Báo cáo khoa học: Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis potx

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Identification of multiple isoforms of the cAMP-dependentprotein kinase catalytic subunit in the bivalve molluscMytilus galloprovincialisJose´R. Bardales1, Ulf Hellman2and J. A. Villamarı´n11 Departamento de Bioquı´mica e Bioloxı´a Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain2 Ludwig Institute for Cancer Research, Uppsala, SwedenThe cAMP-dependent protein kinase (PKA; EC2.7.11.11) plays a crucial role in the regulation ofseveral physiological processes, as it is the main media-tor of the effects of cAMP in eukaryotic organisms.Inactive PKA is a tetrameric holoenzyme composed oftwo functionally distinct subunits: a dimeric regulatorysubunit (R-subunit) and two monomeric catalyticsubunits (C-subunits). The main function of the R-sub-unit is to inhibit the phosphotransferase activity of theC-subunit. The transitory increase of cAMP levelsinside the cell, induced by an extracellular signal, andthe binding of cyclic nucleotide to R-subunits causethe dissociation of C-subunits which, once free, canphosphorylate protein substrates, mainly in the cyto-plasm, but also in the nucleus [1,2].It has been widely reported that PKA is involved inthe regulation of some physiological events that specifi-cally occur in bivalve molluscs as a consequence ofenvironmental adaptation. For example, the relaxationof mollusc ‘catch’ muscles, induced by serotonin,occurs through the PKA-mediated phosphorylation oftwitchin, a high molecular mass protein present in thethick filaments [3,4]. The mollusc ‘catch’ muscles, suchas the posterior adductor muscle (PAM), are special-ized muscles that can sustain high tension for verylong periods with low energy expenditure [5]. On theKeywordscAMP-dependent protein kinase;catalytic subunit; C-subunit isoforms;MALDI-TOF ⁄ TOF MS; MytilusCorrespondenceJ. A. Villamarı´n, Departamento deBioquı´mica e Bioloxı´a Molecular, Facultadede Veterinaria, Universidade de Santiago deCompostela, Campus de Lugo, 27002 Lugo,SpainFax: +34 82 252 195Tel: +34 82 285 900E-mail: antonio.villamarin@usc.es(Received 28 March 2008, revised 4 July2008, accepted 10 July 2008)doi:10.1111/j.1742-4658.2008.06591.xSeveral isoforms of the cAMP-dependent protein kinase catalytic subunit(C-subunit) were separated from the posterior adductor muscle and themantle tissues of the sea mussel Mytilus galloprovincialis by cationexchange chromatography, and identified by: (a) protein kinase activity; (b)antibody recognition; and (c) peptide mass fingerprinting. Some of the iso-zymes seemed to be tissue-specific, and all them were phosphorylated atserine and threonine residues and showed slight but significant differencesin their apparent molecular mass values, which ranged from 41.3 to44.5 kDa. The results from the MS analysis suggest that at least some ofthe mussel C-subunit isoforms arise as a result of alternative splicingevents. Furthermore, several peptide sequences from mussel C-subunits,determined by de novo sequencing, showed a high degree of homology withthe mammalian Ca-isoform, and contained some structural motifs that areessential for catalytic function. On the other hand, no significant differ-ences were observed in the kinetic parameters of C-subunit isoforms, deter-mined by using synthetic peptides as substrate and inhibitor. However, theC-subunit isoforms separated from the mantle tissue differed in their abilityto phosphorylate in vitro some proteins present in a mantle extract.AbbreviationsCAF-PSD, chemically assisted fragmentation–post-source decay; C-subunit, catalytic subunit of cAMP-dependent protein kinase; PAM,posterior adductor muscle; PKA, cAMP-dependent protein kinase; PKI(5–24),protein kinase inhibitor peptide; PMF, peptide massfingerprinting; PTM, post-translational modification; R-subunit, regulatory subunit of cAMP-dependent protein kinase.FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4479other hand, phosphofructokinase from the sea musselMytilus galloprovincialis, unlike that from mammals,was clearly activated when phosphorylated by PKA ata serine residue [6]; moreover, the enzyme activitychanged seasonally in parallel with its phosphorylationdegree [7]. These and other results reported by otherauthors [8] suggest that PKA activation contributes tothe regulation of carbohydrate metabolism duringbivalve gametogenic development, through the revers-ible phosphorylation of key regulatory enzymes.Finally, various authors have argued that PKA-medi-ated protein phosphorylation could be responsible formetabolic rate depression, a strategy that bivalve mol-luscs use to survive during the long periods of aerialexposure causing environmental hypoxia [9,10].Therefore, to understand the biochemical basis ofthese molluscan regulatory events, the diverse forms ofPKA in these organisms must be defined. Over the lastfew years, we have identified and purified two differentisoforms of the PKA R-subunit from the sea musselM. galloprovincialis, which were named Rmyt1andRmyt2[11–13]. Interestingly, both isoforms have iden-tical apparent molecular masses of 54 kDa, but theydiffer in: (a) their isoelectric point; (b) their biochemi-cal properties; (c) their antigenicity; and (d) their tissuedistribution [12–14]. According to its physicochemicaland biochemical properties, a partial amino acidsequence from Rmyt1showed a clear homology withthe type I R-subunits from both mammalian andinvertebrate sources [13]; likewise, Rmyt2was shown tobe homologous to the type II R-subunits from thesame species [14].The purpose of the work described in this articlewas to investigate the possible existence of differentisoforms of the PKA C-subunit in the sea musselM. galloprovincialis.ResultsSeparation of different isoforms of the C-subunitIn order to demonstrate the presence of different iso-forms of the PKA C-subunit in mussels, the proteinwas partially purified from the PAM and the mantletissues of the mollusc, and then subjected to cationexchange chromatography on a Mono-S column.Figure 1A shows the elution profile corresponding tothe PAM C-subunit. The application of a salt gradientresulted in separation of four absorbance peaks. Threeof them – labelled peak I, peak II and peak III –showed protein kinase activity; they eluted at 0.13,0.16 and 0.25 m NaCl, respectively. SDS ⁄ PAGE analy-sis and Coomassie staining revealed the presence of aprotein with apparent molecular mass  40 kDa in thefractions corresponding to peak I, peak II andpeak III (Fig. 1B). This protein band was recognizedby an antibody raised against the human Ca-isoformof the C-subunit (Fig. 1C). Therefore, peak I, peak IIand peak III correspond to three different isoforms ofthe C-subunit, which we named C1,C2and C3, respec-tively. On the other hand, fraction 18, correspondingto the first absorbance peak, without protein kinaseactivity, contained an unidentified protein < 30 kDa,and fractions 21–23 also contained an unidentifiedhigh molecular mass protein (Fig. 1B). None of theseproteins was recognized by the Ca-isoform antibody inthe western blot analysis (Fig. 1C).Figure 2A shows a representative elution pattern ofthe C-subunit preparation obtained from the mantletissue. Two absorbance peaks, associated with proteinkinase activity, were separated; these eluted at 0.19 and0.25 m NaCl, and were labelled peak I and peak II,respectively. Coomassie staining of an SDS ⁄ PAGEgel revealed that fractions corresponding to peak IABCFig. 1. Separation and identification of PKA C-subunit isoformsfrom mussel PAM. (A) Elution profile of C-subunit from a Mono-SHR 5 ⁄ 5 column. A sample (2 mL, 1.5 mg of protein) of C-subunitpurified from PAM as described in Experimental procedures wasapplied to the column and eluted with a linear salt gradient. Frac-tions of 0.5 mL were collected and assayed for protein kinase activ-ity. Three distinct peaks associated with protein kinase activitywere separated: I, II and III. Aliquots of fractions were also analy-sed by (B) Coomassie-stained SDS ⁄ PAGE, and (C) western blottingwith an antibody against the human Ca-isoform.Mussel C-subunit isoforms J. R. Bardales et al.4480 FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBScontained only a  40 kDa protein, whereas those cor-responding to peak II contained two different proteinbands of  41 and  43 kDa (Fig. 2B). The three pro-tein bands showed reactivity with the human Ca-iso-form antibody in the western blot analysis (Fig. 2C).In summary, three different isoforms of C-subunit wereseparated from the mantle tissue preparation: theisoform named C4, which corresponded to peak I ofthe Mono-S chromatogram, and the isoforms namedC5and C6, which coeluted together at peak II. C4was3–4-fold more abundant than C5and C6together.Characterization of C-subunit isoformsSamples of purified C1–C6were analysed by SDS ⁄PAGE, using a 16 · 16 cm polyacrylamide gel. Asshown in Fig. 3A, slight but significant differenceswere observed in the migration behaviour among mus-sel isozymes. Only C3(from PAM) and C5(from man-tle) have identical apparent mobilities, which suggeststhat they could be the same isoform present in bothtissues. The values of the apparent molecular massranged between 41.3 kDa for C4and 44.5 kDa for C6.All the mussel isoforms were slightly heavier than thebovine C-subunit used as a control (lane 2), whosemolecular mass, determined by MS, was exactly40 855.7 Da [15].On the other hand, samples of purified musselC1–C6were probed with both phosphoserine and phos-phothreonine antibodies, and they were all serine andthreonine phosphorylated, as shown in Fig. 3B,C,respectively. Moreover, incubation of C-subunitisoforms with MgATP did not change their mobilityon SDS ⁄ PAGE (not shown).Structural analysis of C-subunit isoformsIn order to determine possible structural differencesamong mussel C-subunit isoforms, samples of purifiedC1–C6proteins were subjected to ‘in-gel’ trypticdigestion, and peptide mixtures were analysed byMALDI-TOF MS. The corresponding peptide massfingerprinting (PMF) spectra are shown in Fig. 4.Furthermore, a sample of C-subunit purified frombovine heart (fraction CB), consisting mainly of theA B C Fig. 2. Separation and identification of PKA C-subunit isoformsfrom mussel mantle tissue. (A) Elution profile of C-subunit from aMono-S HR 5 ⁄ 5 column. A sample (2 mL, 1.8 mg of protein) ofC-subunit purified from mantle tissue as described in Experimentalprocedures was applied to the column and eluted with a linear saltgradient. Fractions of 0.5 mL were collected and assayed for pro-tein kinase activity. Two distinct peaks associated with proteinkinase activity were separated: I and II. Aliquots of fractions werealso analysed by (B) Coomassie-stained SDS ⁄ PAGE, and (C) wes-tern blotting with an antibody against the human Ca-isoform.ABCFig. 3. Characterization of mussel C-subunit isoforms. (A) Determi-nation of molecular mass by SDS ⁄ PAGE. Samples (1–2 lg of pro-tein) were subjected to 10% SDS ⁄ PAGE in a 16 · 16 cmpolyacrylamide gel which was Coomassie stained. Lane 1: molecu-lar mass standards. Lane 2: sample of bovine heart C-subunit, frac-tion CB[17]. Lanes 3–5: samples of fractions 20, 22 and 29 ofFig. 1A chromatogram, respectively. Lanes 6 and 7: fractions 24and 28 of Fig. 2A chromatogram, respectively. The apparent molec-ular mass of mussel C-subunit isoforms was estimated from thepositions of molecular mass standards and bovine C-subunit. (B, C)Western blot analysis of mussel C-subunit isoforms. Samples (140–300 ng of protein) of the same fractions were subjected to 10%SDS ⁄ PAGE, and C-subunit was detected by western blotting usingmonoclonal antibodies against phosphoserine (B) or phosphothreo-nine (C).J. R. Bardales et al. Mussel C-subunit isoformsFEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4481Ca-isoform [15,16], was also digested and analysed. Adetailed analysis of data allowed us to draw the fol-lowing conclusions. (a) Eight peptide masses werefound to be common to the bovine Ca-isoform and allmussel C-subunit isozymes; these are (in Da): 734.5,744.5, 759.4, 895.5, 1138.6, 1419.8, 1661.9, and 1917.1.The partial sequences with theoretical masses identicalto those measured are marked with dashed lines in thewhole sequence of the bovine Ca-isoform in Fig. 5A.Furthermore, two of these common peptides, whichyielded peaks at m ⁄ z 744.5 and 895.5, were sequencedde novo by chemically assisted fragmentation–post-source decay (CAF-PSD) (Table 1: peptides 1 and 2),and exactly matched the sequences of the bovine.4 4 * C 1 8 59.453 9 4.89 2 64 0 5 8 1.25 5 x1 0 e ns. [a.u.] 791 * C 2 8 14 9 1059 . 1172. 7 5 1253.716 1917.072 734.499 1661.911 804.339 1014.650 1713.88 4 1855.033 1138.649 1338.785 1419.79 7 966.537 2213.147 1970.996 1582.823 2152.230 2109.072 2283.220 2621.455 2833.537 2556.224 0.25 0.50 0.75 1.00 In t e 7.058 8 97 70 1.25 5 x1 0 e ns. [a.u.] 1230.6 4 C 3 19 1 1661. 8 1713.8 859.45 1 1855.016 1494.882 1172.75 4 1605.878 804.333 1040.614 1253.712 734.496 1419.78 3 959.070 2212.978 2109.05 4 2166.932 1745.860 1970.975 1118.577 1338.783 2621.471 2833.535 2283.218 2556.215 0.25 0.50 0.75 1.00 In t e 4 2.446 1. 5 5 x1 0 ns. [a.u.] 8 4 1916.857 1713.684 1661.717 1854.82 1 895.38 6 1172.619 774.28 8 1605.705 1494.720 2151.991 1014.508 2621.197 1253.577 958.924 2212.749 713.38 9 1419.625 2108.832 1970.785 1118.434 2308.088 2849.241 1338.62 7 2555.97 9 0. 5 1. 0 Inte 8 98 1 7 1. 0 5 x1 0 n s. [a.u.] C 4 1916. 8 1713. 7 1 1854.85 7 1661.745 859.33 1 2152.041 1172.63 3 1059.513 2386.96 2 1014.523 1494.73 7 2212.806 734.385 804.21 0 2621.27 5 1419.65 0 1253.59 4 2575.086 1582.658 959.43 9 2092.888 2833.334 1768.624 2308.15 9 1970.81 6 0. 2 0. 4 0. 6 0. 8 Inte n 65 2. 0 5 x1 0 n s. [a.u.] * * C 5 842.4 1916.904 1661.75 7 1713.724 1854.867 895.402 2152.047 1172.64 6 774.29 2 1014.53 5 1605.744 1494.752 2621.281 1419.65 7 1253.60 6 2108.88 8 2198.78 7 959.450 1065.99 3 2308.16 6 1970.83 0 2849.328 0. 5 1. 0 1. 5 Inte n 49 1 1. 0 5 x1 0 s . [a.u.] C 6 842. 1916.952 1661.81 0 895.434 1713.780 774.322 1172.682 1864.886 1014.569 713.435 1605.786 1494.79 4 2152.088 1419.706 2108.941 1253.636 1066.030 2212.893 2591.142 1970.869 2849.451 0. 0 0. 2 0. 4 0. 6 0. 8 Inte n s 750 1000 125 0 150 0 175 0 2000 2250 2500 275 0 m/z* 2623.3 4 Fig. 4. Peptide mass fingerprints of mussel C-subunit isoforms after tryptic digestion. The asterisk(s) indicate the peak(s) observed only inthe spectrum from a particular isoform. Arrowheads indicate the peak at 1059.5 Da, common to C1and C4, and arrows indicate the peak at1605.7 Da, common to the remaining isoforms: C2,C3,C5and C6.Mussel C-subunit isoforms J. R. Bardales et al.4482 FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBSCa-isoform corresponding to amino acids 73–78 and48–56, respectively (Fig. 5A). (b) Most peptide masseswere common to all mussel isoforms, C1–C6. Severalof these peptides were also sequenced de novo (Table 1:peptides 3–12), showing high amino acid sequenceidentities to the bovine Ca-isoform (Fig. 5A). (c) Withregard to the C1,C2,C4and C6spectra, there was atleast one peptide mass that was unique for each iso-form, being absent in the remainder. This was particu-larly true for m ⁄ z peaks labelled with asterisks in thespectra of Fig. 5: 791.4 (only in C1); 1230.6 (only inC2); 1768.6 and 2386.9 (only in C4); and 2623.3 (onlyin C6). (d) There was one peak at 1059.5 Da observedonly in the C1and C4spectra, whereas another peakat 1605.7 Da was found in the spectra of the remainingisoforms: C2,C3,C5and C6. Interestingly, an incom-plete sequence derived from this last peak (Table 1:peptide 13) matches a sequence lying at the N-terminusof a C-subunit (N1-isoform) from the mollusc Aplysia(Fig. 5B). This result indicates that mussel C1and C4differ from C2,C3,C5and C6at the N-terminalregion. (e) When spectra from C3and C5were com-pared, no significant difference was observed, whichsuggests that both C3and C5are the same C-subunitisoforms present in the PAM and the mantle tissuerespectively.Kinetic characterization of C-subunit isoformsand protein phosphorylationIn order to determine possible functional differencesamong mussel C-subunit isoforms, the kinetic parame-ters were determined for each purified isozyme. Itshould be noted that C5and C6coeluted from theMono-S column, and therefore, samples containing amix of both isoforms were used in the kinetic experi-ments. No significant differences among C-subunitisoforms regarding the values of apparent KmforKemptide and Vmaxwere observed. Furthermore, allthe mussel isozymes were inhibited by the proteinkinase inhibitor peptide [PKI(5–24)] with similar I50values (Table 2).The ability of mussel C-subunit isoforms to phos-phorylate proteins in vitro was also investigated. Thus,Fig. 5. Comparison of amino acidsequences from mussel C-subunit isoforms(in bold) with homologous regions of (A)bovine Ca-isoform (UniProtKB P00517), and(B) Aplysia C-subunit (UniProtKB Q16958).Identical residues are in black boxes. Aster-isks indicate residues playing a key role inthe catalytic function (see text). Dashedlines show the partial sequences of thebovine Ca-isoform corresponding to theeight measured m ⁄ z peaks that were com-mon to bovine and all mussel C-subunitisoforms.Table 1. Peptides from Mytilus C-subunit isoforms identified byde novo sequencing.PeptideMeasuredm ⁄ z (Da)Present inspectrafrom De novo peptide sequence1 744.47 Bovine Ca,C1–C6[I ⁄ L][I ⁄ L]DKQK2 895.47 Bovine Ca,C1–C6T[I ⁄ L]GTGSFGR3 859.44 C1–C6FSEPHSR4 870.51 C1–C6VF[I ⁄ L]VQHK5 884.48 C1–C6VTDFGFAK6 1014.64 C1–C6KVDAPF[I ⁄ L]PK7 1016.65 C1–C6S[I ⁄ L][I ⁄ L]QVD[I ⁄ L]TK8 1040.61 C1–C6VTDFGFAKR9 1172.75 C1–C6S[I ⁄ L][I ⁄ L]QVD[I ⁄ L]TKR10 1338.80 C1–C6[I ⁄ L]KQVEHT[I ⁄ L]NEK11 1494.89 C1–C6[I ⁄ L]KQVEHT[I ⁄ L]NEKR12 1910.82 C1–C6GPGDASNFDDYEEEP[I ⁄ L]R13 1605.88 C2,C3,C5,C6KGDVPMNVKE(x)Kaax Dmass = 360.3 Da.J. R. Bardales et al. Mussel C-subunit isoformsFEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4483C1,C2and C3(purified from PAM) were individuallyincubated, in the presence of labelled ATP, with aliqu-ots of a PAM extract. In the same way, C4and themixture of C5and C6were incubated with samples ofa mantle tissue extract. Densitometric analysis of auto-radiographs corresponding to the PAM samplesshowed identical protein phosphorylation patterns forC1,C2and C3(Fig. 6A). Three protein bands, markedwith arrows in Fig. 6A, were mainly phosphorylatedby each C-subunit isoform. The protein with the high-est molecular mass ( 600 kDa) was identified as twit-chin, whose PKA-mediated phosphorylation had beenpreviously demonstrated [3,4]. The intermediate pro-tein band was identified as actin by PMF and de novosequence analysis (not shown). The protein band withthe lowest molecular mass could not be identified byPMF; a correct sequence of 14 amino acids (RESE-FQSGDLWEVR) was then obtained by de novosequencing, although no clear identity could be drawnfrom the databases.For the mantle extract (Fig. 6B), the patterns ofproteins phosphorylated by C4and the mixture of C5and C6were also apparently similar, although densito-metric analysis of the autoradiograph showed someprotein bands, marked by asterisks, that seemed tobe phosphorylated by C4but not by the mix of C5and C6. Thus, it is possible that mantle isoforms havedifferent abilities to phosphorylate some proteins ofmantle tissue.DiscussionIn this article, we describe the separation and identifi-cation of several catalytically active isoforms of thePKA C-subunit from the sea mussel M. galloprovin-cialis. The isozymes named C1,C2and C3wereisolated from the PAM tissue, whereas C4,C5and C6were separated from the mantle tissue. However, itTable 2. Kinetic parameters of mussel C-subunit isoforms. Theapparent Kmfor Kemptide and Vmaxvalues were determined at0.2 mM ATP. The I50values for PKI(5–24) were determined at100 lM Kemptide and 0.2 mM ATP. The data are expressed asmeans ± SE of three independent experiments.Isoform Km(lM)Vmax(nmol PÆmin)1Ælg)1) I50(nM)C115.8 ± 8.0 6.6 ± 1.9 8.9 ± 1.7C224.7 ± 9.5 4.9 ± 2.2 7.8 ± 3.3C314.7 ± 5.3 4.3 ± 1.3 9.5 ± 2.1C418.6 ± 2.4 3.6 ± 0.5 6.9 ± 2.9C5+C612.5 ± 5.2 4.0 ± 1.3 7.2 ± 2.7CCCC+CCtwitchin132–+ –+ –+564–+ – +PAABMextractactinmantleextract?0.4C1C2C30.4C4C5+C6**0.0 0.5 0.5*RfRfFig. 6. In vitro phosphorylation of proteinsfrom mussel extracts by C-subunit isoforms.Aliquots of a crude extract from PAM,100 lg of protein (A), and from mantle tis-sue, 120 lg of protein (B), were individuallyincubated with MgCl2and [32P]ATP[cP] inthe absence and presence of each C-subunitisoform isolated from the correspondingtissue (5 unitsÆmg)1protein). Samples of C1,C2and C3were from fractions 20, 22 and29 of the Fig. 1A chromatogram, respec-tively, and samples of C4and C5+C6werefrom fractions 24 and 28 of the Fig. 2Achromatogram, respectively. At 20 min, allthe reactions were stopped by adding SDSsample buffer and boiling for 5 min.Samples were then analysed by 10%SDS ⁄ PAGE, and the gel was Coomassiestained, destained, dried and exposed forautoradiography at )80 °C. ?, unidentifiedprotein. The lower figures show the densito-metric analysis of the autoradiographs.Mussel C-subunit isoforms J. R. Bardales et al.4484 FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBSseems highly likely that C3and C5are the same iso-form present in both tissues, as they showed identicalapparent molecular masses, were eluted from a Mono-S column at the same salt concentration, indicatingsimilar pI values, and yielded near-identical PMFresults.In essence, mussel C-subunits could be: (a) encodedby various different genes; (b) generated by alternativesplicing from a single gene; or (c) produced by post-translational modifications (PTMs). Several authorshave reported that the purified C-subunits from differ-ent mammalian species can be separated into two frac-tions, called CAand CB, by means of cation exchangechromatography [17,18]. CAarises from CB, as a resultof the in vivo deamidation of the Asn2 residue, andtherefore the only difference between CAand CBwasthe presence of aspartic acid or asparagine, respec-tively, at position 2 of their sequences [15]. Unlikemammalian CAand CB, all the mussel C-subunitsshowed significant differences in their molecularmasses, as revealed by SDS ⁄ PAGE mobility. This find-ing rules out the possibility that some of them areproduced by a similar PTM to that generating mam-malian CAand CB, despite the fact that they were alsoseparated by cation exchange chromatography. On theother hand, all the mussel C-subunits are phosphory-lated at serine and threonine residue(s), and they couldnot be interconverted by treatment with MgATP,which suggests that the differences were not due toautophosphorylation. Finally, the comparison of PMFresults from tryptic digests showed that, with theexception of C3and C5, there was at least one peptidemass that was unique for each mussel C-subunit.Therefore, taken together, these results clearly indi-cated that mussel C-subunits are not generated as aconsequence of the PTMs typical of the PKA C-sub-unit, but rather they differ in their amino acidsequences.In most mammalian species, two principal genes forthe C-subunit have been identified and termed Ca andCb [19,20]; additionally, the human genome containsa third gene encoding the Cc-isoform, which appearsto be expressed only in testis [21]. Among inverte-brates, the nematode Caenorhabditis elegans also hastwo genes for the PKA C-subunit: the kin-1 gene,with potential to generate several C-subunit isoformsby alternative splicing, and the F47F2.1b gene, encod-ing a catalytic subunit-like protein [22,23]. Otherinvertebrate species, such as the fruit fly Drosophilamelanogaster [24], the mollusc Aplysia californica [25],the honeybee Apis mellifera [26] and the tick Ambly-omma americanum [27], seem to have a single geneencoding the C-subunit. Our results from MS analysisrevealed that almost all tryptic peptide masses werecommon to all C-subunit isoforms, and only a fewm ⁄ z peaks were specific for a particular isoform,which indicates that amino acid differences are notscattered over the whole sequences, but rather limitedto a particular region of the proteins. On the otherhand, the presence of a peak at 1605.8 Da wasobserved in the spectra of C2,C3⁄ C5and C6that wasabsent in those of C1and C4; moreover, a partialamino acid sequence derived from this peak matches asequence located at the N-terminal region of an alter-natively spliced C-subunit isoform from the molluscAplysia. Thus, taken together, these results indicatethat C2,C3⁄ C5and C6differ from C1and C4at theN-termini; that is, both sets of isoforms are likely tobe encoded by two alternative first exons. Interest-ingly, C1and C4also had a common peptide (m ⁄ zpeak 1059.5 Da), absent in the remaining isoforms,which would be the equivalent to that of 1605.8 Da,although, unfortunately, its sequence could not bedetermined. In conclusion, structural data stronglysuggest that at least some of the C-subunits identi-fied in mussel arise as a result of differentialsplicing events involving various forms of the firstexon, as has been widely reported for C-subunits fromboth mammalian and invertebrate sources [22,23,25,26,28–32].Sequence alignments of tryptic peptides from musselC-subunit isoforms with the bovine Ca-isoformshowed a degree of sequence identity near to 90%,which confirms that the PKA C-subunit is a highlyconserved protein. As expected, mussel sequences con-tain some structural motifs, conserved throughout theprotein kinase family, that are crucial for Mg2+andATP binding [2]. For example: (a) the glycine-rich loopor nucleotide positioning motif (GxGxxG), which isparticularly important for positioning the phosphatesof ATP; (b) the glutamic acid residue occupying posi-tion 91 in the bovine Ca-isoform, which suitably posi-tions Lys72, which, in turn, binds to the a-phosphateand b-phosphate of ATP; and (c) the Mg2+position-ing loop or DGF motif, with the aspartic acid residuechelating the primary Mg2+ion that bridges theb-phosphate and c-phosphate of ATP.Various authors have proposed that the functionalsignificance of C-subunit diversity could be related tothe different ability of C-subunit isoforms to phos-phorylate cellular proteins, and ⁄ or to interact withpartner proteins that determine the subcellular distri-bution of PKA activity [23,33,34]. In mussel, theC-subunit isoforms isolated from the PAM tissuedisplayed an identical pattern of protein phospho-rylation; however, the C-subunit isoforms from theJ. R. Bardales et al. Mussel C-subunit isoformsFEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4485mantle tissue showed minor but reproducible differ-ences in this pattern, despite the fact that they phos-phorylated a synthetic peptide substrate with similarapparent affinity. Specifically, certain proteins from amantle tissue extract were phosphorylated in vitro byC4, the main C-subunit isoform present in that tissue,but not by C5or C6. Therefore, this finding suggeststhat some of the mussel C-subunit isoforms differ intheir ability to phosphorylate cellular proteins, as hasalso been reported for Aplysia C-subunit isoforms[33].In summary, in this work we demonstrate the pres-ence of several structurally different isoforms of thePKA C-subunit in mussel tissues. In principle, thecombination of these catalytically active C-subunitswith the two types of R-subunit previously identified(Rmyt1and Rmyt2[11–13]) could potentially generatemultiple PKA holoenzymes. In order to establish thefunctional differences among these PKA isoforms, itwould now be interesting to investigate the ability ofC-subunits to interact with partner proteins, includingRmyt1and Rmy2, and to examine the cellular distribu-tion of both R-subunit and C-subunit isoforms in themussel tissues.Experimental proceduresMolluscsSea mussels of the species M. galloprovincialis Lmk. werecollected from a sea farm located at the Rı´a de Betanzos(Galicia, north-west Spain). Molluscs were placed in tankscontaining seawater and transported to the laboratory.Tissues were dissected out and immediately frozen at)20 °C until use.Mussel extractsMantle tissue was homogenized 1 : 3 (m ⁄ v) in ice-cold buf-fer A (pH 7.0) [55 mm potassium phosphate, 2 mm EDTA,1mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride,1mgÆL)1leupeptin and 1 mgÆL)1pepstatin A (Sigma-Aldrich Quı´mica, Madrid, Spain)], using a Potter-Elvehjemhomogenizer. PAM tissue was homogenized 1 : 6 (m ⁄ v) inice-cold buffer B (pH 7.0) (30 mm potassium phosphate,2mm EDTA, 1 mm dithiothreitol, 1 mm phenyl-methanesulfonyl fluoride, 1 mgÆL)1leupeptin and 1 mgÆL)1pepstatin A), using a blade homogenizer (VirTis TempestIQ2; SP Industries, Warminster, PA, USA). The homogen-ates were centrifuged at 35 000 g for 30 min at 4 °Cinarefrigerated centrifuge (Beckman Coulter, Fullerton, CA,USA), and the supernatants, once filtered through glasswool, constituted the crude extracts.Separation of C-subunit isoformsFirst, C-subunit was purified from PAM and mantle tissuesas described previously [35,36]. Briefly, the procedure isbased on the binding of PKA, through its R-subunit, toDEAE–cellulose, and the specific elution of the C-subunitby addition of cAMP, which causes the dissociation ofholoenzyme. The crude extract obtained from each tissuewas mixed with DEAE–cellulose (DE52; Whatman Interna-tional, Maidstone, UK) at 30 mL gel per gram of protein.After 2 h of gentle stirring, the gel was allowed to settle –to allow the supernatant containing unbound proteins to bediscarded – and then packed into a chromatographiccolumn. Next, the gel was extensively washed with thehomogenization buffer, and then C-subunit was specificallyeluted with the same buffer containing 0.12 mm cAMP(Sigma-Aldrich Quı´mica). The fractions showing proteinkinase activity were pooled and concentrated to  3mLbyultrafiltration through a PM-30 membrane (Millipore,Bedford, MA, USA). This procedure allows enzymatic prep-arations containing mainly C-subunit together with minorcontaminant proteins to be obtained. The separation ofC-subunit isoforms was performed by means of cationexchange chromatography on a Mono-S HR 5 ⁄ 5 FPLCcolumn (GE Healthcare Bioscience, Uppsala, Sweden). Sam-ples (2 mL) of the enzymatic preparations obtained from thePAM and the mantle tissues were applied to the column,previously equilibrated with buffer C (pH 6.8) (45 mmpotassium phosphate, 1 mm dithiothreitol). The column wasthen washed with buffer C to eliminate most contaminantproteins, and C-subunit isoforms were eluted by applying acontinuous NaCl gradient (0–0.4 m in buffer C). Thecollected fractions of 0.5 mL were assayed for protein kinaseactivity, and also analysed by SDS ⁄ PAGE and western blot-ting.C-subunit from bovine heart was purified following theprocedure of Pepperkok et al. [16], and purified enzymewas separated into fractions CAand CBby cation exchangechromatography on a Mono-S HR 5 ⁄ 5 column (GEHealthcare Bioscience) [16].Assay of C-subunit activity and determination ofkinetic parametersC-subunit activity was assayed using the synthetic peptideKemptide (Sigma-Aldrich Quı´mica) as substrate. In a totalvolume of 50 lL, the assay contained 50 mm Tris ⁄ HCl(pH 7.0), 1 mm dithiothreitol, 5 mm MgCl2, 0.2 mm[32P]ATP[cP] ( 100 c.p.m.Æpmol)1) (Hartmann Analytic,Braunschweig, Germany), and a sample, suitably diluted,containing C-subunit. The reactions were started by addi-tion of 100 lm Kemptide. In the kinetic experiments, theconcentrations of Kemptide ranged from 5 to 150 lm andthe concentrations of PKI(5–24) (Sigma-Aldrich Quı´mica)Mussel C-subunit isoforms J. R. Bardales et al.4486 FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBSranged from 5 to 200 nm. After 10 min at 25 °C, the reac-tions were stopped by addition of 10 lL of 300 mm phos-phoric acid. Next, 30 lL of the mixture was spotted onto aphosphocellulose disc paper, and the discs were: (a) washedthree times with 75 mm phosphoric acid and gently shakento remove free ATP; (b) dried under a lamp; and (c)counted with 5 mL of scintillation liquid Ecoscint H(National Diagnostics, Hessle, UK) in a scintillation coun-ter. One activity unit was defined as the quantity of enzymethat transfers 1 nmol of phosphate to Kemptide per min.Experimental data describing the dependence of proteinkinase activity on Kemptide concentrations were fitted tothe Michaelis–Menten equation, and the values of theMichaelis–Menten (Km) and maximum velocity (Vmax) con-stants were determined from the plots 1 ⁄ Voversus 1 ⁄ [S],where Vois the initial rate at a given substrate concentra-tion [S]. The I0.5for PKI(5–24) (concentration of peptidethat reduces enzyme activity by 50%) was determined fromplots of Voversus [PKI(5–24)] at saturating concentrationsof Kemptide and ATP.Phosphorylation of mussel proteins by purifiedC-subunit isoformsAliquots of the crude extract from PAM (100 lg of pro-tein) or from mantle tissue (120 l g of protein) were individ-ually incubated at 25 °C with each tissue-purified C-subunitisoform (5 unitsÆmg)1protein) in the presence of 5 mmMgCl2and 0.2 mm [32P]ATP[cP] ( 500 c.p.m.Æpmol)1). At20 min, reactions were stopped by adding a one-quartervolume of SDS sample buffer [250 mm Tris ⁄ HCl (pH 6.8),8% (m ⁄ v) SDS, 20% (v ⁄ v) 2-mercaptoethanol, 40% (v ⁄ v)glycerol] and boiled for 5 min. Samples were then analysedby 10% SDS ⁄ PAGE, and the gel was stained with CoomassieBrilliant Blue R (Sigma-Aldrich Quı´mica), destained, dried,and exposed for autoradiography at )80 °C. Densitometricevaluation of the autoradiographs was carried out using theversadoc imaging system (Bio-Rad Laboratories, Hercules,CA, USA).SDS/PAGE and western blottingSDS ⁄ PAGE was carried out according to Laemmli [37],using 10% polyacrylamide slab-gels of size 16 · 16 cm(Protean II xi cell) or 8.2 · 6.2 cm (Mini Protean 3 cell)(Bio-Rad Laboratories). For performance of western blotanalysis, the proteins were transferred to a poly(vinylidenedifluoride) membrane (Immobilon-P; Millipore) by applyinga 400 mA current for 2 h at 4 °C. After blocking for 6 h atroom temperature with 5% nonfat dry milk in 20 mmTris ⁄ HCl with Tween-20 (Tris ⁄ HCl, pH 7.5, 0.15 m NaCl,0.1% Tween-20), membranes were washed with Tris ⁄ HClwith Tween-20 and then incubated overnight at 4 °C withthe primary antibodies: (a) polyclonal antibody againsthuman Ca-isoform (sc903; Santa Cruz Biotechnology,Santa Cruz, CA, USA) diluted 1 : 2500 in Tris ⁄ HCl withTween-20; (b) monoclonal antibody against phosphoserine(P3430; Sigma-Aldrich Quı´mica) diluted 1 : 2000 inTris ⁄ HCl with Tween-20; or (c) monoclonal antibodyagainst phosphothreonine (P6623; Sigma-Aldrich Quı´mica)diluted 1 : 1500 in Tris ⁄ HCl with Tween-20. After washingwith Tris ⁄ HCl with Tween-20, the blots were incubatedfor 1 h at room temperature with secondary antibodies(anti-rabbit IgG or anti-mouse IgG, diluted 1 : 50 000 and1 : 25 000 in Tris ⁄ HCl with Tween-20, respectively) conju-gated to horseradish peroxidase (Sigma-Aldrich Quı´mica).Next, the blots were: (a) extensively washed; (b) developedwith the chemiluminiscent horseradish peroxidase substrate(Millipore); and (c) exposed to X-ray film (Curix RP2 Plus;Agfa-Gevaert, Mortsel, Belgium) for a few seconds.MSSamples of mussel C-subunit isoforms and bovine C-sub-unit (fraction CB[16,17]) were first reduced by adding aone-quarter volume of SDS sample buffer supplementedwith dithiothreitol to a final concentration of 10 mm, andthen alkylated with 20 mm iodoacetamide (Sigma-AldrichQuı´mica) for 30 min in the dark. Next, proteins were sepa-rated by SDS ⁄ PAGE and subjected to ‘in-gel’ digestionprocedure with modified trypsin, sequence grade (Promega,Madison, WI, USA) [38]. Digested samples were analysedusing a MALDI-TOF ⁄ TOF Ultraflex instrument (BrukerDaltonics, Bremen, Germany) in reflector mode to obtainPMF spectra. Sequences of tryptic peptides from C-subun-its were determined using the CAF-PSD approach [39].After the peptide mixture was sulfonated by the CAFreagent (GE Healthcare Bioscience), PMF was performedagain and peptide masses that had increased their massesby 136 or 204 Da were searched for. The former representtryptic peptides with a C-terminal arginine, and the latterthose with a C-terminal lysine, where the e-amino groupsof lysine residues were blocked with 2-methoxy-4,5-dihy-dro-1H-imidizole (Lys Tag 4H; Agilent Technologies, SantaClara, CA, USA) in order to prevent lysine from beingsulfonated. The MALDI instrument was switched to PSDmode and the ion selector was adjusted for the appropriatemass. PSD analysis was performed according to the manu-facturer’s instructions, and the generated spectra, mainlyconsisting of a clean y-ion series, were interpreted manu-ally. A sequence homology search for the mussel C-subunitisoforms was conducted using the ‘short, nearly exactmatches’ option of blast [40].AcknowledgementsThis work was supported by grant PGIDIT02-RMA26101PR from the Autonomous Government ofGalicia (Xunta de Galicia).J. R. Bardales et al. Mussel C-subunit isoformsFEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4487References1 Taylor SS, Buechler JA & Yonemoto W (1990) cAMP-dependent protein kinase: framework for a diversefamily of regulatory enzymes. Annu Rev Biochem 59,971–1005.2 Smith CM, Radzio-Andzelm E, Madhusudan, AkamineP & Taylor SS (1999) The catalytic subunit of cAMP-dependent protein kinase: prototype for an extendednetwork of communication. Prog Biophys Mol Biol 71,313–341.3 Siegman MJ, Funabara D, Kinoshita S, Watabe S,Hartshorne D & Butler TM (1998) Phosphorylationof a twitchin-related protein controls catch andcalcium sensitivity of force production in invertebratesmooth muscle. Proc Natl Acad Sci USA 95, 5383–5388.4 Yamada A, Yoshio M, Kojima H & Oiwa K (2001) Anin vitro assay reveals essential protein components forthe catch state of invertebrate smooth muscle. Proc NatlAcad Sci USA 98, 6635–6640.5 Ishii N, Simpson AWM & Ashley CC (1989) Free cal-cium at rest during catch in single smooth muscle cells.Science 243, 1367–1368.6 Ferna´ndez M, Cao J, Vega FV, Hellman U, Wernstedt C& Villamarı´n JA (1997) cAMP-dependent phosphoryla-tion activates phosphofructokinase from mantle tissue ofthe mollusc Mytilus galloprovincialis. Identification of thephosphorylated site. Biochem Mol Biol Int 43, 173–181.7 Ferna´ndez M, Cao J & Villamarı´n JA (1998) In vivophosphorylation of phosphofructokinase from thebivalve mollusk Mytilus galloprovincialis. Arch BiochemBiophys 353, 251–256.8 Sanjuan-Serrano F, Ferna´ndez-Gonza´lez M, Sa´nchez-Lo´pez JL & Garcı´a-Martı´n LO (1995) Molecularmechanism of the control of glycogenolysis by cal-cium ions and cyclic AMP in the mantle of Mytilusgalloprovincialis Lmk. Comp Biochem Physiol B 110,577–582.9 Storey KB & Storey JM (1990) Facultative metabolicrate depression: molecular regulation and biochemicaladaptation in anaerobiosis, hibernation and aestivation.Q Rev Biol 65, 145–174.10 Storey KB & Storey JM (2004) Metabolic rate depres-sion in animals: transcriptional and translational con-trols. Biol Rev 79, 207–233.11 Cao J, Ramos-Martı´nez JI & Villamarı´n JA (1995)Characterization of a cAMP-binding protein from thebivalve mollusc Mytilus galloprovincialis. Eur J Biochem232, 664–670.12 Rodrı´guez JL, Barcia R, Ramos-Martı´nez JI & Villa-marı´n JA (1998) Purification of a novel isoform of theregulatory subunit of cAMP-dependent protein kinasefrom the bivalve mollusk Mytilus galloprovincialis. ArchBiochem Biophys 359, 57–62.13 Dı´az-Enrich MJ, Ibarguren I, Hellman U & Villamarı´nJA (2003) Characterization of a type I regulatory sub-unit of cAMP-dependent protein kinase from thebivalve mollusk Mytilus galloprovincialis. Arch BiochemPhysiol 416, 119–127.14 Bardales JR, Hellman U & Villamarı´n JA (2007) CK2-mediated phosphorylation of type II regulatory subunitof cAMP-dependent protein kinase from the molluscMytilus galloprovincialis. Arch Biochem Physiol 461,130–137.15 Jedrzejewski PT, Girod A, Tholey A, Ko¨nig N, Thull-ner S, Kinzel V & Bossemeyer D (1998) A conserveddeamidation site at Asn 2 in the catalytic subunit ofmammalian cAMP-dependent protein kinase detectedby capillary LC-MS and tandem mass spectrometry.Protein Sci 7, 457–469.16 Pepperkok R, Hotz-Wagenblatt A, Ko¨nig N, Girod A& Bossemeyer D (2000) Intracellular distribution ofmammalian protein kinase A catalytic subunit alteredby conserved Asn2 deamidation. J Cell Biol 148, 715–726.17 Kinzel V, Hotz A, Ko¨nig N, Gagelmann M, Pyerin W,Reed J, Ku¨bler D, Hofmann F, Obst C, GensheimerHP et al. (1987) Chromatographic separation of twoheterogeneous forms of the catalytic subunit of cyclicAMP-dependent protein kinase holoenzyme type I andtype II from striated muscle of different mammalianspecies. Arch Biochem Biophys 253, 341–349.18 Herberg FW, Bell S & Taylor SS (1993) Expression ofthe catalytic subunit of cAMP-dependent protein kinasein Escherichia coli: multiple isozymes reflect differentphosphorylation states. Protein Eng 6, 771–777.19 Uhler MD, Carmichael DF, Lee DC, Chrivia JC, KrebsEG & McKnight GS (1986) Isolation of cDNA clonescoding for the catalytic subunit of mouse cAMP-depen-dent protein kinase. Proc Natl Acad Sci USA 83, 1300–1304.20 Uhler MD, Chrivia JC & McKnight GS (1986)Evidence for a second isoform of the catalytic subunitof cAMP-dependent protein kinase. J Biol Chem 261,15360–15363.21 Beebe SJ, Oyen O, Sandberg M, Froysa A, HanssonV & Jahnsen T (1990) Molecular cloning of a tissue-specific protein kinase (Cc) from human testis –representing a third isoform for the catalytic subunitof cAMP-dependent protein kinase. Mol Endocrinol 4 ,465–475.22 Gross RE, Bagchi S, Lu X & Rubin CS (1990) Cloning,characterization and expression of the gene for thecatalytic subunit of cAMP-dependent protein kinase inCaenorhabditis elegans. J Biol Chem 265, 6896–6907.23 Tabish M, Clegg RA, Rees HH & Fischer MJ (1999)Organization and alternative splicing of the Caenor-habditis elegans cAMP-dependent protein kinase cata-lytic-subunit gene (kin-1). Biochem J 339, 209–216.Mussel C-subunit isoforms J. R. Bardales et al.4488 FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS[...]... (2006) Expression of multiple isoforms of the cAMP-dependent protein kinase (PK-A) catalytic subunit in the nematode Caenorhabditis elegans Cell Signal 18, 2230–2237 ´ Bejar P & Villamarı´ n JA (2006) Catalytic subunit of cAMP-dependent protein kinase from a match muscle of the bivalve mollusk Mytilus galloprovincialis: purification, characterization, and phosphorylation of muscle proteins Arch Biochem... Menzel R (2001) Cloning of a catalytic subunit of cAMP-dependent protein kinase from the honeybee (Apis mellifera) and its localization in the brain Insect Mol Biol 10, 173–181 27 Palmer MJ, McSwain JL, Spatz MD, Tucker JS, Essenberg RC & Sauer JR (1999) Molecular cloning of cAMP-dependent protein kinase catalytic subunit isoforms from the lone star tick, Amblyomma americanum (L) Insect Biochem Mol... RamosMartinez JI & Villamarı´ n JA (1995) Purification and characterization of the catalytic subunit of cAMPdependent protein kinase from the bivalve mollusc Mytilus galloprovincialis Comp Biochem Physiol B 111, 453–462 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 277, 680–685 ´ Hellman U, Wernstedt C, Gomez J & Heldin CH (1995) Improvement of. .. Skalhegg BS (2001) Identification of novel splice variants of the human catalytic subunit Cb of Mussel C -subunit isoforms 33 34 35 36 37 38 39 40 cAMP-dependent protein kinase Eur J Biochem 268, 5066–5073 Panchal RG, Cheyley S & Bayley H (1994) Differential phosphorylation of neuronal substrates by catalytic subunits of Aplysia cAMP-dependent protein kinase with alternative N termini J Biol Chem 269,... Expression of a nonmyristylated variation of the catalytic subunit of protein kinase A during male germ-cell development Proc Natl Acad Sci USA 97, 6433–6438 ´ 31 Reinton N, Orstavik S, Haugen TB, Jahnsen T, Tasken T & Skalhegg BS (2000) A novel isoform of human cyclic 3¢,5¢-adenosine monophosphate-dependent protein kinase, Ca-s, localizes to sperm midpiece Biol Reprod 63, 607–611 32 Orstavik S, Reinton... 28 Beushausen S, Lee E, Walker B & Bayley H (1992) Catalytic subunits of Aplysia neuronal cAMP-dependent protein kinase with two different N termini Proc Natl Acad Sci USA 89, 1641–1645 29 Tabish M, Clegg RA, Turner CP, Jonczy J, Rees HH & Fisher MJ (2006) Molecular characterization of cAMPdependent protein kinase (PK-A) catalytic subunit isoforms in the male tick, Amblyomma hebraeum Mol Biochem Parasitol...J R Bardales et al 24 Kalderon D & Rubin GM (1998) Isolation and characterization of Drosophila cAMP-dependent protein kinase genes Genes Dev 2, 1539–1556 25 Beushausen S, Bergold P, Sturner S, Elste A, Roytenberg V, Schwartz H & Bayley H (1988) Two catalytic subunits of cAMP-dependent protein kinase generated by alternative RNA splicing are expressed in Aplysia neurons Neuron 1, 853–864 26 Eisenhardt... Wernstedt C, Gomez J & Heldin CH (1995) Improvement of an In- Gel’ digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing Anal Biochem 224, 451–455 Hellman U & Bhikhabhai R (2002) Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption ⁄ ionization time -of- flight mass spectrometer equipped with a variable... Spectrom 16, 1851–1859 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W & Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 25, 3389–3402 FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4489 . Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis Jose´R C -subunit, catalytic subunit of cAMP-dependent protein kinase; PAM,posterior adductor muscle; PKA, cAMP-dependent protein kinase; PKI(5–24), protein kinase
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Xem thêm: Báo cáo khoa học: Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis potx, Báo cáo khoa học: Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis potx, Báo cáo khoa học: Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis potx