The 35-45-kDa range M, 30 “C. The reactions were terminated by the addition of SDS-PAGE the gel is shown. Assignments are:N, nonmyristylated C,,,,NlAl; myristylated C,,,NIAl. B , an RNA transcript encoding C4~,,,N1A1 sample buffer (36),followed by heating at100 “C for 5 min. was translated in a rabbit reticulocyte lysate containing [‘ Slmethlonine in the presenceof fatty acid-free BSA (lane I ) or the 2-hydroxyDicine-SDS Gel Electrophoresis myristic acid-BSA complex (5 mg of BSNml; lane 2). The translation To resolve polypeptides ranging in mass from 60 to 8 kDa, samples products were subjectedto SDS-PAGE and visualized by autoradiograwere separated in a Tricine-SDS gel system (39). The gels contained phy as in A, but without PKIP-MI-Gel enrichment. C , Sf9cells infected at 3% ofthe monomer (10% T, with AcMNPV-C,,N2Al 10% total acrylamide, with bisacrylamide were treated asdescribed in A, except that 3% C). Molecular weight markers (Bio-Rad) were ( M J : rabbit muscle the concentration of the BSA-2-hydroxymyristic acid complex was 5 mg phosphorylase b (97,400), bovine serumalbumin (66,2001, henegg of BSNml (lane 2). white ovalbumin (45,000),bovine carbonic anhydrase (31,000), soybean trypsin inhibitor (21,5001, and hen egg white lysozyme (14,400). I4CLabeled markers (Life Technologies, Inc.) were ( M J : myosin heavy 100 A b (97,400), bovine serumalbumin chain(200,000),phosphorylase (68,000). ovalbumin(43,000),carbonicanhydrase (29,000), p-lacto% *O globulin (18,400), and lysozyme (14,300).Theapparentmolecular N1 ?weights of the phosphoproteins identified in this paper are based on 3 60 N2 ‘1 electrophoretic mobility in the Laemmli SDS-PAGE system (36). m.
8 40
Proteolysis of Phosphorylated Aplysia RII Subunits in aNeuronal Membrane Extract The detergent-solubilized membrane fraction from Aplysia nervous tissue (160 pg of protein) was phosphorylated in the presence of 13.2 pmol of [y-”P]ATP (3000 Ci/mmol) and C,plc,NIA1 (6.9 ng) for 5 min a t 30 “C in a volume of 200 pl. The solution was then added to 100 p1 (settledvolume) of CAMP-agarose beads(C8-linked,Sigma A0144), which had been preequilibrated with binding buffer (10 mM Tris.HC1, 100 mM NaCI, pH 7.4). The mixture was placed on a rotator for 1 h at 4 “C. The beads were thencollected by centrifugation a t 16,000 x g for mM Tris.HC1, pH 7.4, containing 1.5M NaCl(3 30 s and washed with 10 x 1 ml) followed by 10 mM Tris.HC1, pH 7.4, without salt (3 x 1 ml). Phosphorylated RII subunits were elutedfrom the washed beads with 10 mM CAMPin 10mM Tris.HC1, pH 7.4 (2.5 ml). The eluate was rotary evaporated to a volume of 100 pl. A sample (10 pl) was mixed with unfractionated Aplysia neuronal extract (10 pg of protein) in buffer containing 10 mM Tris.HC1, pH 7.4,l mM EDTA, 1mM MgCI,, 1mM DTT (100 1.11). The mix was incubateda t 30 “C and samples(10 pl) were taken a t 5, 15.30 and 45 min,mixed with an equal volume of 2 x SDS-PAGE sample buffer (36), and heated a t 100 “C for 5 min before electrophoresis in theTricine-SDS gel system (39).
Phosphopeptide Mapping by Limited Proteolysis Aplysia neuronalmembraneproteinswerephosphorylatedwith CAP,,N1A1 kinase as described above and separated ina 10% T, 3% C
-
rp
20
1 / 7 ; ”n/
0 -3
-2
100-
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0
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-
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-
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3 60‘1 -u
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FIG.2. CmLANIA1and CmLAN2A1bind R subunits with similar A, titration with recombinantAplysia RAPLN4 (a type I subaffinities. Tricine-SDS gel. The dried gel was subjected to autoradiography. The unit); B , titrationwithrecombinantmurineRIIa.Recombinant bands corresponding to the24-kDa phosphoprotein (see “Results”) and CApLANIA1 (0) and CAp,,N2A1 ( . were ) treated with R subunits as the 50- and 47-kDa phosphorylated forms of the RII subunit (24) were described in thetext. Residual kinase activity was assayed with 100 p~ cut from the gel and rehydrated in the wells of a second Tricine-SDS gel, Kemptide. The data displayed are from representative experiments. containing 16% total acrylamide with bisacrylamidea t 6% of the monomer (16% T, 6% C), in buffer (100p1) containing 100mM Tris.HC1, pH forms of the RII subunit (24) and the 8-kDa protein (see “Results”) were 6.8, 10% glycerol, and 20 pg of staphylococcal V8 protease. After 1 h, located by autoradiography and cut from the dried blot. The nitroceltwice for 10 minwith 20% aqueous electrophoresis was carried out a t 30-mA constant currentfor 24 h. The lulose fragments were washed gel was fixed, dried, and subjected to autoradiography. L-1-tosylamido-2-phenylethyl methanol,air-dried,andtreatedwith chloromethyl ketone-trypsin (10 pg, Promega V5111) in 200 pl of 100 Phosphopeptide Mapping afcer Exhaustive Dypsin Deatment mM ammonium bicarbonate and acetonitrile(955). After 72 h a t 37 “C, Aplysia neuronal membrane proteins were phosphorylated C with , the supernatants were lyophilized, redissolved in water (10 pl), and a 10% ANlAl (see above). The samples were electrophoresed in T, 3% C applied to prewashed silicagel TLC plates. Phosphopeptides were reMcine-SDS gel and the separated polypeptides transferred to a nitro- solved by ascending chromatography for 1h in ethanol:water:15M amcellulose membrane by electroblotting. Bands corresponding to the 24 monium hydroxide (7:2:1 by volume). The dried plate was subjected to kDa phosphoprotein (see “Results”), the 50- and 47-kDa phosphorylated autoradiography.
Neuronal Substrates of Aplysia PKA
C
B
A
23725
I
M
1
2
3
M
+I
4 m-
+
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1
M
+ CAMP 2
45
66 97
45
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22 31
66 -
14 -
-
45 -
+-
45
22 -
-
22 14 31
14 -
D
PH
31 -
b
22
-
14 -
D
FIG.3. Phosphorylationof neuronal substrates byC subunits of ApZysia PKA. Key: V, pp24; D, pp8; M,low molecular weight markers. A , differential phosphorylation of neuronal substrates by C,,NlAl and C,,NPAl. Autoradiogram of a 10% T, 3% C Tricine-SDS gel. A solubilized membrane fraction from Aplysia nervous tissue was phosphorylatedin presence of [Y-~*P]ATP and in the absence (lane 1)or presence of recombinant C,,,NlAl (lane 2) or CAP,N2A1 (lane 3). Equal amountsof membrane protein(5 pg) and equal Kemptide units of each enzyme were usedin thephosphorylation reactions,which were performed as described under "Experimental Procedures."B, two-dimensional separations of neuronal substrates phosphorylated by C,,,NlAl and CML,N2A1. Autoradiograms of isoelectric focusing-PAGE gels displaying solubilized neuronal membrane proteins (5 pg)phosphorylated in presence of [Y-~~PIATP and equal kemptide unitsof purified recombinant C,,,NlAl (upper panel) or CApL,N2A1 (lower panel).C , phosphorylation of neuronal substratesby endogenous PKA. Autoradiogram of a 10% T,3%C Tricine -SDS gel showing the generationof pp24 and pp8 by endogenous PKA activated by CAMP. A solubilized membrane fractionfrom Aplysia nervous tissue was phosphorylated withoutexogenous kinase in the presenceof [ Y - ~ ~ P ~ A and T Pin the absence(lane 1) or presence (lane 2 ) of 100 PM CAMP. RESULTS
The C , , , N I A l Subunit of AplysiaPKAIs Myristylated when Expressed in Insect Sf9 Cells-Although proteins have been reported tobe correctly N-myristylated when expressedin Sf9 cells (40), it was important to check this for C,,,NlAl. Furthermore, theincorporation of [3H]myristate,which is often the sole criterion for myristylation, does not revealthe extentof acylation. Because insufficient C,,NlAl was available for analysis by mass spectrometry, we chose to determine the extent of N-myristylation by seeking a diagnostic shift inelectrophoretic mobility between the myristylated and unmyristylated forms of the C subunit. This approach has been used ~ previously. For example, when a cell line expressing ~ 5 6 ' 'is treated with the N-myristyltransferase inhibitor 2-hydroxymyristate, a nonmyristylated form of ~ 5 is expressed, 6 ~ ~ which has reduced electrophoreticmobility on SDS-PAGE (34). In this case, the shift is probably accentuated, because phosphorylation of the protein is altered (34) and palmitylation is prevented (41). With the aid of PKIP-Affi-Gel, C,,,NlAl, pulse-labeled with [35Slmethionine, was recovered from Sf9 cells that had been treated with 2-hydroxymyristate. After extended SDSPAGE, this polypeptide migrated slightly moreslowly than the The majority of the C,,,NlAl from untreated cells (Fig. a). same shift in mobility was seen when 35S-labeledC,,,NlAl was prepared by in vitro translation in a rabbit reticulocyte lysate in the presence or absence of 2-hydroxymyristate (Fig. 1 B ) . N-Myristyltransferase is highly active in reticulocyte ly-
sates (42, 43). Based on densitometer scans, we estimate that 280% of the recombinant C,,,NlAl subunits from Sf9 cells are acylated. Because C,,,NPAl exhibited no electrophoretic shifts inparallel experiments (e.g. Fig. lC), itis unlikely that it is myristylated, which is not surprising as the penultimate amino acid residue is alanine (41). Both C,,,NlAl and C,NBAl expressed in vitro with an Escherichia coli S30 transcriptiodtranslation system exhibited slightly greater mobilities than the Sf9 or reticulocyte lysate proteins expressed in theabsence of 2-hydroxymyristate (data not shown). It is possible that thisreflects the lack of autophosphorylation of the Aplysia proteins when expressed in the E. coli cell-free system. Accordingly, attempts to obtain active C,,,NlAl and Cm,,N2A1 from E. coli cells have been unsuccessful. Phosphorylation of the C subunits from Sf9 cells is likely to be on Thr-199 and Ser-340 (44),which correspond to residues Thr-197 and Ser-338 of the mammalian Ca subunit (27). C,,,NlAl lacks Ser-10 found in Ca, which slowly autophosphorylates undercertain conditions (44). Interestingly, Ser-10 is replaced with the negatively charged asparate in C,,,NlAl. C Subunits ofAplysia PKA with Alternative N Termini Phosphorylate a Synthetic Peptide Substrate with Similar Kinetic Parameters and Bind R Subunits with Similar Affinities in Vitro-Kinetic constants for purified recombinant C,,NlAl and C,,,N2Al were measured withthe syntheticpeptide substrate Kemptide in a standard assay (29). The two forms of C had similar K,, values (k,,Nl/kC,,N2 = 1.12 * 0.02, n = 4). The
Neuronal Substrates of Aplysia PKA
23726
1.00 4
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PP24
A
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5B 0.80
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lime k=ecl
60
FIG.4. Relative rates of phosphorylation of substrates by C,,,NlAl and CmL,N2A1 in neuronal membrane extracts. A , typical densitometry scan after phosphorylation by C,,,NlAI (top) and C,,,NSAI (bottom). Key: V, pp 24; V, pp 8. B , rate of formation of pp24; C , formation of pp8; D, formation of phosphorylated RII subunits. Recombinant C,,,NlAl (0) and C,,.,NSAI (W) were used to phosphorylate neuronal membrane extracts in the presence of [y3'P1ATP. Kinase in samples removed at 10, 30, and 60 s was inactivated by the rapid addition of electrophoresis sample buffer and heating. After SDS-PAGE, the extent of incorporation of 32Pinto the bands of interest was estimated by densitometry of autoradiograms (see A ) . For each phosphoprotein, the curves are normalizedfor the extent of incorporation after 60 s in the presence of the N1 kinase.The incorporation into the two type I1 R subunits have been combined (in D l . Representative data are shown.
K, valueswerealsosimilar: C,,.,NlAl (32 2 p ~ n, = 4; two-dimensional isoelectric focusing-PAGE (Fig. 3B). The same literature value 36 p~ (29)); Cm,.,N2A1 (39 f 1 PM, n = 4). patterns wereobserved whether or not the membrane proteins C,,.,NlAl and C,,,N2Al also bound purified recombinant were solubilized in detergent before phosphorylation. The 24regulatory subunits with similar affinities (Fig. 2). For Aplysia and 8-kDa polypeptides were phosphorylated at the same rate (a type I subunit) theIC,, values were: 143 T 53 PM (n = by C,,,NlAl, in the presence of C,,,NBAl. 6) for C,,,NlAl (literature value 110 PM using bovine RIa In the absence of recombinant C subunits, there was no (29)) and 93 22 PM ( n = 6) for Cm,,N2A1. For murine RIIa, phosphorylation of the 24- and 8-kDa proteins under normal the IC,, values were: 18 f 5 PM ( n = 4) for C,,,NlAl and 16 f assay conditions (Fig.36, lane 1).However, pp24 and pp8were 7 PM (n = 4) for Cm,,N2A1. detected in the absence of added kinaseswhen the reaction mix C Subunits of Aplysia PKA with Alternative N Termini Phos- was supplemented with CAMP (Fig. 3C, lane 2). Therefore, phorylate 24- a n d 8-kDa Substrates at Digerent Rates in Exendogenous PKA is capable of phosphorylating the samesubtracts of Neural Tissue--Treatment of an Aplysia neuronal strates as exogenous kinase.Furthermore,this shows that membrane protein fraction with equal Kemptide units of puripp24 and pp8 must derive from Aplysia neuronal membranes fied recombinant C,,,NlAl or CML,N2A1 and [Y-~~PIATP and not the enzyme preparations. The50- and 47-kDa type I1 R produced distinct patterns of phosphorylated proteinsafter subunits are phosphorylated, albeit less rapidly, in the absence (Fig. 3A, lane separation by SDS-PAGE (Fig. 3A). C,,,NlAl 2) phosphorylated two polypeptides of 24 and 8 kDa2 more of exogenous enzyme or added CAMPby undissociated endograpidly thandid C,,,NSAl (Fig. 3A, lane 3). Differential phos- enous C subunits (Fig. 3 A , lane 1 , and Ref. 24). pp24 and pp8 were quantitated by densitometric scanningof phorylation of the two polypeptides was also apparent after autoradiograms (Fig. 4A).Analyses of the timecourse of phosphorylation showed that initial rates and not end points of Phosphorylation canproduce shifts in electrophoretic mobility upon phosphorylation were being measured (Fig. 4, B-D). SDS-PAGE. Nevertheless, for convenience of description, substrates catalyzed the formation of pp24 and pp8 at rates here are assigned the same M , values as the observed phosphoproteins. C,,,NlAl
Neuronal Substrates of Aplysia PKA
C
B
A M
0
5
15
30
45
min
23727
M
3I
2
1
2
3
4
200 -
97 68
-
43
10.6
-
29 -
b 1814 -
D FIG.5.Neither pp24 nor pp8 is a proteolysis product of an Aplysia type II regulatory subunit.Key: b, pp24; D, pp8; M ," C molecular weight markers. A , autoradiogram of a 10% T, 3% C Tricine-SDS gel showing the proteolytic breakdown products of purified phosphorylatedAplysia RII subunits in a crude neuronal homogenate. Phosphorylated Aplysia RII subunits purified by CAMP-agarose affinity chromatography were incubated with a crude neural homogenate,at 30 "C, for the times indicated.B , phosphopeptide maps of pp24 (lane 11, the BO-kDa RII (lane 2), and the 47-kDa RII (lane 3 ) obtained by limited proteolysis with staphylococcal V8 protease after phosphorylation in thepresence of Ly-"'PIATP and recombinant C,,,NlAl. Phosphorylated polypeptides were separated by SDS-PAGE and digested in the wells of a second gel, before re-electrophoresis to separate the fragments in one dimension. C , phosphopeptide maps generatedby exhaustive digestion with trypsin.Polypeptides phosphorylated in the presence of [y-"2P1ATPand recombinant CAPL,N1Alwere separated by SDS-PAGE and transferred to nitrocellulose. After digestion in situ, phosphopeptides were separated by one-dimensional TLC: lane 1, 50-kDa RII; lane 2, 47-kDa RII; lane 3, pp24; and lane 4, P P ~ .
that were, respectively, 2.21 k 0.02 and 6.9 k 0.4 ( n = 2 sets of data scanned)-fold higher than the ratesobserved in the presence of equal Kemptide units of C,,,N2Al. In contrast, C, ANlAl and C,,,N2A1 phosphorylated neuronal RII subunits (24) at about the same rates(Fig. 40). pp24 and pp8 Are Not Fragments of the 50- and 47-kDa Phosphorylated 9 p e II R Subunits-It was essential to demonstrate that pp24 and pp8 are not proteolytic degradation products of the 50- and 47-kDa R subunits, since the latter are the majorphosphorylated polypeptides of greater apparent mass (24). To test this possibility, phosphorylated Aplysia RII subunits, affinity-purified by using CAMP-agarose beads, were incubated with a crude nervous tissue homogenate. This preparation, which was used in the absence of protease inhibitors, contains far more endogenous protease activity than the extracts generallyused inthe phosphorylation experiments, which were made from membranes treated with the inhibitors PMSF and leupeptin and then washed before solubilization. Although the phosphorylated R subunits were broken down homogenate, yielding 3 5 , upon extended digestion in the crude 30-, and 15-kDa products, no phosphorylated polypeptides in the 24- and 8-kDa regionsof the gel were observed (Fig. 5A 1. In addition, phosphopeptide mapping of pp24 and pp8 after limited digestion with V8 protease (Fig. 5 B ) or after exhaustive trypsin treatment (Fig. 5 C ) revealed no peptides in common with digests of the 50- and 47-kDa RII subunits (Fig. 5, B and C) or the 30-kDa proteolysis product of the RII subunits (data not shown). Furthermore, pp24 and pp8 neither bound to CAMP-agarose nor to Ht 31-Affi-Gel (data not shown), both of
which avidly bind the 50- and 47-kDa RII subunits, through their CAMP- and anchoring protein (-)-binding sites, respectively (24). These sites span almost the entireRII subunit polypeptide chain. Therefore, these experiments further reduce the likelihood that pp24 and pp8 are RII fragments. The possibility that pp24 and pp8 are contaminatingproteins present in thepurified C,,,NlAl and C,,NBAl preparations is also ruled out, asno radiolabeled proteins were detected when the phosphorylation reactions were carried out without added Aplysia neural extract. Furthermore, pp24 and pp8can be generated by endogenous kinase activity (Fig. 3C). pp24 and pp8 Are Found Only in Neuronal Extracts-To determine the tissue distribution of pp24 and pp8, PKA substrates were examined in various tissues by phosphorylation in the presence of [y32P]ATP and recombinant vitro in C,,,NlAl. First, neuronal components were compared with the connective sheath thatenvelopes them. pp24 and pp8 were of relatively low abundance in the latter (Fig. 6A).Next, membrane fractions from other Aplysia tissues, including ovotestis and buccal muscle, were screened for pp24 and pp8. Again, these phosphoproteins were found only inneural tissue (Fig. 6 B ) . Distribution of pp24 and pp8 in Subcellular Fractions of Neural Tissue-Subcellular fractions comprising Aplysia neuronal membranes, cytoskeleton, and taxol-stabilized microtubules were analyzed for the presence of pp24 and pp8, after phosphorylation with C,,,NlAl. pp24 was absent from the cytoskeleton pellet and enriched in thecorresponding supernatant (Fig. 7A, lanes l and 2 ) . By contrast, pp24 was present in
Neuronal Substrates of Aplysia PKA
23728
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2
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-
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~
N2
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2
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”
3
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N2 5
6
*
9766
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-
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45 31 -
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31
-
31
-
b 31
-
b
22
-
14
-
-
b 22
-
Id.
22 14-
14
D
0
D
FIG.6. Both pp24 and pp8 are neural-specific. Key: F, pp24; D, pp8; M ,low molecular weight markers. A, autoradiogram of a 10% T,3% C Tricine-SDS gel showing the enrichment of pp24 and pp8 in the microdissected neuronalcomponents of thecentralganglia.Equal amounts of membrane protein from microdissected neuronal components (NZ? lane 1 ) and the surroundingconnective sheath (Sh: lane 2 ) were phosphorylated in presence of [y-”PIATP and equal Kemptide units of CAp,,NIA1. B , autoradiogram of a 10% T, 3%C Tricine-SDS gel designed to detect pp24 or pp8 in various tissues. Solubilized membrane fractions ( 5 pg of protein) from nervous tissue (NT:lane 1 ), ovotestis (0110: lane 2), and buccal muscle (BM: lane 3 ) were phosphorylated in presence of [Y-~~PIATP and CA,,,,NIA1.
both the membranepellet and its supernatant (Fig. 7A, lanes 3 and 4). pp24 was found in taxol-stabilized microtubules, but it was absentfrom the corresponding supernatant (Fig. 7A, lanes 5 and 6).pp8 wasfound in both the supernatants and pellets of the membrane and cytoskeleton fractions (Fig. 7 , A and B ) . Interestingly, an even greater difference in therate of phosphorylation of the 24-kDa substrate was observed when the cytoskeleton supernatant was treated with C,,,NlAl, as opposed to CmLAN2A1, thanwasseenwiththe solubilized neuronal membrane fraction (Fig. 7B). In addition, a 55-kDa protein was observed in thecytoskeleton supernatant thatwas specifically phosphorylated by C,,N2A1 (Fig. 7B). The 55kDa species was not observed consistently, which might be attributed to genetic or seasonal variation in native Aplysia, whereas the more rapid phosphorylation of the 24- and 8-kDa proteins by CAPL,N1A1 was seen in all cases examined (n 2 10). DISCUSSION
The present findings demonstrate differential phosphorylation of neuronal substrates by C subunits ofAplysia PKA with the alternativeN termini N1 andN2. The addition of purified recombinant C,,NlAl to homogenates of neural tissue generates two phosphoproteins, pp24 and pp8, more rapidly than does C,,N2A1. Furthermore, in some preparations of detergentsupernatants, C,,N2A1 produced a phosphoprotein,
FIG.7. pp24 and ppS are differentially distributed in subcellular fractions of nervous tissue. Key: b, pp24; D, pp8; M, low molecular weight markers. A, autoradiogram of a 10% T,3% C Tricine-SDS gel showing the distribution of pp24 and pp8 in cytoskeleton a (C) pellet (P: lane 1 ) and the corresponding supernatant (S: lane 2 ) , a membrane ( M ) pellet and supernatant (lanes 3 and 4 ) and a taxol-stabilized microtubule (MT) pellet and supernatant(lanes 5 and 6 ) . Equal amounts of protein (5 pg) were phosphorylated in the presence of [y-”PIATP and C,,NIAI. The 8-kDa region of the gel is shown a s a longer exposure in the bottom panel. B, autoradiogram of a 10% T, 3% C Tricine-SDS showing differences in the amounts of pp55 (arrow),pp24, and pp8 generated by C,,NlAI and C,,NBAl in cytoskeletal fractions (all 5 pg): the initial homogenate (H: lanes 1 and 2 1, the supernatantfrom the cytoskeleton fraction (S: lanes 3 and 4 ) , and the cytoskeleton pellet (P: lanes 5 and 6 ) were phosphorylated in the presenceof [y-”P]ATP and equal Kemptide units ofC,,,NIAI (lanes 1, 3, and 5) or C,,N2Al (lanes 2, 4 , and 6). The 8-kDa region of the gel is shown as a longer exposure in thebottom panel.
pp55, that was not seen with C,,NlAl. Numerous experiments were done to show that pp24 and pp8 are not proteolysis products of phosphoproteins of higher mass, in particulartype I1 R subunits of PKA (24). Additional experiments eliminated the possibility that pp24 and pp8 are contaminants of the recombinant C subunit preparations. pp24 and pp8 are found only in the centralnervous system of Aplysia and derive from neuronal components rather than the surroundingconnective sheath. pp24 may be a membrane protein as it is both detergent-soluble and enriched in “membrane” pellets. However, pp24 is also associated with taxol-stabilized microtubules. Therefore, it is possible that pp24 is capable of linking membranes and cytoskeleton, even though little of it is found in a conventional cytoskeleton pellet. pp8 was found in both cytoskeleton and membrane pellets, as well as their supernatants. Attempts were made to determine the identities of pp24 and pp8. Each nervous system of Aplysia contains only -1 mg of protein, and neitherpp24 nor pp8was sufficiently abundant to permit sequence analysis by Edman degradation. Therefore, we couldonly guess at the identityof the polypeptides and then test our ideas by experiment. For example, the possibility that pp24 is the -28-kDa Aplysia synaptophysin homolog reported by Chin et al. (45) was given serious consideration. However, although pp24 was detected in synaptic vesicle subfractions of Aplysia nervous tissue (data not shown), it was not enriched
Neuronal Substrates of Aplysia PKA therein compared with other fractions, and it was not recognized on Western blots by the monoclonal antibody against bovine brain synaptophysin, SY38, which cross-reacts with the Aplysia protein (45). The basis for differential substrate phosphorylation by the N1 and N2 forms of the C subunit has also been considered. One possibility is adifference in substratespecificity mediated by the enzyme active sites. This was observed forthe A1 andA2 forms of the C subunit, where alternative splicing of a central exon cassette produces alternative sequences in the catalytic loop region (27,29). However, this possibility seemedless likely for the N1 and N2 forms tested here, which have identical active site residues. Indeed, the two forms phosphorylated the small synthetic peptide Kemptide with similar kinetic constants. Asecond possibilityis differential binding to R subunits in the homogenates. For example, this could sequester and inactivate the N2 form so that it cannot phosphorylate substrates that remain accessible to the N1 form. Because the primary interaction of R with C is through a pseudosubstrate sequence in R thatbinds to the active site of C, the differential binding would have to be through secondary interactions outside the identical active sites of the N1 and N2 forms. Secondary interactionsof R with C have been demonstrated, but they do not involve the N terminus of the C subunit (46). Accordingly, type I or type I1 R subunits inhibited the N1 and N2 forms of C with similar IC,, values. In addition, in some detergent supernatants, the N2 form produced a phosphoprotein (pp55) not seen with the N1form, further implying that theN2 form was not differentially inactivated. A third possibility, which we favor, is that the N1 and N2 termini bind t o specific substrates or to specific receptors located near substrates. If the latter is the case, the receptor-substrate interaction must be resistant to the detergent treatment used to solubilize neuronal membranes. Substrate targetingby protein kinases is not unprecedented. For example, the N terminus of rhodopsin kinase is involved in selective binding to photolyzed rhodopsin (47) and one of the functions of src-homology (SH2) domains is t o tether substrates to tyrosine kinases (48). It is also conceivable that N1 and N2 act as negative regulatory elements, to prevent the phosphorylation of certain substrates. In the case of pp60""", it was long felt that themyristylated N terminuswas bound to a receptor on the inner surface of the plasma membrane. This view has fallen from favor, following the failure to isolate a bona fide receptor protein for the Nterminal segment (49) and has been replaced with the idea that the N-terminal fatty acid and a number of proximal positively charged amino acids are sufficient to endow a direct interaction with the lipid bilayer and its headgroups (41, 50). A two-step mechanism has been proposed in which the relatively weak interaction with the bilayer promotes a second stronger interaction between another domain on pp60v-sm and a protein receptor (49, 50). Although it has been assumed that the myristylated C subunits of PKA are completelysoluble inthe cytoplasm after release from the holoenzyme, a weak interaction with lipid bilayers that directs the kinase to key residues on important substrates cannot be ruled out. Indeed, the N terminus of mammalian C a is able to replace the N terminusof ~p60"""~ in mediating binding to plasma membrane preparations (43). However, because C,,,NlAl can phosphorylate specific substrates in detergent, as well as in membranes, an interaction of the N terminus with the lipid bilayer is unlikely t o provide the sole explanation for the substrate targeting observed in the present work, which may be analogous t o the second of the two steps proposed for pp60""". Nevertheless, in vivo, an interaction of C,,,NlAl with bilayers may facilitate targeting of membrane protein substrates by reducing the
23729
search to two dimensions (49, 50). Interestingly, the N terminus of nonmyristylated recombinant mouse Ca is disordered or mobile in crystals (30). This finding, the attachment of the N terminus at a point distant from the active site (301, and the existence of multiple N termini prompted our suggestion that theN terminiof C might act as molecular handles for substrate targeting(28).Recently, the structure of the myristylated pig Ca subunit has been solved, revealing the fatty acid buried in a pocket near the surface of the protein, thereby immobilizing the N-terminal amino acids (51). There may be circumstances when the fatty acid jackknifes out into contact with solvent (501, revealing its hydrophobic surface or presenting a previously occludedaspect of the N terminus. Regulated membrane attachment requiring N-myristylation has been noted for several other signal transduction proteins, including myristylated alanine-rich C kinase substrate (phosphorylation-dependent binding, Ref. 521, ADPribosylation factor (GTP-dependent binding, Ref. 53), and recoverin (Ca2+-dependentbinding, Ref. 54). The many unresolved issues concerning N-myristylation in central signaling pathways are worthy of continued examination. Furthermore, the possible existence of binding sites for N2 and other termini of autonomous catalytic subunits of protein kinases should also be explored. The catalytic subunit of a close relative of PKA in Aplysia, spermatozoon-associated kinase, sak ( 5 9 , has anN terminusthat differs yet again from the N1 and N2 sequences. C subunits of PKA with alternative N termini (Cp and Cp2) have also been noted in mammals (56). As yet, no functional significance has been ascribed to these forms. Nevertheless, the findings suggest that a family of alternative small N termini with a variety of functions, originating at the exon 1-exon 2 splice boundary (281, may exist for C subunits of PKA and their relatives. Evidence for functional differences between Aplysia C subunits with different central cassettes was obtained earlier. These kinases differ in substrate specificity toward synthetic peptides. They also bindcommon R subunitswith different affinities, thereby forming holoenzymes that areactivated at different CAMP concentrations (29). Furthermore, PKA holoenzymes may be located in different parts of the cell through their interactions with a variety of binding proteins (24,25).Together with the present findings, these results indicate that functional diversity of PKA is likely to contribute to spatial andtemporal control of phosphorylation in neurons. For example, in thepresent case, if N2subunits were located in one compartment of the cell t o the exclusion of N1 subunits, auniform cell-wide elevation in CAMPlevels would lead to spatially organized phosphorylation. We are presently examining the spatialdistribution of N1 and N2 subunits inneurons and their possible translocation upon activation of holoenzyme byCAMP. In in vitro experiments: we have also shown that Cm,,N2A1, but not CMLANlAl, can be phosphorylated on Tyr-28 by c-src kinase, which is highly enriched in synaptic vesicle preparations (57). CML.,N2A1also binds Zn2+, which is presumably coordinated by two or more of the four histidine residues in the N2 sequence. The physiological significanceof these findings is not yet clear. Acknowledgments-We thank Dan Cam and John Scott for recombinant RIIa subunits, John Leszyk for the synthesis of peptides in the W. M. Keck Protein Chemistry Facility at the Worcester Foundation, and Susan Taylor for a stimulating discussion. Tax01 was obtained from the National Cancer Institute. REFERENCES 1. Kandel, E. R., and Schwartz, J. H. (1982) Science 2 1 8 , 4 3 3 4 3 2. Bailey, C. H., and Kandel, E. R. (1993)Annu. Reu. Physiol. 66, 397426
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