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

Identification of multiple isoforms of the cAMP-dependent
protein kinase catalytic subunit in the bivalve mollusc
Mytilus galloprovincialis
Jose
´
R. Bardales
1
, Ulf Hellman
2
and J. A. Villamarı
´
n
1
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;
MALDI-TOF ⁄ TOF MS; Mytilus
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:
(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,
posterior adductor muscle; PKA, cAMP-dependent protein kinase; PKI(5–24)
,
protein kinase inhibitor peptide; PMF, peptide mass
fingerprinting; 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 4479
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 R
myt1
and
R
myt2
[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 R
myt1
showed a clear homology with
the type I R-subunits from both mammalian and
invertebrate sources [13]; likewise, R
myt2
was 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 C
1
,C
2
and C
3
, 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
BC
Fig. 1. Separation and identification of PKA C-subunit isoforms
from mussel PAM. (A) Elution profile of C-subunit from a Mono-S

HR 5 ⁄ 5 column. A sample (2 mL, 1.5 mg of protein) of C-subunit
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 analy-
sed by (B) Coomassie-stained SDS ⁄ PAGE, and (C) western blotting
with 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 FEBS
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 C
4
, which corresponded to peak I of
the Mono-S chromatogram, and the isoforms named
C
5
and C
6
, which coeluted together at peak II. C
4
was
3–4-fold more abundant than C
5

and C
6
together.
Characterization of C-subunit isoforms
Samples of purified C
1
–C
6
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 C
3
(from PAM) and C
5
(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 C
4
and 44.5 kDa for C
6
.
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

C
1
–C
6
were 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
C
1
–C
6
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 C
B
), 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

Mono-S HR 5 ⁄ 5 column. A sample (2 mL, 1.8 mg of protein) of
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 protein
kinase activity were separated: I and II. Aliquots of fractions were
also analysed by (B) Coomassie-stained SDS ⁄ PAGE, and (C) wes-
tern blotting with an antibody against the human Ca-isoform.
A
BC
Fig. 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 cm
polyacrylamide gel which was Coomassie stained. Lane 1: molecu-
lar mass standards. Lane 2: sample of bovine heart C-subunit, frac-
tion C
B
[17]. Lanes 3–5: samples of fractions 20, 22 and 29 of
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%
SDS ⁄ PAGE, and C-subunit was detected by western blotting using
monoclonal antibodies against phosphoserine (B) or phosphothreo-
nine (C).
J. R. Bardales et al. Mussel C-subunit isoforms
FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4481
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
.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 in
the spectrum from a particular isoform. Arrowheads indicate the peak at 1059.5 Da, common to C
1
and C
4
, and arrows indicate the peak at
1605.7 Da, common to the remaining isoforms: C
2
,C
3
,C
5
and C
6
.
Mussel C-subunit isoforms J. R. Bardales et al.
4482 FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS
Ca-isoform corresponding to amino acids 73–78 and
48–56, respectively (Fig. 5A). (b) Most peptide masses
were common to all mussel isoforms, C
1
–C
6
. 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 C
1
,C
2
,C
4
and C
6
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 C
1
); 1230.6 (only in
C
2
); 1768.6 and 2386.9 (only in C
4
); and 2623.3 (only
in C
6
). (d) There was one peak at 1059.5 Da observed
only in the C
1
and C
4
spectra, whereas another peak
at 1605.7 Da was found in the spectra of the remaining
isoforms: C

2
,C
3
,C
5
and C
6
. 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 C
1
and C
4
differ from C
2
,C
3
,C
5
and C
6
at the N-terminal
region. (e) When spectra from C
3
and C
5
were com-
pared, no significant difference was observed, which

suggests that both C
3
and C
5
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 C
5
and C
6
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 K
m
for
Kemptide and V
max
were observed. Furthermore, all
the mussel isozymes were inhibited by the protein
kinase inhibitor peptide [PKI(5–24)] with similar I
50
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
eight measured m ⁄ z peaks that were 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
m ⁄ z (Da)
Present in
spectra
from De novo peptide sequence
1 744.47 Bovine Ca,
C
1
–C
6
[I ⁄ L][I ⁄ L]DKQK
2 895.47 Bovine Ca,

C
1
–C
6
T[I ⁄ L]GTGSFGR
3 859.44 C
1
–C
6
FSEPHSR
4 870.51 C
1
–C
6
VF[I ⁄ L]VQHK
5 884.48 C
1
–C
6
VTDFGFAK
6 1014.64 C
1
–C
6
KVDAPF[I ⁄ L]PK
7 1016.65 C
1
–C
6
S[I ⁄ L][I ⁄ L]QVD[I ⁄ L]TK

8 1040.61 C
1
–C
6
VTDFGFAKR
9 1172.75 C
1
–C
6
S[I ⁄ L][I ⁄ L]QVD[I ⁄ L]TKR
10 1338.80 C
1
–C
6
[I ⁄ L]KQVEHT[I ⁄ L]NEK
11 1494.89 C
1
–C
6
[I ⁄ L]KQVEHT[I ⁄ L]NEKR
12 1910.82 C
1
–C
6
GPGDASNFDDYEEEP[I ⁄ L]R
13 1605.88 C
2
,C
3
,

C
5
,C
6
KGDVPMNVKE(x)K
a
a
x Dmass = 360.3 Da.
J. R. Bardales et al. Mussel C-subunit isoforms
FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4483
C
1
,C
2
and C
3
(purified from PAM) were individually
incubated, in the presence of labelled ATP, with aliqu-
ots of a PAM extract. In the same way, C
4
and the
mixture of C
5
and C
6
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
C

1
,C
2
and C
3
(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 C
4
and the mixture of C
5
and C
6
were also apparently similar, although densito-
metric analysis of the autoradiograph showed some
protein bands, marked by asterisks, that seemed to
be phosphorylated by C
4

but not by the mix of C
5
and C
6
. 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 C
1
,C
2
and C
3
were
isolated from the PAM tissue, whereas C
4
,C
5
and C
6
were separated from the mantle tissue. However, it
Table 2. Kinetic parameters of mussel C-subunit isoforms. The
apparent K
m
for Kemptide and V
max

values were determined at
0.2 m
M ATP. The I
50
values for PKI(5–24) were determined at
100 l
M Kemptide and 0.2 mM ATP. The data are expressed as
means ± SE of three independent experiments.
Isoform K
m
(lM)
V
max
(nmol PÆmin
)1
Ælg
)1
) I
50
(nM)
C
1
15.8 ± 8.0 6.6 ± 1.9 8.9 ± 1.7
C
2
24.7 ± 9.5 4.9 ± 2.2 7.8 ± 3.3
C
3
14.7 ± 5.3 4.3 ± 1.3 9.5 ± 2.1
C

4
18.6 ± 2.4 3.6 ± 0.5 6.9 ± 2.9
C
5
+C
6
12.5 ± 5.2 4.0 ± 1.3 7.2 ± 2.7
CCCC+CC
twitchin
132
–+ –+ –+
564
–+ – +
PA
AB
M
extract
actin
mantle
extract
?
0.4
C
1
C
2
C
3
0.4
C

4
C
5
+C
6
*
*
0.0 0.5 1.0
0.0
0.2
A
A
0.0 0.5 1.0
0.0
0.2
*
Rf
Rf
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
incubated with MgCl
2
and [
32
P]ATP[cP] in
the absence and presence of each C-subunit
isoform isolated from the corresponding

tissue (5 unitsÆmg
)1
protein). Samples of C
1
,
C
2
and C
3
were from fractions 20, 22 and
29 of the Fig. 1A chromatogram, respec-
tively, and samples of C
4
and C
5
+C
6
were
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%
SDS ⁄ PAGE, and the gel was Coomassie
stained, destained, dried and exposed for
autoradiography at )80 °C. ?, unidentified
protein. 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 FEBS

seems highly likely that C
3
and C
5
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 C
A
and C
B
, by means of cation exchange
chromatography [17,18]. C
A
arises from C
B
, as a result
of the in vivo deamidation of the Asn2 residue, and
therefore the only difference between C
A
and C

B
was
the presence of aspartic acid or asparagine, respec-
tively, at position 2 of their sequences [15]. Unlike
mammalian C
A
and C
B
, 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 C
A
and C
B
, 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 C
3
and C
5
, 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 C
2

,C
3
⁄ C
5
and C
6
that was
absent in those of C
1
and C
4
; 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 C
2
,C
3
⁄ C
5
and C
6
differ from C
1
and C
4
at the
N-termini; that is, both sets of isoforms are likely to

be encoded by two alternative first exons. Interest-
ingly, C
1
and C
4
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 Mg
2+
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 posi-
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 Mg
2+
position-
ing loop or DGF motif, with the aspartic acid residue
chelating the primary Mg
2+
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
J. R. Bardales et al. Mussel C-subunit isoforms
FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4485
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
C
4
, the main C-subunit isoform present in that tissue,
but not by C
5

or C
6
. 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
(R
myt1
and R
myt2
[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
R
myt1
and R
my2
, 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
Mantle 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
)1
leupeptin and 1 mgÆL
)1
pepstatin A (Sigma-
Aldrich Quı
´
mica, Madrid, Spain)], using a Potter-Elvehjem
homogenizer. PAM tissue was homogenized 1 : 6 (m ⁄ v) in
ice-cold buffer B (pH 7.0) (30 mm potassium phosphate,
2mm EDTA, 1 mm dithiothreitol, 1 mm phenyl-
methanesulfonyl fluoride, 1 mgÆL
)1
leupeptin and 1 mgÆL
)1
pepstatin A), using a blade homogenizer (VirTis Tempest
IQ
2

; SP Industries, Warminster, PA, USA). The homogen-
ates were centrifuged at 35 000 g for 30 min at 4 °Cina
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
kinase activity were pooled and concentrated to  3mLby
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
exchange chromatography on a Mono-S HR 5 ⁄ 5 FPLC

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
activity, and also analysed by SDS ⁄ PAGE and western blot-
ting.
C-subunit from bovine heart was purified following the
procedure of Pepperkok et al. [16], and purified enzyme
was separated into fractions C
A
and C
B
by cation exchange
chromatography on a Mono-S HR 5 ⁄ 5 column (GE
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
volume of 50 lL, the assay contained 50 mm Tris ⁄ HCl
(pH 7.0), 1 mm dithiothreitol, 5 mm MgCl
2
, 0.2 mm

[
32
P]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, the
concentrations of Kemptide ranged from 5 to 150 lm and
the 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 FEBS
ranged 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 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
Michaelis–Menten (K
m
) and maximum velocity (V

max
) con-
stants were determined from the plots 1 ⁄ V
o
versus 1 ⁄ [S],
where V
o
is the initial rate at a given substrate concentra-
tion [S]. The I
0.5
for PKI(5–24) (concentration of peptide
that reduces enzyme activity by 50%) was determined from
plots of V
o
versus [PKI(5–24)] at saturating concentrations
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 l g of protein) were individ-
ually incubated at 25 °C with each tissue-purified C-subunit
isoform (5 unitsÆmg
)1
protein) in the presence of 5 mm
MgCl
2
and 0.2 mm [
32
P]ATP[cP] ( 500 c.p.m.Æpmol
)1

). At
20 min, reactions were stopped by adding a one-quarter
volume 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 analysed
by 10% SDS ⁄ PAGE, and the gel was stained with Coomassie
Brilliant Blue R (Sigma-Aldrich Quı
´
mica), destained, dried,
and exposed for autoradiography at )80 °C. Densitometric
evaluation of the autoradiographs was carried out using the
versadoc imaging system (Bio-Rad Laboratories, Hercules,
CA, USA).
SDS/PAGE and western blotting
SDS ⁄ 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 blot
analysis, the proteins were transferred to a poly(vinylidene
difluoride) membrane (Immobilon-P; Millipore) by applying
a 400 mA current for 2 h at 4 °C. After blocking for 6 h at
room temperature with 5% nonfat dry milk in 20 mm
Tris ⁄ HCl with Tween-20 (Tris ⁄ HCl, pH 7.5, 0.15 m NaCl,
0.1% Tween-20), membranes were washed with Tris ⁄ HCl
with Tween-20 and then incubated overnight at 4 °C with
the primary antibodies: (a) polyclonal antibody against
human Ca-isoform (sc903; Santa Cruz Biotechnology,
Santa Cruz, CA, USA) diluted 1 : 2500 in Tris ⁄ HCl with
Tween-20; (b) monoclonal antibody against phosphoserine
(P3430; Sigma-Aldrich Quı

´
mica) diluted 1 : 2000 in
Tris ⁄ HCl with Tween-20; or (c) monoclonal antibody
against phosphothreonine (P6623; Sigma-Aldrich Quı
´
mica)
diluted 1 : 1500 in Tris ⁄ HCl with Tween-20. After washing
with Tris ⁄ HCl with Tween-20, the blots were incubated
for 1 h at room temperature with secondary antibodies
(anti-rabbit IgG or anti-mouse IgG, diluted 1 : 50 000 and
1 : 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) 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 C-sub-
unit (fraction C
B
[16,17]) were first reduced by adding a
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 sepa-
rated by SDS ⁄ PAGE and subjected to ‘in-gel’ digestion

procedure with modified trypsin, sequence grade (Promega,
Madison, WI, USA) [38]. Digested samples were analysed
using a MALDI-TOF ⁄ TOF Ultraflex instrument (Bruker
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).
J. R. Bardales et al. Mussel C-subunit isoforms
FEBS Journal 275 (2008) 4479–4489 ª 2008 The Authors Journal compilation ª 2008 FEBS 4487
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