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

On the Keywords cAMP-dependent protein kinase; catalytic subunit; C-subunit isoforms; Correspondence J.. Villamarı´n, Departamento de Bioquı´mica e Bioloxı´a Molecular, Facultade de Vete

protein kinase catalytic subunit in the bivalve mollusc

Mytilus galloprovincialis

Jose´ R Bardales1, Ulf Hellman2and J A Villamarı´n1

1 Departamento de Bioquı´mica e Bioloxı´a Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain

2 Ludwig Institute for Cancer Research, Uppsala, Sweden

The cAMP-dependent protein kinase (PKA; EC

2.7.11.11) plays a crucial role in the regulation of

several physiological processes, as it is the main

media-tor of the effects of cAMP in eukaryotic organisms

Inactive PKA is a tetrameric holoenzyme composed of

two functionally distinct subunits: a dimeric regulatory

subunit (R-subunit) and two monomeric catalytic

subunits (C-subunits) The main function of the

R-sub-unit is to inhibit the phosphotransferase activity of the

C-subunit The transitory increase of cAMP levels

inside the cell, induced by an extracellular signal, and

the binding of cyclic nucleotide to R-subunits cause

the dissociation of C-subunits which, once free, can

phosphorylate protein substrates, mainly in the cyto-plasm, but also in the nucleus [1,2]

It has been widely reported that PKA is involved in the regulation of some physiological events that specifi-cally occur in bivalve molluscs as a consequence of environmental adaptation For example, the relaxation

of mollusc ‘catch’ muscles, induced by serotonin, occurs through the PKA-mediated phosphorylation of twitchin, a high molecular mass protein present in the thick filaments [3,4] The mollusc ‘catch’ muscles, such

as the posterior adductor muscle (PAM), are special-ized muscles that can sustain high tension for very long periods with low energy expenditure [5] On the

Keywords

cAMP-dependent protein kinase;

catalytic subunit; C-subunit isoforms;

Correspondence

J A Villamarı´n, Departamento de

Bioquı´mica e Bioloxı´a Molecular, Facultade

de Veterinaria, Universidade de Santiago de

Compostela, Campus de Lugo, 27002 Lugo,

Spain

Fax: +34 82 252 195

Tel: +34 82 285 900

E-mail: antonio.villamarin@usc.es

(Received 28 March 2008, revised 4 July

2008, accepted 10 July 2008)

doi:10.1111/j.1742-4658.2008.06591.x

Several isoforms of the cAMP-dependent protein kinase catalytic subunit (C-subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange 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 at serine and threonine residues and showed slight but significant differences

in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa The results from the MS analysis suggest that at least some of the mussel C-subunit isoforms arise as a result of alternative splicing events Furthermore, several peptide sequences from mussel C-subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Ca-isoform, and contained some structural motifs that are essential 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, the C-subunit isoforms separated from the mantle tissue differed in their ability

to phosphorylate in vitro some proteins present in a mantle extract

Abbreviations

CAF-PSD, chemically assisted fragmentation–post-source decay; C-subunit, catalytic subunit of cAMP-dependent protein kinase; PAM,

fingerprinting; PTM, post-translational modification; R-subunit, regulatory subunit of cAMP-dependent protein kinase.

other hand, phosphofructokinase from the sea mussel

Mytilus galloprovincialis, unlike that from mammals,

was clearly activated when phosphorylated by PKA at

a serine residue [6]; moreover, the enzyme activity

changed seasonally in parallel with its phosphorylation

degree [7] These and other results reported by other

authors [8] suggest that PKA activation contributes to

the regulation of carbohydrate metabolism during

bivalve 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 for

metabolic rate depression, a strategy that bivalve

mol-luscs use to survive during the long periods of aerial

exposure causing environmental hypoxia [9,10]

Therefore, to understand the biochemical basis of

these molluscan regulatory events, the diverse forms of

PKA in these organisms must be defined Over the last

few years, we have identified and purified two different

isoforms of the PKA R-subunit from the sea mussel

M galloprovincialis, which were named Rmyt1 and

Rmyt2 [11–13] Interestingly, both isoforms have

iden-tical apparent molecular masses of 54 kDa, but they

differ in: (a) their isoelectric point; (b) their

biochemi-cal properties; (c) their antigenicity; and (d) their tissue

distribution [12–14] According to its physicochemical

and biochemical properties, a partial amino acid

sequence from Rmyt1 showed a clear homology with

the type I R-subunits from both mammalian and

invertebrate sources [13]; likewise, Rmyt2was shown to

be homologous to the type II R-subunits from the

same species [14]

The purpose of the work described in this article

was to investigate the possible existence of different

isoforms of the PKA C-subunit in the sea mussel

M galloprovincialis

Results

Separation of different isoforms of the C-subunit

In order to demonstrate the presence of different

iso-forms of the PKA C-subunit in mussels, the protein

was partially purified from the PAM and the mantle

tissues of the mollusc, and then subjected to cation

exchange chromatography on a Mono-S column

Figure 1A shows the elution profile corresponding to

the PAM C-subunit The application of a salt gradient

resulted in separation of four absorbance peaks Three

of 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 a

protein with apparent molecular mass  40 kDa in the fractions corresponding to peak I, peak II and peak III (Fig 1B) This protein band was recognized

by an antibody raised against the human Ca-isoform

of the C-subunit (Fig 1C) Therefore, peak I, peak II and peak III correspond to three different isoforms of the C-subunit, which we named C1, C2 and C3, respec-tively On the other hand, fraction 18, corresponding

to the first absorbance peak, without protein kinase activity, contained an unidentified protein < 30 kDa, and fractions 21–23 also contained an unidentified high molecular mass protein (Fig 1B) None of these proteins was recognized by the Ca-isoform antibody in the western blot analysis (Fig 1C)

Figure 2A shows a representative elution pattern of the C-subunit preparation obtained from the mantle tissue Two absorbance peaks, associated with protein kinase activity, were separated; these eluted at 0.19 and 0.25 m NaCl, and were labelled peak I and peak II, respectively Coomassie staining of an SDS⁄ PAGE gel revealed that fractions corresponding to peak I

A

Fig 1 Separation and identification of PKA C-subunit isoforms from mussel PAM (A) Elution profile of C-subunit from a Mono-S

purified from PAM as described in Experimental procedures was applied 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 activity were separated: I, II and III Aliquots of fractions were also

with an antibody against the human Ca-isoform.

contained only a 40 kDa protein, whereas those

cor-responding to peak II contained two different protein

bands 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 were

separated from the mantle tissue preparation: the

isoform named C4, which corresponded to peak I of

the Mono-S chromatogram, and the isoforms named

C5 and C6, which coeluted together at peak II C4 was

3–4-fold more abundant than C5and C6together

Characterization of C-subunit isoforms

Samples of purified C1–C6 were analysed by SDS⁄

PAGE, using a 16· 16 cm polyacrylamide gel As

shown in Fig 3A, slight but significant differences

were observed in the migration behaviour among

mus-sel isozymes Only C3(from PAM) and C5 (from

man-tle) have identical apparent mobilities, which suggests

that they could be the same isoform present in both

tissues The values of the apparent molecular mass ranged between 41.3 kDa for C4 and 44.5 kDa for C6 All the mussel isoforms were slightly heavier than the bovine C-subunit used as a control (lane 2), whose molecular mass, determined by MS, was exactly

40 855.7 Da [15]

On the other hand, samples of purified mussel

C1–C6were probed with both phosphoserine and phos-phothreonine antibodies, and they were all serine and threonine phosphorylated, as shown in Fig 3B,C, respectively Moreover, incubation of C-subunit isoforms with MgATP did not change their mobility

on SDS⁄ PAGE (not shown)

Structural analysis of C-subunit isoforms

In order to determine possible structural differences among mussel C-subunit isoforms, samples of purified

C1–C6 proteins were subjected to ‘in-gel’ tryptic digestion, and peptide mixtures were analysed by MALDI-TOF MS The corresponding peptide mass fingerprinting (PMF) spectra are shown in Fig 4 Furthermore, a sample of C-subunit purified from bovine heart (fraction CB), consisting mainly of the

A

B C

Fig 2 Separation and identification of PKA C-subunit isoforms

from mussel mantle tissue (A) Elution profile of C-subunit from a

C-subunit purified from mantle tissue as described in Experimental

procedures was applied to the column and eluted with a linear salt

gradient Fractions of 0.5 mL were collected and assayed for

pro-tein kinase activity Two distinct peaks associated with propro-tein

kinase activity were separated: I and II Aliquots of fractions were

wes-tern blotting with an antibody against the human Ca-isoform.

A

Fig 3 Characterization of mussel C-subunit isoforms (A)

polyacrylamide gel which was Coomassie stained Lane 1: molecu-lar mass standards Lane 2: sample of bovine heart C-subunit,

Fig 1A chromatogram, respectively Lanes 6 and 7: fractions 24 and 28 of Fig 2A chromatogram, respectively The apparent molec-ular mass of mussel C-subunit isoforms was estimated from the positions 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%

monoclonal antibodies against phosphoserine (B) or phosphothreo-nine (C).

Ca-isoform [15,16], was also digested and analysed A

detailed analysis of data allowed us to draw the

fol-lowing conclusions (a) Eight peptide masses were

found to be common to the bovine Ca-isoform and all

mussel 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 identical

to those measured are marked with dashed lines in the whole sequence of the bovine Ca-isoform in Fig 5A Furthermore, two of these common peptides, which yielded peaks at m⁄ z 744.5 and 895.5, were sequenced

de novo by chemically assisted fragmentation–post-source decay (CAF-PSD) (Table 1: peptides 1 and 2), and exactly matched the sequences of the bovine

1.25

5

x1 0

*

C 2

0.25

0.50

0.75

1.00

1.25

5

x1 0

C 3

0.25

0.50

0.75

1.00

1 5

5

x1 0

0 5

1 0

1 0

5

x1 0

734.385 804.21

0 2

0 4

0 6

0 8

2 0

5

x1 0

C 5

959.450 1065.99

0 5

1 0

1 5

1 0

5

x1 0

0 0

0 2

0 4

0 6

0 8

*

Fig 4 Peptide mass fingerprints of mussel C-subunit isoforms after tryptic digestion The asterisk(s) indicate the peak(s) observed only in

Ca-isoform corresponding to amino acids 73–78 and

48–56, respectively (Fig 5A) (b) Most peptide masses

were common to all mussel isoforms, C1–C6 Several

of these peptides were also sequenced de novo (Table 1:

peptides 3–12), showing high amino acid sequence

identities to the bovine Ca-isoform (Fig 5A) (c) With

regard to the C1, C2, C4 and C6 spectra, there was at

least 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 the

spectra of Fig 5: 791.4 (only in C1); 1230.6 (only in

C2); 1768.6 and 2386.9 (only in C4); and 2623.3 (only

in C6) (d) There was one peak at 1059.5 Da observed only in the C1 and C4 spectra, whereas another peak

at 1605.7 Da was found in the spectra of the remaining isoforms: C2, C3, C5 and C6 Interestingly, an incom-plete sequence derived from this last peak (Table 1: peptide 13) matches a sequence lying at the N-terminus

of a C-subunit (N1-isoform) from the mollusc Aplysia (Fig 5B) This result indicates that mussel C1 and C4 differ from C2, C3, C5 and C6 at the N-terminal region (e) When spectra from C3 and C5 were com-pared, no significant difference was observed, which suggests that both C3 and C5 are the same C-subunit isoforms present in the PAM and the mantle tissue respectively

Kinetic characterization of C-subunit isoforms and protein phosphorylation

In order to determine possible functional differences among mussel C-subunit isoforms, the kinetic parame-ters were determined for each purified isozyme It should be noted that C5 and C6 coeluted from the Mono-S column, and therefore, samples containing a mix of both isoforms were used in the kinetic experi-ments No significant differences among C-subunit isoforms regarding the values of apparent Km for Kemptide and Vmax were observed Furthermore, all the mussel isozymes were inhibited by the protein kinase inhibitor peptide [PKI(5–24)] with similar I50 values (Table 2)

The ability of mussel C-subunit isoforms to phos-phorylate proteins in vitro was also investigated Thus,

Fig 5 Comparison of amino acid

sequences 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 in

the catalytic function (see text) Dashed

lines show the partial sequences of the

bovine Ca-isoform corresponding to the

com-mon to bovine and all mussel C-subunit

isoforms.

Table 1 Peptides from Mytilus C-subunit isoforms identified by

de novo sequencing.

Peptide

Measured

Present in spectra

C1, C2 and C3 (purified from PAM) were individually

incubated, in the presence of labelled ATP, with

aliqu-ots of a PAM extract In the same way, C4 and the

mixture of C5 and C6 were incubated with samples of

a mantle tissue extract Densitometric analysis of

auto-radiographs corresponding to the PAM samples

showed identical protein phosphorylation patterns for

C1, C2and C3(Fig 6A) Three protein bands, marked

with arrows in Fig 6A, were mainly phosphorylated

by each C-subunit isoform The protein with the

high-est molecular mass ( 600 kDa) was identified as

twit-chin, whose PKA-mediated phosphorylation had been

previously demonstrated [3,4] The intermediate pro-tein band was identified as actin by PMF and de novo sequence analysis (not shown) The protein band with the lowest molecular mass could not be identified by PMF; a correct sequence of 14 amino acids (RESE-FQSGDLWEVR) was then obtained by de novo sequencing, although no clear identity could be drawn from the databases

For the mantle extract (Fig 6B), the patterns of proteins phosphorylated by C4 and the mixture of C5 and C6were also apparently similar, although densito-metric analysis of the autoradiograph showed some protein bands, marked by asterisks, that seemed to

be phosphorylated by C4 but not by the mix of C5 and C6 Thus, it is possible that mantle isoforms have different abilities to phosphorylate some proteins of mantle tissue

Discussion

In this article, we describe the separation and identifi-cation of several catalytically active isoforms of the PKA C-subunit from the sea mussel M galloprovin-cialis The isozymes named C1, C2 and C3 were isolated from the PAM tissue, whereas C4, C5 and C6 were separated from the mantle tissue However, it

Table 2 Kinetic parameters of mussel C-subunit isoforms The

means ± SE of three independent experiments.

twitchin

– + – + – +

4

– + – +

PA

M

extract

actin

mantle extract

?

0.4

C 1

C 2

C 3

0.4

C 4

C 5 +C 6

*

*

0.0

0.2

0.0

Fig 6 In vitro phosphorylation of proteins from 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 individually

the absence and presence of each C-subunit isoform isolated from the corresponding

29 of the Fig 1A chromatogram,

from fractions 24 and 28 of the Fig 2A chromatogram, respectively At 20 min, all the reactions were stopped by adding SDS sample buffer and boiling for 5 min Samples were then analysed by 10%

stained, destained, dried and exposed for

protein The lower figures show the densito-metric analysis of the autoradiographs.

seems highly likely that C3 and C5 are the same

iso-form present in both tissues, as they showed identical

apparent molecular masses, were eluted from a

Mono-S column at the same salt concentration, indicating

similar pI values, and yielded near-identical PMF

results

In essence, mussel C-subunits could be: (a) encoded

by various different genes; (b) generated by alternative

splicing from a single gene; or (c) produced by

post-translational modifications (PTMs) Several authors

have reported that the purified C-subunits from

differ-ent mammalian species can be separated into two

frac-tions, called CA and CB, by means of cation exchange

chromatography [17,18] CAarises from CB, as a result

of the in vivo deamidation of the Asn2 residue, and

therefore the only difference between CA and CB was

the presence of aspartic acid or asparagine,

respec-tively, at position 2 of their sequences [15] Unlike

mammalian CA and CB, all the mussel C-subunits

showed significant differences in their molecular

masses, as revealed by SDS⁄ PAGE mobility This

find-ing rules out the possibility that some of them are

produced by a similar PTM to that generating

mam-malian CA and CB, despite the fact that they were also

separated by cation exchange chromatography On the

other hand, all the mussel C-subunits are

phosphory-lated at serine and threonine residue(s), and they could

not be interconverted by treatment with MgATP,

which suggests that the differences were not due to

autophosphorylation Finally, the comparison of PMF

results from tryptic digests showed that, with the

exception of C3 and C5, there was at least one peptide

mass that was unique for each mussel C-subunit

Therefore, taken together, these results clearly

indi-cated that mussel C-subunits are not generated as a

consequence of the PTMs typical of the PKA

C-sub-unit, but rather they differ in their amino acid

sequences

In most mammalian species, two principal genes for

the C-subunit have been identified and termed Ca and

Cb [19,20]; additionally, the human genome contains

a third gene encoding the Cc-isoform, which appears

to be expressed only in testis [21] Among

inverte-brates, the nematode Caenorhabditis elegans also has

two genes for the PKA C-subunit: the kin-1 gene,

with potential to generate several C-subunit isoforms

by alternative splicing, and the F47F2.1b gene,

encod-ing a catalytic subunit-like protein [22,23] Other

invertebrate species, such as the fruit fly Drosophila

melanogaster [24], the mollusc Aplysia californica [25],

the honeybee Apis mellifera [26] and the tick

Ambly-omma americanum [27], seem to have a single gene

encoding the C-subunit Our results from MS analysis

revealed that almost all tryptic peptide masses were common to all C-subunit isoforms, and only a few

m⁄ z peaks were specific for a particular isoform, which indicates that amino acid differences are not scattered over the whole sequences, but rather limited

to a particular region of the proteins On the other hand, the presence of a peak at 1605.8 Da was observed in the spectra of C2, C3⁄ C5 and C6 that was absent in those of C1 and C4; moreover, a partial amino acid sequence derived from this peak matches a sequence located at the N-terminal region of an alter-natively spliced C-subunit isoform from the mollusc Aplysia Thus, taken together, these results indicate that C2, C3⁄ C5 and C6 differ from C1 and C4 at the N-termini; that is, both sets of isoforms are likely to

be encoded by two alternative first exons Interest-ingly, C1 and C4 also had a common peptide (m⁄ z peak 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 be determined In conclusion, structural data strongly suggest that at least some of the C-subunits identi-fied in mussel arise as a result of differential splicing events involving various forms of the first exon, as has been widely reported for C-subunits from both mammalian and invertebrate sources [22,23,25, 26,28–32]

Sequence alignments of tryptic peptides from mussel C-subunit isoforms with the bovine Ca-isoform showed a degree of sequence identity near to 90%, which confirms that the PKA C-subunit is a highly conserved protein As expected, mussel sequences con-tain some structural motifs, conserved throughout the protein kinase family, that are crucial for Mg2+ and ATP binding [2] For example: (a) the glycine-rich loop

or nucleotide positioning motif (GxGxxG), which is particularly important for positioning the phosphates

of ATP; (b) the glutamic acid residue occupying tion 91 in the bovine Ca-isoform, which suitably posi-tions Lys72, which, in turn, binds to the a-phosphate and b-phosphate of ATP; and (c) the Mg2+ position-ing loop or DGF motif, with the aspartic acid residue chelating the primary Mg2+ ion that bridges the b-phosphate and c-phosphate of ATP

Various authors have proposed that the functional significance of C-subunit diversity could be related to the different ability of C-subunit isoforms to phos-phorylate cellular proteins, and⁄ or to interact with partner proteins that determine the subcellular distri-bution of PKA activity [23,33,34] In mussel, the C-subunit isoforms isolated from the PAM tissue displayed an identical pattern of protein phospho-rylation; however, the C-subunit isoforms from the

mantle tissue showed minor but reproducible

differ-ences in this pattern, despite the fact that they

phos-phorylated a synthetic peptide substrate with similar

apparent affinity Specifically, certain proteins from a

mantle tissue extract were phosphorylated in vitro by

C4, the main C-subunit isoform present in that tissue,

but not by C5 or C6 Therefore, this finding suggests

that some of the mussel C-subunit isoforms differ in

their ability to phosphorylate cellular proteins, as has

also been reported for Aplysia C-subunit isoforms

[33]

In summary, in this work we demonstrate the

pres-ence of several structurally different isoforms of the

PKA C-subunit in mussel tissues In principle, the

combination of these catalytically active C-subunits

with the two types of R-subunit previously identified

(Rmyt1 and Rmyt2 [11–13]) could potentially generate

multiple PKA holoenzymes In order to establish the

functional differences among these PKA isoforms, it

would now be interesting to investigate the ability of

C-subunits to interact with partner proteins, including

Rmyt1 and Rmy2, and to examine the cellular

distribu-tion of both R-subunit and C-subunit isoforms in the

mussel tissues

Experimental procedures

Molluscs

Sea mussels of the species M galloprovincialis Lmk were

collected from a sea farm located at the Rı´a de Betanzos

(Galicia, north-west Spain) Molluscs were placed in tanks

containing seawater and transported to the laboratory

Tissues were dissected out and immediately frozen at

)20 C until use

Mussel extracts

buf-fer A (pH 7.0) [55 mm potassium phosphate, 2 mm EDTA,

1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride,

(Sigma-Aldrich Quı´mica, Madrid, Spain)], using a Potter-Elvehjem

ice-cold buffer B (pH 7.0) (30 mm potassium phosphate,

pepstatin A), using a blade homogenizer (VirTis Tempest

refrigerated centrifuge (Beckman Coulter, Fullerton, CA,

USA), and the supernatants, once filtered through glass

wool, constituted the crude extracts

Separation of C-subunit isoforms

First, C-subunit was purified from PAM and mantle tissues

as described previously [35,36] Briefly, the procedure is based on the binding of PKA, through its R-subunit, to DEAE–cellulose, and the specific elution of the C-subunit

by addition of cAMP, which causes the dissociation of holoenzyme The crude extract obtained from each tissue was 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 be discarded – and then packed into a chromatographic column Next, the gel was extensively washed with the homogenization buffer, and then C-subunit was specifically eluted with the same buffer containing 0.12 mm cAMP (Sigma-Aldrich Quı´mica) The fractions showing protein

ultrafiltration through a PM-30 membrane (Millipore, Bedford, MA, USA) This procedure allows enzymatic prep-arations containing mainly C-subunit together with minor contaminant proteins to be obtained The separation of C-subunit isoforms was performed by means of cation

column (GE Healthcare Bioscience, Uppsala, Sweden) Sam-ples (2 mL) of the enzymatic preparations obtained from the PAM and the mantle tissues were applied to the column, previously equilibrated with buffer C (pH 6.8) (45 mm potassium phosphate, 1 mm dithiothreitol) The column was then washed with buffer C to eliminate most contaminant proteins, and C-subunit isoforms were eluted by applying a continuous NaCl gradient (0–0.4 m in buffer C) The collected fractions of 0.5 mL were assayed for protein kinase

blot-ting

C-subunit from bovine heart was purified following the procedure of Pepperkok et al [16], and purified enzyme

Healthcare Bioscience) [16]

Assay of C-subunit activity and determination of kinetic parameters

C-subunit activity was assayed using the synthetic peptide Kemptide (Sigma-Aldrich Quı´mica) as substrate In a total

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, the concentrations of Kemptide ranged from 5 to 150 lm and the concentrations of PKI(5–24) (Sigma-Aldrich Quı´mica)

ranged from 5 to 200 nm After 10 min at 25C, 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 a

phosphocellulose disc paper, and the discs were: (a) washed

three times with 75 mm phosphoric acid and gently shaken

to 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 enzyme

that transfers 1 nmol of phosphate to Kemptide per min

Experimental data describing the dependence of protein

kinase activity on Kemptide concentrations were fitted to

the Michaelis–Menten equation, and the values of the

that reduces enzyme activity by 50%) was determined from

of Kemptide and ATP

Phosphorylation of mussel proteins by purified

C-subunit isoforms

Aliquots of the crude extract from PAM (100 lg of

pro-tein) or from mantle tissue (120 lg of propro-tein) were

20 min, reactions were stopped by adding a one-quarter

glycerol] and boiled for 5 min Samples were then analysed

Brilliant Blue R (Sigma-Aldrich Quı´mica), destained, dried,

evaluation of the autoradiographs was carried out using the

CA, USA)

SDS/PAGE and western blotting

(Bio-Rad Laboratories) For performance of western blot

analysis, the proteins were transferred to a poly(vinylidene

difluoride) membrane (Immobilon-P; Millipore) by applying

room temperature with 5% nonfat dry milk in 20 mm

0.1% Tween-20), membranes were washed with Tris⁄ HCl

the primary antibodies: (a) polyclonal antibody against

human Ca-isoform (sc903; Santa Cruz Biotechnology,

Tween-20; (b) monoclonal antibody against phosphoserine

against phosphothreonine (P6623; Sigma-Aldrich Quı´mica)

for 1 h at room temperature with secondary antibodies (anti-rabbit IgG or anti-mouse IgG, diluted 1 : 50 000 and

conju-gated to horseradish peroxidase (Sigma-Aldrich Quı´mica) Next, the blots were: (a) extensively washed; (b) developed with the chemiluminiscent horseradish peroxidase substrate (Millipore); and (c) exposed to X-ray film (Curix RP2 Plus; Agfa-Gevaert, Mortsel, Belgium) for a few seconds

MS

Samples of mussel C-subunit isoforms and bovine

one-quarter volume of SDS sample buffer supplemented with dithiothreitol to a final concentration of 10 mm, and then alkylated with 20 mm iodoacetamide (Sigma-Aldrich Quı´mica) for 30 min in the dark Next, proteins were

procedure with modified trypsin, sequence grade (Promega, Madison, WI, USA) [38] Digested samples were analysed

Daltonics, Bremen, Germany) in reflector mode to obtain PMF 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 CAF reagent (GE Healthcare Bioscience), PMF was performed again and peptide masses that had increased their masses

by 136 or 204 Da were searched for The former represent tryptic peptides with a C-terminal arginine, and the latter those with a C-terminal lysine, where the e-amino groups

of lysine residues were blocked with 2-methoxy-4,5-dihy-dro-1H-imidizole (Lys Tag 4H; Agilent Technologies, Santa Clara, CA, USA) in order to prevent lysine from being sulfonated The MALDI instrument was switched to PSD mode and the ion selector was adjusted for the appropriate mass PSD analysis was performed according to the manu-facturer’s instructions, and the generated spectra, mainly consisting of a clean y-ion series, were interpreted manu-ally A sequence homology search for the mussel C-subunit isoforms was conducted using the ‘short, nearly exact matches’ option of blast [40]

Acknowledgements

This work was supported by grant PGIDIT02-RMA26101PR from the Autonomous Government of Galicia (Xunta de Galicia)

1 Taylor SS, Buechler JA & Yonemoto W (1990)

cAMP-dependent protein kinase: framework for a diverse

family of regulatory enzymes Annu Rev Biochem 59,

971–1005

2 Smith CM, Radzio-Andzelm E, Madhusudan, Akamine

P & Taylor SS (1999) The catalytic subunit of

cAMP-dependent protein kinase: prototype for an extended

network of communication Prog Biophys Mol Biol 71,

313–341

3 Siegman MJ, Funabara D, Kinoshita S, Watabe S,

Hartshorne D & Butler TM (1998) Phosphorylation

of a twitchin-related protein controls catch and

calcium sensitivity of force production in invertebrate

smooth muscle Proc Natl Acad Sci USA 95, 5383–

5388

4 Yamada A, Yoshio M, Kojima H & Oiwa K (2001) An

the catch state of invertebrate smooth muscle Proc Natl

Acad 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 of

the mollusc Mytilus galloprovincialis Identification of the

phosphorylated site Biochem Mol Biol Int 43, 173–181

7 Ferna´ndez M, Cao J & Villamarı´n JA (1998) In vivo

phosphorylation of phosphofructokinase from the

bivalve mollusk Mytilus galloprovincialis Arch Biochem

Biophys 353, 251–256

8 Sanjuan-Serrano F, Ferna´ndez-Gonza´lez M,

Sa´nchez-Lo´pez JL & Garcı´a-Martı´n LO (1995) Molecular

mechanism of the control of glycogenolysis by

cal-cium ions and cyclic AMP in the mantle of Mytilus

577–582

9 Storey KB & Storey JM (1990) Facultative metabolic

rate depression: molecular regulation and biochemical

adaptation 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 the

bivalve mollusc Mytilus galloprovincialis Eur J Biochem

232, 664–670

12 Rodrı´guez JL, Barcia R, Ramos-Martı´nez JI &

Villa-marı´n JA (1998) Purification of a novel isoform of the

regulatory subunit of cAMP-dependent protein kinase

from the bivalve mollusk Mytilus galloprovincialis Arch

Biochem Biophys 359, 57–62

13 Dı´az-Enrich MJ, Ibarguren I, Hellman U & Villamarı´n

JA (2003) Characterization of a type I regulatory sub-unit of cAMP-dependent protein kinase from the bivalve mollusk Mytilus galloprovincialis Arch Biochem Physiol 416, 119–127

14 Bardales JR, Hellman U & Villamarı´n JA (2007) CK2-mediated phosphorylation of type II regulatory subunit

of cAMP-dependent protein kinase from the mollusc Mytilus 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 conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected

by 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 of mammalian protein kinase A catalytic subunit altered

by 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, Gensheimer

HP et al (1987) Chromatographic separation of two heterogeneous forms of the catalytic subunit of cyclic AMP-dependent protein kinase holoenzyme type I and type II from striated muscle of different mammalian species Arch Biochem Biophys 253, 341–349

18 Herberg FW, Bell S & Taylor SS (1993) Expression of the catalytic subunit of cAMP-dependent protein kinase

in Escherichia coli: multiple isozymes reflect different phosphorylation states Protein Eng 6, 771–777

19 Uhler MD, Carmichael DF, Lee DC, Chrivia JC, Krebs

EG & McKnight GS (1986) Isolation of cDNA clones coding 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 subunit

of cAMP-dependent protein kinase J Biol Chem 261, 15360–15363

21 Beebe SJ, Oyen O, Sandberg M, Froysa A, Hansson

V & Jahnsen T (1990) Molecular cloning of a

representing a third isoform for the catalytic subunit

of 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 the catalytic subunit of cAMP-dependent protein kinase in Caenorhabditis elegans J Biol Chem 265, 6896–6907

23 Tabish M, Clegg RA, Rees HH & Fischer MJ (1999) Organization and alternative splicing of the

cata-lytic-subunit gene (kin-1) Biochem J 339, 209–216