Download Classification and Phylogenetic Analysis of the cAMP

J Mol Evol (2002) 54:17–29
DOI: 10.1007/s00239-001-0013-1
© Springer-Verlag New York Inc. 2002
Classification and Phylogenetic Analysis of the cAMP-Dependent Protein
Kinase Regulatory Subunit Family
Jaume M. Canaves, Susan S. Taylor
Department of Chemistry and Biochemistry, 0654, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0654, USA
Received: 2 November 2000/Accepted: 14 June 2001
Abstract. The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple
sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic
trees generated from multiple sequence alignment of the
complete sequences, the PKA-R family was divided into
four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II),
fungi (type III), and alveolates (type IV). Application of
the same methodology to the cAMP-binding domains,
and subsequently to the region delimited by ␤-strands 6
and 7 of the crystal structures of bovine RI␣ and rat RII␤
(the phosphate-binding cassette; PBC), proved that this
highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single
signature sequence, F–G–E–[LIV]–A–L–[LIMV]–x(3)–
[PV]–R–[ANQV]–A, corresponding to the PBC was
identified which is characteristic of the PKA-R family
and is sufficient to distinguish it from other members of
the cyclic nucleotide-binding protein superfamily. Specific determinants for the A and B domains of each Rsubunit type were also identified. Conserved residues
defining the signature motif are important for interaction
with cAMP or for positioning the residues that directly
interact with cAMP. Conversely, residues that define
subfamilies or domain types are not conserved and are
mostly located on the loop that connects ␣-helix B⬘ and
␤ strand 7.
Key words:
PKA — cAMP — cAPK — Regulatory
Correspondence to: Susan S. Taylor; email: [email protected]
subunit — Motif — Classification — Phylogeny — Signature
Introduction
The cyclic AMP (cAMP)-dependent protein kinase
(PKA) regulatory subunit family (PKA-R family) is part
of the cyclic nucleotide-binding protein (cNMP) superfamily. Among others, the cNMP superfamily includes a
variety of bacterial regulators, such as the catabolite gene
activator protein (CAP) (Eron et al. 1971; Weber and
Steitz 1987), cyclic nucleotide-gated ion channels (Nakamura and Gold 1987; Ludwig et al. 1990), guanine
nucleotide exchange factors (Kawasaki et al. 1998), and
the regulators of PKA and PKG. The distinctive feature
of the members of the superfamily is the presence of a
common structural domain of about 120 residues (Shabb
and Corbin 1992) capable of binding cyclic nucleotides.
In eukaryotes, PKA mediates a wide range of cellular
responses to external stimuli (Taylor et al. 1990). Of the
protein kinases, PKA is also one of the simplest and best
characterized (Taylor et al. 1990), primarily because the
catalytic (C) and regulatory (R) components are the
products of distinct genes and the proteins are separated
upon activation. In the absence of cAMP, PKA exists as
an equimolar tetramer of R and C subunits. In addition to
its role as an inhibitor of the C subunit, the R subunit
anchors the holoenzyme to specific intracellular locations (DellÁcqua and Scott 1997) and prevents the C
subunit from entering the nucleus (Fantozzi et al. 1994).
All R-subunit isoforms have a conserved and welldefined domain structure (Taylor et al. 1990) (Fig. 1).
18
Fig. 1. Summary of the general
domain structure of a representative
dimeric PKA regulatory subunit. A
Schematic linear domain structure
of bovine RI␣. The N-terminal
dimerization domain, inhibitory
region, cAMP-binding domain A,
and cAMP-binding domain B are
shown. The expanded view of the
inhibitory sequence shows the
different motifs identified for this
region. B and C Detailed views of
PBCs A and B in the presence of
cAMP. A ribbon representation
showing the structural elements of
the PBCs has been overlaid to a
stick representation of the sequence.
Each region has its own function, and it also communicates with other regions as part of the conformational
changes that are induced by the binding of cAMP. R
subunits interact primarily with C subunits through the
inhibitory site. PKA regulatory subunits contain two tandem cAMP-binding domains at the C terminus, designated A and B (Takio et al. 1982) (Fig. 1). These cAMPbinding domains, presumably resulting from gene
duplication, show extensive sequence similarity and bind
cAMP cooperatively (Døskeland and Øgreid 1984; Robinson-Steiner and Corbin 1983).
PKA regulatory subunits have typically been classified according to their physicochemical properties,
namely, overall charge and molecular mass, and the presence or absence of a serine residue susceptible to phosphorylation in the inhibitory site. In the present work, we
propose a new classification based on analysis of the
most conserved region of the R subunits: the phosphatebinding cassette (PBC) of each cAMP-binding domain.
Also, we have generated a highly specific signature pattern capable of unequivocally identifying the PBCs of
PKA regulatory subunits and identified type- and subtype-specific residues. This provides a means for fast
assignment of new sequences to specific R subfamilies
and could be used as a valuable tool for R-subunit identification and automatic sequence annotation of short
protein fragments.
Experimental Procedures
Sequences and Database Searches. Sources of sequences analyzed in
this study are shown in Table 1. Sequence retrieval was performed
using BLAST. Initial searches were carried out with PSI-BLAST (po-
sition-specific iterated BLAST) (Altschul et al. 1997) set to search the
nonredundant database using human RI␣ as the query sequence and a
BLOSUM62 weight matrix (Henikoff and Henikoff 1992). Sequences
identified in this first stage were used as query sequences for subsequent searches. Sequences unequivocally identified as corresponding to
cAMP-dependent kinase regulatory subunits were downloaded from
SwissProt and TrEMBL Release 38. In a later stage of the project, a
consensus sequence pattern common to all the detected R subunits was
used with human RI␣ as template sequence to try to detect additional
R subunits using PHI-BLAST (Pattern Hit Initiated BLAST) (Zheng et
al. 1998). Search for unidentified R subunits was performed using
BLAST on several major genomic databases. The sequences matching
the signature pattern were compared to the signature subpatterns for
PBC A and B, as well as the different type-specific subpatterns, and
classified accordingly. DNA sequences were translated with the Translate utility at Expasy (http://www.expasy.ch).
Multiple Sequence Alignment. Multiple sequence alignments were
performed using ClustalW 1.6 (Thompson et al. 1994). Priority was set
to placing gaps within loops connecting secondary structure elements.
The cAMP-binding regions were manually curated with the program
GeneDoc (Nicholas et al. 1997), taking into consideration the crystal
structures of bovine RI␣ (Su et al. 1995) and rat RII␤ (Diller et al.
2001). The Gonnet weight matrix (Gonnet et al. 1994) was used in the
sequence alignments. The complete sequence alignments are available
from the authors upon request.
Phylogenetic Tree Construction. Phylogenetic trees based on the
amino acid alignments of the complete sequences, the cAMP-binding
domains, and PBCs were reconstructed using the neighbor-joining
method (Saitou and Nei 1987). The statistical significance of the phylogenetic trees obtained was tested by bootstrap analysis with 100
replicates of random additions (Felsenstein 1985). The resulting trees
were visualized with the program Treeview (Page 1996). Amino acid
sequence distances were corrected for multiple amino acid substitutions
according to Kimura (Kimura 1983).
Motif Identification and Signature Sequence Generation. Amino
acids that were conserved within the entire family of PKA regulatory
19
Table 1.
cAMP-dependent protein kinase regulatory subunit family members analyzed in this study: (A) full-length sequences; (B) fragmentsa
A
Abbreviation
b
hRI␣
bRI␣
pRI␣
rRI␣
hRI␤
mRI␤
rRI␤
hRII␣
bRII␣
mRII␣
hRII␤
bRII␤
rRII␤
AplyR
StroR
HemiR
CaenR
DrosR
DictR
Sch1R
Sch2R
BlasR
UstiR
SaccR
EmerR
MagnR
NeurR
CollR
EuplR
ParaR
Source species
Accession No.
No. AAc
MWd
pIe
Homo sapiens
Bos taurus
Sus scrofa
Rattus norvegicus
Homo sapiens
Mus musculus
Rattus norvegicus
Homo sapiens
Bos taurus
Mus musculus
Homo sapiens
Bos taurus
Rattus norvegicus
Aplysia californica
Strongylocentrotus purpuratus
Hemicentrotus pulcherrimus
Caenorhabditis elegans
Drosophila melanogaster
Dictyostelium discoideum
Schizosaccharomyces pombe
Schizosaccharomyces pombe
Blastocladiella emersonii
Ustilago maydis
Saccharomyces cerevisiae
Emericella nidulans
Magnaporthe grisea
Neurospora crassa
Colletotrichum trifolii
Euplotes octocarinatus
Paramecium tetraurelia
P10644
P00514
P07082
P09456
P31321
P12849
P81377
P13861
P00515
P12367
P31323
P31322
P12369
P31319
Q26619
Q25114
P30625, A21820
P16905
P05987
P36600
O14272
P31320
P49605
P07278
O59922
O14448
Q01386
O42794
Q9XTM6
Q94725
381
379
379
380
380
380
380
403
400
400
417
417
415
377
369
368
376
377
327
411
347
403
522
415
412
390
385
404
338
325
42,981.6
42,761.5
42,790.4
42,963.7
43,026.8
43,092.9
43,150.9
45,387.2
44,962.6
45,257.9
46,214.9
46,204.8
45,991.7
42,606.2
41,788.8
41,679.8
42,649.3
42,367.2
36,835.7
46,363.3
38,917.1
44,467.6
55,957.9
47,087.9
44,903.7
42,270.5
42,156.2
44,778.0
38,786.3
37,275.5
5.27
5.27
5.27
5.27
5.64
5.71
5.60
4.96
4.80
4.78
4.82
4.85
4.90
5.36
4.61
4.66
4.99
5.07
5.65
5.06
5.85
4.82
8.88
7.82
5.09
5.05
5.45
9.17
8.11
7.61
B
Abbreviationb
Source species
Accession no.
Comments
pRI
RbRI
RET/TyrK
pRII␣
rRII␣
mRII␤
hRII␤
MucoR
Sus scrofa
Oryctolagus cuniculus
Homo sapiens
Sus scrofa
Rattus norvegicus
Mus musculus
Homo sapiens
Mucor rouxii
Q29083
O77795
Q15300
P05207
P12368
P31324
O60380
AAF44694 (GenBank)
DD domain
A domain
A domain
A domain
A and B domains
DD domain
B domain
A and B domains
a
The Comments column indicates the structural modules that are contained in the fragment: dimerization-docking domain (DD domain),
cAMP-binding domain A (A domain), or cAMP-binding domain B (B
domain). Unless specified, all the accession numbers correspond to
Swissprot entries.
b
subunits or only within subfamilies were identified based on the final
alignment of all the regulatory subunits. Signature sequences were
created manually from the sequence logos corresponding to the cAMPbinding cassettes. Amino acid patterns are abbreviated in one-letter
code, where “X” indicates any of the 20 essential amino acids. The
signature sequence was evaluated for specificity by carrying out motif
searches with the ScanProsite tool (Appel et al. 1994) for searching the
Swissprot and TrEMBL protein databases.
were generated via the WebLogo server maintained by Steven E. Brenner (http://www.bio.cam.ac.uk/cgi-bin/seqlogo/logo.cgi). In sequence
logos, the horizontal axis represents the position of the residue, whereas
the vertical axis represents the information content of that position in
bits. The height of the one-letter residue symbol at each position is
proportional to the information content of the residue at that position.
Entropy plots (Schneider and Stephens 1990) were created with the
BioEdit sequence alignment editor (Hall 1999). Entropy plots are a
measure of the lack of predictability for each alignment position. The
entropy or uncertainty (H) was calculated according to the expression
Sequence Logos and Entropy Plots. Sequence logos (Schneider and
Stephens 1990) corresponding to R-subunit cAMP-binding domains
Abbreviation used in this study.
Number of amino acids in the protein.
d
Molecular mass (Da).
e
Theoretical isoelectric point (pH units).
c
Hx ⳱ −⌺f(r,x) ⭈ ln(f(r,x))
20
Fig. 2. Clustering of the members
of the PKA-R family listed in Table
I according to physicochemical
parameters. The estimated
molecular mass calculated from the
amino acid sequence was plotted
against the theoretical isoelectric
point for each subunit. Inset: An
expanded view of the region with
the highest clustering density.
where x represents each position along the alignment, r represents each
choice of amino acid for the position in question, and f(r,x) is the
frequency at which residue r is found at position x.
Results
Sequence Identification. Abbreviations in the first column in Table 1 are used in all subsequent figures and
tables. Thirty full-length and eight fragments of eukaryotic R-subunit sequences were identified by an extensive
search of all major databases (Table 1) using known
regulatory subunits as query sequences. Only 30 complete and 8 incomplete eukaryotic sequences previously
identified and annotated as PKA regulatory subunits
were considered. Of the 30 complete sequences, 13 belonged to Chordata and 9 to Fungi. The rest of the sequences were from Echinodermata (2), Alveolata (2),
Arthopoda (1), Mollusca (1), Nematoda (1), and Dictyosteliida/slime molds (1). Two entries from Fungi corresponded to R-subunit isoforms from Schizosaccharomyces pombe, one monomeric and one dimeric, both
available from Swissprot. Although there are four isoforms of the type I R subunit from Drosophila, three
monomeric and one dimeric (Kalderon and Rubin 1988),
only the representative sequence (dimeric) incorporated
into Swissprot was used for our analysis.
Physicochemical Properties of Full-Sequence R Subunits. The size of the subunits used in the present study
ranged between 325 amino acids, with a theoretical molecular mass (MW) of 37 kDa (Paramecium tetraurelia;
Q94725), and 522 amino acids with a MW of 56 kDa
(Ustilago maydis; P49605) (Table 1). The theoretical isoelectric points covered a range from 4.82 (Blastocladiella emersonii; P31320) to 9.17 (Colletotrichum trifolii;
O42794) Most of the sequence variation occurred in the
N-terminal region preceding the inhibitory sequence,
which contains the variable region and, in most cases,
contains a dimerization/docking domain.
Plotting the predicted molecular mass versus the theoretical isoelectric point for each subunit showed a considerable degree of diversity within the PKA-R family
(Fig. 2). The vertebrate type I and II subunits, and their
␣ and ␤ subtypes formed tight and well-defined clusters
(Fig. 2, inset). The nonvertebrate type I subunits from
Drosophila, Aplysia, and C. elegans clustered closer to
type RI␣ than to RI␤. The type I subunit from Dictyostelium was practically equidistant from the RI␣ and RI␤
clusters. Vertebrate type II subunits also formed two
well-defined clusters. In contrast, subunits from Echinodermata, typically considered type II subunits, formed a
separate cluster that was equidistant from those for vertebrate types I and II. Finally, the subunits from Fungi, in
contrast with the well-defined clustering of vertebrate
type I and type II regulatory subunits, were completely
dispersed. Based on these observations, it is clear that a
classification of the PKA-R family based on their physicochemical properties is appropriate only for the vertebrate subunits; the approach cannot be generalized.
Inhibitory Sequence Motif Identification. An inhibitory sequence conforming to the RRx[AG]⌿ motif,
where ⌿ is a hydrophobic residue, was identified in each
type I subunit (Fig. 1A). Conversely, an RRxS⌿ motif
21
Fig. 3. Radial phylogenetic tree of the PKA-R family. Protein abbreviations are provided in Table 1. Phylogenetic distance is approximately proportional to branch length. Analysis was performed using the
neighbor-joining algorithm. The tree is based on a complete sequence
alignment of the proteins listed in Table 1A. Clustering patterns are
shaded in gray. ␣ and ␤ subtypes are also indicated for type I and type
II subunits. A bar for calibration of phylogenetic distances is provided
at the bottom.
was identified in vertebrate RII type subunits as well as
R subunits from Fungi. In all the subunits from Fungi
and type I subunits, the ⌿ residue was followed by a
serine, whereas in all type II subunits that position was
occupied by a cysteine. The R subunit from the ciliate
Paramecium tetraurelia contains an atypical inhibitory
sequence conforming to the pattern TRxS⌿ (Carlson and
Nelson 1996). When the R subunit from Euplotes octocarinatus was scanned for suitable PKA phosphorylation
sites that could function as inhibitory sequences, a novel
sequence corresponding to the motif KxS⌿ was identified (Fig. 1A).
Sequence Alignment and Phylogenetic Analysis. A
multiple alignment of all the full-sequence subunits was
performed (not shown) and a radial phylogenetic tree
was built from it as described under Experimental Procedures (Fig. 3). The general clustering of proteins derived from organisms of the same kingdoms is noteworthy. For that reason, the PKA-R family was subdivided
22
Fig. 4. Phylogeny of the PKA-R family. The phylogenetic tree shown in Fig. 3 was rooted using the sequence of Euplotes octocarinatus (Eup1R)
as the outgroup. Protein abbreviations are provided in Table 1. Bootstrapping values are provided.
into the “type I,” “type II,” “fungi,” and “alveolate” subfamilies. In the type I and II clusters, two subclusters can
be identified corresponding to the subtypes ␣ and ␤.
Tentatively, we designated the subunits from fungi as
type III and those from alveolates as type IV.
When an unrooted tree was constructed based on a
sequence alignment of the complete cNMP superfamily
(not shown), the deepest-branching clade in the PKA R
subunit group was the subunit from Euplotes (Eupl R).
Consequently, it was used as outgroup to root the previously described radial tree. The rooted and bootstrapped
tree (Fig. 4) showed the clustering of the subunits in
separate groups which were consistent with accepted
phylogenetic relationships. Although bootstrapping values were relatively low (49%) for the branching between
type I and type II subunits, the use of different methodologies and parameters always yielded trees with the
same topology in which Fungi were always the first divergent group. Additionally, bootstrapping values were
low for the branching point of the sequences of Blastocladiella (BlasR) and Ustilago (UstiR).
Upon observation of the complete sequence alignment
of all the R subunits (not shown), there was a remarkable
lack of conservation of the dimerization/docking domains, the linker regions, and the inhibitory sequences in
the most primitive organisms. Accordingly, we focused
our study on the most conserved region, which corresponded to cAMP-binding domains A and B (Fig. 5).
The alignment of the cAMP-binding domains was curated based on structural evidence from the crystal structures of bovine RI␣ (Su et al. 1995) and rat RII␤ (Diller
et al. 2001). Most of the variability in the cAMP-binding
domains corresponded to the loop between ␤-strand 4
and ␤-strand 5 of both domain A and domain B, and the
C-terminal region. Two distinct blocks, which corresponded to the phosphate-binding cassettes (PBCs), contained the majority of invariant residues (asterisks in Fig.
5). The PBCs are defined as the segments of each cAMPbinding domain that contain most of the key residues for
cAMP-mediated activation of PKA (Figs. 1B and C and
5) (Su et al. 1995; Diller et al. 2001).
Signature Sequences and Profiles. Sequence logos for
PBC A and B and a logo defining the consensus PBC
23
were generated based on the sequence alignment of the
cAMP-binding domains. Residue conservation was
lower in positions not critical for the interaction with
cAMP. Residue conservation (dots in Fig. 6A) was
higher for PBC A. The occurrence of an invariant tyrosine residue in position 9 of each PBC A was especially
striking, since position 9 is the most variable in PBC B,
as shown by the entropy plots in Fig. 6B. The entropy
plots also show that conservation is higher in the Nterminal region than in the C-terminal part of the PBC.
Signature sequences for the R subunits were generated from the consensus sequence logo shown in Fig. 6.
Swissprot and TreMBL were scanned with signature patterns of different length. Shortening the N terminus of
the original 19-residue-long signature produced a dramatic loss of specificity. In contrast, shortening of the C
terminus of the signature sequence still kept the original
specificity. The minimal length of a signature capable to
retain enough stringency to detect all the R subunits
without any false positives was 14 residues.
The signature F–G–E–[LIV]–A–L–[LIMV]–X(3)–
[PV]–R–[ANVQ]–A (Table 2 and Fig. 7) can specifically identify a protein belonging to the R-subunit family
(X ⳱ any residue; alternative residues at any one position are presented in brackets). When Swissprot and
TrEMBL were scanned for proteins matching the signature, all the sequences and fragments of R subunits that
contained a cAMP-binding domain were identified.
There were no false positives. Thus, it is a genuine signature sequence and can be used to identify new members of the family as they become sequenced. Futhermore, by integrating this pattern with unique residues
identified from the sequence logos that define the different PBCs (Fig. 6), it was possible to define subpatterns
(Table 2). Those derived signatures are based on the
three central residues in the PBC, which are the residues
most distant from the cAMP molecule (Figs. 1B and C
and 7).
Identification of Potential R Subunits. The signature
pattern generated for the PKA-R family was used to scan
different databases. When the genome of Drosophila melanogaster was scanned, a new putative PKA regulatory
subunit was identified (Swissprot/TreMBL AC:
Q9V5E8).1 Scanning the complete genome of Drosophila revealed the presence of only one type I and one
type II subunit. Additional searches were performed on
the nonredundant database, dbEST, and also diverse genomic databases (Caenorhabditis elegans database at the
Sanger Center, Dictyostelium discoideum database at the
1
The RII subunit from Drosophila melanogaster was deposited and
annotated as a type II PKA regulatory subunit in the Swissprot and the
Celera Genomics Flybase databases after the completion of these studies. Therefore, the sequence was not included in our analysis.
Sanger Center, TIGR, and the collection of finished and
unfinished microbial genomes at the NCBI). As a result,
we identified several fragments previously not annotated
as belonging to PKA regulatory subunits (data not
shown), as well as two new possibly complete sequences. The putative regulatory subunit from Plasmodium falciparum (clone 3D7) corresponds to Contig
04.000625 in chromosome 12. The potential subunit
from Candida albicans corresponds to Contig 5-2380.
No additional subunits were identified in the partial genomes of C. elegans and Dictyostelium. No recognizable
homologues were identified in Bacteria or Archaea.
Discussion
Two types of R subunits are generally accepted: type I
and type II. While all R subunits share the same general
domain organization, type I and type II subunits differ in
molecular weight, isoelectric point, amino acid sequence,
antigenicity, autophosphorylation capacity, cellular location, and tissue distribution. Also, they differ in their
affinities for cAMP and C-subunit isoforms (Corbin et al.
1975; Øgreid et al. 1989). The original criterion to assign
R subunits to types I and II was the order of elution
following ion-exchange chromatography. Subsequently,
types I and II were subdivided into ␣ and ␤ subtypes
based on their SDS-PAGE apparent mobilities. This classification was formulated from a very limited number of
mammalian sequences available at the time. After that,
nonmammalian subunits were included into one of those
groups based on sequence similarity. While this methodology is adequate in some cases, in the PKA-R family
it leads to significant inconsistencies. Our analysis indicates that traditional classifications based on physicochemical properties such as isoelectric point/charge and
molecular weight are not adequate.
Inhibitory sequence motifs are another criterion used
to classify the PKA-R family. The inhibitory site for the
mammalian type II isoform contains the “RRxS⌿” consensus sequence for phosphorylation by the PKA catalytic subunit (Rosen and Erlichman 1975). In contrast,
the inhibitory site for type I isoforms contains a nonphosphorylatable pseudo-substrate site in which the serine found in type II motif is replaced by a small hydrophobic amino acid residue. One important characteristic
of the inhibitory site of animal type II isoforms is a
conserved cysteine residue that can form intermolecular
disulfide bonds with cysteine residues at the active sites
of C subunits (First et al. 1988). To fit the R subunit from
Fungi in the classic type I/type II schema, the length of
the inhibitory sequence motif is conventionally set to
five residues. If the motif is expanded to include the
residue following the ⌿ residue, it is apparent that in all
the subunits from Fungi, the cysteine residue charac-
24
Fig. 5. Partial multiple sequence alignment of the sequences shown in Table 1A. For method of alignment see text. Alignments are restricted to the C-terminal region of the R subunits that contains the
cAMP-binding domains. Dashes indicate gaps introduced during the alignment process. Straight arrows indicate ␤-strands and curvy arrows indicate ␣-helices, according to the bovine RI␣ crystal structure (Su
et al. 1995). The boundaries of the phosphate-binding cassettes (PBC) A an B are indicated. All sequences are listed under the abbreviated names indicated in Table 1. Dots under the PBC bars indicate residues
that are critical for the activation of the RI␣ subunits. Residues indicated by arrows in domains A and B may be important for interdomain communication. Sequence conservation is shown under the alignment
according to the following key: (*) invariant; (:) conserved; (.) partially conserved residue.
25
26
Fig. 6. Sequence logos and entropy plots. A Sequence logos defining
PBC A and B, and PBC consensus describing the general residue
distribution in any PBC belonging to a protein from the PKA-R family.
The bar over the PBC consensus logo indicates the region that was
used for the generation of the PKA-R family signature pattern. Dots in
the PBC A and B logos indicate residues that are invariant in each PBC.
The horizontal axis represents the position of the residue within the
PBC motif. The vertical axis represents the amount of information (in
bits) that this position holds. The height of the one-letter residue symbol at each position is proportional to the information content of the
residue at that position. B Entropy plots corresponding to the sequences
depicted in column A. The horizontal axis represents the position of the
residue within the PBC motif, whereas the vertical axis represents the
entropy or lack of predictability for each alignment position.
teristics of all animal type II subunits is replaced by an
invariant serine, as in type I subunits. Thus, based on the
extended six-residue motif, the subunits from Fungi
could be classified as a separate type. However, if differences in the inhibitory were applied as a classification
criterion was to the R subunits from Alveolates, each one
should be included in a separate group (Paramecium
TRxS⌿, Euplotes IKxS⌿, Plasmodium KKxS⌿), or they
should all be included in a separate group for “nonconforming” R subunits. In conclusion, the inhibitory sequence cannot be consistently used as a satisfactory criterion to classify the PKA-R family.
Phylogenetic trees are more revealing than raw homology data, because they integrate the data of all pairs
of protein sequences. Therefore, in the present study, we
have used clustering analysis based on phylogenetic trees
to classify the PKA-R family. Clustering based on phy-
logenetic analysis revealed the existence of four groups/
types. Two of them corresponded to classical type I and
II subunits, and the other two to Fungi (designated type
III) and Alveolates (designated type IV). The proposed
designations are a compromise between the most commonly used nomenclature and the fact that the Fungi and
Alveolate clusters have characteristics that clearly set
them apart from the type I/type II classification.
Our phylogenetic analysis indicates that it is likely
that an ancestral R subunit gave rise to type IV subunits
before the Metazoa and Fungi lineages separated. All
alveolate subunits contain atypical inhibitory sequence
motifs and lack dimerization domains. These characteristics suggest that these subunits descended early from an
ancestral R subunit. Phylogenetic trees indicate that the
emergence of multiple paralogous R subunits (types I
and II and subtypes ␣ and ␤) occurred late in the evo-
27
Table 2. Signature sequence for the cAMP-dependent protein kinase regulatory subunit family (PKA-R) and variable residues in the phosphatebinding cassette defining PKA-R subfamilies
Family
Signature sequence
PKA-R
1 2 3 4 5 6
7
8 9 10 11 12 13 14
F–G–E–[LIV]–A–L–[LIMV]–X1–X2–X3–[PV]–R–[ANVQ]–A
PBC A
Type & subfamily
X1
X2
PBC B
X3
Type I␣ mammal
X1
X2
X3
M
N
Type I␤ mammal
T
G
R
L
Type I nonmammal
D
Y
Type I Dictyostelium
S
T
Type II (␣/␤)
T
N
K
[NDEH]
[RKTLAE]
N
Types III & IV
[ASV]
Fig. 7. Spatial distribution of the residues that define the signature
sequence for the PKA-R family. The diagram schematically depicts the
residue distribution in the structural elements that define the PBCs and
their relative positions with respect to the cAMP molecule. Lines indicate hydrogen-bonding between the selected residues and cAMP.
lutionary process, after the divergence of Metazoa and
Fungi. The number of paralogous mammalian R subunits
may be explained by multiple gene duplication events.
This phenomenon may have occurred in response to the
need to maintain a stricter homeostasis and elaborate
intercellular communication networks in metazoans.
The rooted phylogenetic tree created using the sequence of Euplotes octocarinatus as outgroup showed a
[NDHKS]
branching pattern that was consistent with known functional differences between R subunits, as well as accepted phylogenetic relationships (Fig. 4, right). According to this tree, Fungi diverged before the Animals, and
before the gene duplication event that resulted in the
emergence of type I and type II subunits. The points of
divergence of Mollusca (Aplysia), Arthropoda (Drosophila), and Nematoda (C. elegans) conformed to accepted phylogeny (Davidson et al. 1995). The presence
of single type I and type II subunits in Drosophila suggests that the subsequent gene duplications that resulted
in the appearance of subtypes ␣ and ␤ occurred after
Arthropoda diverged.
The branching point of Dictyostelium has been the
subject of ample controversy. Most phylogenetic analysis based on rRNA (McCarrol et al. 1983; Hasegawa et
al. 1985; Herzog and Maroteaux 1986; Hendricks et al.
1991; Douglas et al. 1991) and some based on protein
sequence analysis (Kuma et al. 1995; Baldauf and
Doolittle 1997; Baldauf et al. 2000) place Dictyostelium
as outgroup to Fungi and Animals. Conversely, other
models based on protein sequence analysis suggest that
Fungi diverged from the line leading to Animals before
the divergence of Dictyostelium and Animals (Loomis
and Smith 1990, 1995; Roger et al. 1996; Kalhor et al.
1999; Norian et al. 1999; Swigart et al. 2000). Our analysis is consistent with the latter model. It suggests that
Dictyostelium is more closely related to Animals than to
Fungi and that it branched from the line leading to Metazoa after Fungi.
Signature sequences are of great diagnostic and practical value for an immediate assignment of newly sequenced subunits to a common classification scheme.
We have identified a global signature sequence (Table 2)
28
common to all PKA-R members and suitable for discriminating R subunits from other cyclic nucleotidebinding proteins. Since the general structure of the PBC
is very similar between R and other cAMP-binding proteins, such as the bacterial regulator CAP (McKay et al.
1982), it is remarkable that such short signature sequence
is capable of discriminating between members of the
PKA-R family and other cAMP-binding regulators such
as the NFR, CAP, or PKG families. Characteristically,
CAP has a PBC with an extra residue that lies within the
restrained, solvent-accessible loop of the PBC, i.e., in the
X1–X2–X3 region of the signature sequence (Fig. 7).
That part of the PBC, the most distant from the phosphate of the cAMP molecule, is also the most variable
between the R subfamilies. A notable exception is Tyr in
position 8 of PBC A. That residue is invariant in all PBC
A, whereas it corresponds to the most variable position in
PBC B. This is consistent with structural information that
indicates that this tyrosine lies at the center of a complex
network of contacts responsible for interdomain communication.
The residue in position 14 of the signature sequence is
extremely important to determine cyclic nucleotide
specificity. Replacement of an Ala residue in that position with a Thr produces a drastic change in selectivity
for cGMP vs cAMP (Weber et al. 1989). Residue 14 is a
conserved Ala in all PBC A, but only in 27 of 31 PBC B.
The sequences that do not have an Ala in that position,
and could potentially be activated by both cAMP and
cGMP, are Blastocladiella (V), S. pombe (N), and Saccharomyces (Q) (Fig. 7). Experimental evidence indicates that the subunit from Saccharomyces can be activated by both cAMP and cGMP (Cytrynska et al. 1999).
In conclusion, this study shows that R subunits can be
classified taking into consideration their phylogeny and
that the PBC regions can be used to establish family- and
subfamily-specific signatures. Our observations have
profound implications regarding the use of biological
model systems to study cAMP-mediated signaling. Accordingly, Drosophila could provide an ideal model for
the study of type I and type II subunit-mediated regulation of PKA, whereas Dictyostelium constitutes a suitable model for type I subunit-related studies. Attempts to
extrapolate from observations in yeast should take into
consideration that the R subunits from Fungi belong to a
separate type. Most studies classify the R subunits from
yeast as type II, when in fact those R subunits are distinct
and related to both type I and type II subunits. The variability in the residue responsible for cAMP–cGMP
specificity in several fungal subunits also suggests that
caution should be used in the exclusive attribution of
PKA-mediated cellular responses to cAMP.
We are confident that this study will contribute to
establishing a standardized categorical classification and
nomenclature of the PKA-R family and stimulate comparative studies on the evolution of this protein family, in
itself or in the global context of the evolution of cyclic
nucleotide-mediated protein kinase signaling.
Acknowledgments. This work was supported in part by USPHS
Grant GM34921 to S.S.T. Sequence data for Plasmodium falciparum
chromosome 12 was obtained from the Stanford DNA Sequencing and
Technology Center (http://www-sequence.stanford.edu/group/malaria).
Sequencing of Plasmodium falciparum chromosome 12 was accomplished as part of the Malaria Genome Project with support by the
Burroughs Wellcome Fund. Sequence data for Candida albicans were
generated at the Stanford DNA Sequencing and Technology Center
with the support of the NIDR and the Burroughs Wellcome Fund. We
wish to thank Anna Canaves for her assistance in manuscript preparation and graphics design.
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