The Protein Kinase Family: Conserved Features and Deduced Phylogeny of the Catalytic Domains

Abstract

In recent years, members of the protein kinase family have been discovered at an accelerated pace. Most were first described,
not through the traditional biochemical approach of protein purification and enzyme assay, but as putative protein kinase
amino acid sequences deduced from the nucleotide sequences of molecularly cloned genes or complementary DNAs. Phylogenetic
mapping of the conserved protein kinase catalytic domains can serve as a useful first step in the functional characterization
of these newly identified family members.

Full text

The Protein Kinase Family: Conserved Features and Deduced Phylogeny of the Catalytic
Domains
Author(s): Steven K. Hanks, Anne Marie Quinn and Tony Hunter
Source:
Science,
New Series, Vol. 241, No. 4861 (Jul. 1, 1988), pp. 42-52
Published by: American Association for the Advancement of Science
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58. M. L. Alexander,
M. A. Johnson,
W. C. Lineberger,
J. Chem.
Phys.
82, 5288
(1985).
59. D. E. Hunton,
M. Hofmtnann,
T. G. Lindeman,
C. R. Albertoni,
A. W. Castleman,
Jr., ibid., p. 2884.
60. W. D. Reents, Jr., A. M. Mujsce, V. E. Bondybey, M. L. Mandich, ibid.
86, 5568
(1987).
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M. E. Geusic, J. R. Heath,
R. E. Smalley,
ibid.
83, 2293 (1985).
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K. Liu, S. C. Richtsmeier,
L. G. Pobo, S. J. Riley, ibid.
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(1985).
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Phys.
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(1985).
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M. Hofmtnann,
T. G. Lindeman,
C. R. Albertoni,
A. W. Castleman,
Jr., ibid., p. 2884.
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86, 5568
(1987).
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M. E. Geusic, J. R. Heath,
R. E. Smalley,
ibid.
83, 2293 (1985).
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K. Liu, S. C. Richtsmeier,
L. G. Pobo, S. J. Riley, ibid.
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(1985).
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S. L. Anderson,
Chem.
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69. Financial support by the Army Research Office, grant DAAG29-85-K-0215, the
Department of Energy, grant DE-AC02-82ER60055, and the National Science
Foundation, grant ATM-82-04010, is gratefully acknowledged.
64. R. D. Levine, R. B. Bernstein, Eds., Molecular Reaction
Dynamics
and Chemical
Reactivity
(Oxford Univ. Press, New York, 1987).
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69. Financial support by the Army Research Office, grant DAAG29-85-K-0215, the
Department of Energy, grant DE-AC02-82ER60055, and the National Science
Foundation, grant ATM-82-04010, is gratefully acknowledged.
The
Protein
Kinase
Family:
Conserved
Features
and
Deduced
Phylogeny
of
the
Catalytic
Domains
STEVEN
K. HANKS,
ANNE
MARIE
QUINN,
TONY HUNTER
The
Protein
Kinase
Family:
Conserved
Features
and
Deduced
Phylogeny
of
the
Catalytic
Domains
STEVEN
K. HANKS,
ANNE
MARIE
QUINN,
TONY HUNTER
In recent years, members of the protein kinase family have
been discovered at an accelerated pace. Most were first
described, not through the traditional biochemical ap-
proach of protein purification and enzyme assay, but as
putative protein kinase amino acid sequences deduced
from the nucleotide sequences of molecularly cloned
genes or complementary DNAs. Phylogenetic mapping of
the conserved protein kinase catalytic domains can serve
as a useful first step in the functional characterization of
these newly identified family members.
HE PROTEIN KINASES ARE A LARGE
FAMILY
OF ENZYMES,
many of which mediate the response of eukaryotic cells to
external stimuli (1, 2). The number of unique members of
the protein kinase family that have been described has recently risen
exponentially (3) and now approaches 100. The surge in the number
of known protein kinases has been largely due to the advent of gene
cloning and sequencing techniques. Amino acid sequences deduced
from nucleotide sequences are considered to represent protein
kinases if they include certain key residues that are highly conserved
in the protein kinase "catalytic
domain."
Two different molecular approaches have been most instrumental
in the isolation of novel protein kinase-encoding genes or cDNAs:
(i) complementation or suppression of genetic defects in inverte-
brate regulatory mutants, and (ii) screening DNA libraries
by using
protein kinase genes as hybridization probes under low stringency
conditions. Recently, an approach that uses degenerate oligonucleo-
tides as probes has led to the identification of several novel putative
In recent years, members of the protein kinase family have
been discovered at an accelerated pace. Most were first
described, not through the traditional biochemical ap-
proach of protein purification and enzyme assay, but as
putative protein kinase amino acid sequences deduced
from the nucleotide sequences of molecularly cloned
genes or complementary DNAs. Phylogenetic mapping of
the conserved protein kinase catalytic domains can serve
as a useful first step in the functional characterization of
these newly identified family members.
HE PROTEIN KINASES ARE A LARGE
FAMILY
OF ENZYMES,
many of which mediate the response of eukaryotic cells to
external stimuli (1, 2). The number of unique members of
the protein kinase family that have been described has recently risen
exponentially (3) and now approaches 100. The surge in the number
of known protein kinases has been largely due to the advent of gene
cloning and sequencing techniques. Amino acid sequences deduced
from nucleotide sequences are considered to represent protein
kinases if they include certain key residues that are highly conserved
in the protein kinase "catalytic
domain."
Two different molecular approaches have been most instrumental
in the isolation of novel protein kinase-encoding genes or cDNAs:
(i) complementation or suppression of genetic defects in inverte-
brate regulatory mutants, and (ii) screening DNA libraries
by using
protein kinase genes as hybridization probes under low stringency
conditions. Recently, an approach that uses degenerate oligonucleo-
tides as probes has led to the identification of several novel putative
S. K. Hanks is a senior research associate at the Molecular Biology Laboratory, Salk
Institute for Biological Studies, Post Office Box 85800, San Diego, CA 92138. A. M.
Quinn is a scientific applications programmer at the Biocomputing Center, Salk
Institute for Biological Studies, Post Office Box 85800, San Diego, CA 92138. T.
Hunter is a professor at the Molecular Biology and Virology Laboratory, Salk Institute
for Biological Studies, Post Office Box 85800, San Diego, CA 92138.
S. K. Hanks is a senior research associate at the Molecular Biology Laboratory, Salk
Institute for Biological Studies, Post Office Box 85800, San Diego, CA 92138. A. M.
Quinn is a scientific applications programmer at the Biocomputing Center, Salk
Institute for Biological Studies, Post Office Box 85800, San Diego, CA 92138. T.
Hunter is a professor at the Molecular Biology and Virology Laboratory, Salk Institute
for Biological Studies, Post Office Box 85800, San Diego, CA 92138.
42 42
protein kinase genes and cDNAs (4, 5). The oligonucleotide probes
are designed to recognize target sequences that encode short amino
acid stretches highly conserved in protein kinase catalytic domains.
In this article,
we present an alignment of catalytic
domain amino
acid sequences from 65 different members of the protein kinase
family, including many putative protein kinase sequences recently
deduced from nucleotide sequence data. Based on this alignment,
we first identify and discuss conserved features of the catalytic
domains and then provide a visual display of the various interse-
quence relations through construction of a catalytic domain phylo-
genetic tree. Catalytic domains from protein kinases having similar
modes of regulation or substrate specificities are found to cluster
together within the tree. This clustering would appear to be of
predictive value in the determination of the properties and function
of novel protein kinases.
Catalytic
Domain Amino Acid Sequences
Protein kinase catalytic domains range from 250 to 300 amino
acid residues, corresponding to about 30 kD. Fairly precise bound-
aries
for the catalytic
domains have been defined through an analysis
of conserved sequences (1, 6, see below) as well as by assay of
truncated enzymes (7, 8). The location of the catalytic domain
within the protein is not fixed but, in most single subunit enzymes it
lies near the carboxyl
terminus, the amino terminus being devoted to
a regulatory role. In protein kinases having a multiple subunit
structure,
subunit polypeptides consisting almost entirely of catalyt-
ic domain are common. All protein kinases thus far characterized
with regard to substrate specificity fall within one of two broad
classes, serine/threonine-specific and tyrosine-specific. Although
both classes of protein kinase have very similar catalytic domain
primary structures, certain short amino acid stretches appear to
characterize
each class (4), and these regions can be used to predict
whether a putative protein kinase will phosphorylate tyrosine or
serine/threonine.
Members of the protein-serine/threonine kinase and protein-
SCIENCE, VOL. 24I
protein kinase genes and cDNAs (4, 5). The oligonucleotide probes
are designed to recognize target sequences that encode short amino
acid stretches highly conserved in protein kinase catalytic domains.
In this article,
we present an alignment of catalytic
domain amino
acid sequences from 65 different members of the protein kinase
family, including many putative protein kinase sequences recently
deduced from nucleotide sequence data. Based on this alignment,
we first identify and discuss conserved features of the catalytic
domains and then provide a visual display of the various interse-
quence relations through construction of a catalytic domain phylo-
genetic tree. Catalytic domains from protein kinases having similar
modes of regulation or substrate specificities are found to cluster
together within the tree. This clustering would appear to be of
predictive value in the determination of the properties and function
of novel protein kinases.
Catalytic
Domain Amino Acid Sequences
Protein kinase catalytic domains range from 250 to 300 amino
acid residues, corresponding to about 30 kD. Fairly precise bound-
aries
for the catalytic
domains have been defined through an analysis
of conserved sequences (1, 6, see below) as well as by assay of
truncated enzymes (7, 8). The location of the catalytic domain
within the protein is not fixed but, in most single subunit enzymes it
lies near the carboxyl
terminus, the amino terminus being devoted to
a regulatory role. In protein kinases having a multiple subunit
structure,
subunit polypeptides consisting almost entirely of catalyt-
ic domain are common. All protein kinases thus far characterized
with regard to substrate specificity fall within one of two broad
classes, serine/threonine-specific and tyrosine-specific. Although
both classes of protein kinase have very similar catalytic domain
primary structures, certain short amino acid stretches appear to
characterize
each class (4), and these regions can be used to predict
whether a putative protein kinase will phosphorylate tyrosine or
serine/threonine.
Members of the protein-serine/threonine kinase and protein-
SCIENCE, VOL. 24I
I I , I I ,
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tyrosine kinase families with reported catalytic domain amino acid
sequences are listed in Tables 1 and 2, respectively. They are
classified within the tables according to similarities in primary
structure, based on deduced catalytic domain phylogeny. Included
in the tables are all confirmed and putative protein kinases for which
the catalytic domain sequence was available as of November 1987
(9). Presumed functional homologs from different
vertebrate
species
are listed together. Presumed invertebrate functional homologs of
protein kinases also found in vertebrates, however, are given
separate listings as a reflection of greater evolutionary distance and
the possibility of functional divergence. The asterisks indicate
protein kinases that have catalytic domains that are included in the
amino acid sequence alignment. We will use the abbreviated
names
from the tables to refer to individual protein kinases.
Of the 45 unique vertebrate protein kinase family members
included in Tables 1 and 2, 22 are serine/threonine-specific and 23
are tyrosine-specific. Fourteeh of the vertebrate protein-serine/thre-
onine kinases fall within one of the three subgroups that can be
Table 1. Protein-serine/threonine
kinase
family
members.
A. Cyclic
nucleotide-dependent subfamily
cAPK-a:
cAMP-dependent
protein
kinase
catalytic
subunit,
a form
*-bovine
cardiac
muscle
protein
(26)
-mouse S49 lymphoma
cell cDNA (35)
cAPK-P:
cAMP-dependent
protein
kinase
catalytic
subunit,
P form
*-bovine
pituitary
cDNA (36)
-mouse
S49 lymphoma
cell cDNA (37)
SRA3:
cAMP-dependent
protein
kinase from
yeast,
RAS suppressor
*-Saccharomyces
cereviiae
genomic
DNA (38)
TPKI(PK25):
cAMP-dependent protein
kinase from
yeast,
type 1
*-S. cerevisiae
genomic
DNA (39, 40)
TPK2:
cAMP-dependent protein
kinase from
yeast, type 2
*-S. cerevisiae
genomic
DNA (39)
TPK3:
cAMP-dependent
protein
kinase from
yeast, type 3
*-S. cerevisiae
genomic
DNA (39)
cGPK:
guanosine
3',5'-monophosphate (cGMP)-dependent protein
kinase
*-bovine
lung protein
(41)
B. Calcium-phospholipid-dependent
subfamily
PKC-a:
protein
kinase
C, a form
*-bovine brain
cDNA (42)
-rabbit brain cDNA (43)
-human brain
cDNA (partial)
(44)
PKC-P:
protein
kinase
C, 3 form
*-bovine
brain cDNA (44)
-rat brain cDNA (two splice
forms) (45, 46)
-rabbit
brain
cDNA (two splice
forms)
(43)
-human
brain cDNA (44)
PKC-y:
protein
kinase
C, y form
*-bovine brain cDNA (44)
-rat brain
cDNA (45)
-human
brain cDNA (44)
PKC-E:
protein
kinase
C, e form
-rat
brain
cDNA (RP16 clone) (partial)
(46)
DPKC:
Drosophila
gene product
related to protein
kinase
C
*-D. melanogaster
cDNA (47)
C. Calcium-calmodulin-dependent subfamily
CaMII-a:
calcium-calmodulin-dependent
protein
kinase
type II, a
subunit
*-rat brain cDNA (48)
CaMII-3:
calcium-calmodulin-dependent
protein
kinase
type II, P
subunit
*-rat brain
cDNA (49)
PhK-y:
phosphorylase
kinase,
y subunit
*-rabbit skeletal
muscle
protein
and cDNA (50)
-mouse muscle cDNA (51)
MLCK-K:
myosin
light chain
kinase,
skeletal muscle
*-rabbit
skeletal muscle
protein
(52)
MLCK-M:
myosin
light chain
kinase,
smooth
muscle
*-chicken
gizzard
cDNA (53)
PSK-H1:
putative protein-serine
kinase
*-human
HeLa cell cDNA (4, 54)
PSK-C3:
putative protein-serine
kinase
-human
HeLa cell cDNA (partial)
(4)
D. SNF1 subfamily
SNFI: "sucrose
nonfermenting"
mutant
wild-type gene product
*-S. cerevisiae
genomic
DNA (55)
niml+: "new
inducer
of mitosis";
suppressor
of cdc25 mutants
*-Schizosaccharomyces pombe
genomic
DNA (56)
KIN1: putative yeast
protein
kinase
*-Saccharomyces
cerevisiae
genomic
DNA (5)
KIN2: putative yeast
protein
kinase related to KIN1
*-S. cerevisiae
genomic
DNA (5)
E. CDC28-cdc2+
subfamily
CDC28:
"cell-division-cycle"
gene product
in yeast
*-S. cereisiae
genomic
DNA (57)
cdc2+:
"cell-division-cycle"
gene product
in yeast
*-Schizosaccharomyces pombe
genomic
DNA (58)
CDC2Hs: human functional
homolog of cdc2+
*-human
transformed
cell line cDNA (33)
PSK-J3:
putative protein
kinase related
to CDC28-cdc2+
*-human
HeLa cell cDNA (4, 59)
KIN28: putative
protein
kinase
related to CDC28-cdc2+
*-Saccharomyces
cerevisiae
genomic
DNA (60)
F. Casein
kinase
subfamily
CKIIa:
casein kinase
II, a subunit
-bovine
lung protein
(partial)
(61)
DCKII:
Drosophila
casein
kinase
II, a subunit
*-D. melanogaster
cDNA (62)
G. Raf-Mos
proto-oncogene subfamily
Raf: cellular
homolog
of oncogene
products
from 3611 murine
sarcoma virus and
Mill Hill 2 avian acute
leukemia virus
*-human
fetal liver cDNA (63)
A-Raf: cellular
oncogene product closely
related to Raf
*-human
T cell cDNA (64)
-mouse
spleen
cDNA (65)
PKS: cellular
gene product closely
related to Raf
*-human
fetal liver cDNA (66)
Mos: cellular
homolog
of oncogene product
from
Moloney
murine
sarcoma virus
*-human
placenta
genomic
DNA (67)
-mouse NIH 3T3 cell genomic
DNA (68)
-rat
3Y1 cell genomic
DNA (69)
H. STE7
subfamily
STE7: "sterile"
mutant
wild-type
allele
gene product
*-S. cerevisiae
genomic
DNA (70)
PBS2:
polymixin
B antibiotic resistance
gene product
*-S. cerevisiae
genomic
DNA (71)
I. Family
members with no close relatives
CDC7:
"cell-division-cycle"
gene product
*-S. cerevsiae
genomic
DNA (72)
weel+:
"reduced
size at division"
mutant
wild-type
gene product
*-Schizosaccharomyces
pombe
genomic
DNA (73)
ranl+: "meiotic
bypass"
mutant
wild-type
allele
gene product
*-S.
pombe
genomic
DNA (74)
PIM-1:
putative
transforming protein
induced
by murine leukemia
virus integration
*-mouse
BALB/c
cell genomic
DNA (75)
HSVK:
herpes
simplex
virus-US3
gene product
*-herpes simplex
virus
genomic
DNA (76)
*Protein kinases that have catalytic domains included in the amino acid sequence alignment.
ARTICLES 43
I JULY 1988
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classified according to their mode of regulation: cyclic nucleotide-
dependent, calcium-phospholipid-dependent, and calcium-calmo-
dulin-dependent. Two of the serine/threonine kinases, Mos and Raf
(products of the c-mos and c-raf genes, respectively), are cellular
homologs of transforming proteins encoded by the retroviral
onco-
genes. Other members of the serine/threonine group with demon-
strated oncogenic potential are A-Raf (a distinct Raf-related mem-
ber), and PIM-1 (a putative transforming protein activated by viral
integration). Three vertebrate serine/threonine kinases (CDC2Hs,
PSK-J3, and CKIIa) are closely related, by various degrees, to the
yeast cell cycle control protein kinases CDC28 and cdc2+. A
protein-serine/threonine kinase has been described in herpes sim-
plex virus (HSVK) and, like the retroviral oncogenes, probably
originated as a eukaryotic cellular sequence. The protein-tyrosine
kinases can be further grouped as members of either the Src
subfamily or one of three different growth factor receptor subfami-
lies. The protein-tyrosine kinases encoded by the c-abl and c-fes/fps
genes may be considered distant members of the Src subfamily. At
least nine of the protein-tyrosine kinase genes have been transduced
by retroviruses where they encode transforming proteins.
Twenty-five additional sequences listed in Tables 1 and 2 de-
rive from invertebrate species. Eight are from Drosophila, one
from nematode, and the other 16 are from the budding or fission
yeasts. Many of the Drosophila protein kinases, as well as the
nematode protein kinase, were identified by screening DNA librar-
ies with probes from a vertebrate protein kinase gene or cDNA and
thus are likely to represent functional homologs of the vertebrate
enzymes. The Drosophila
"sevenless"
(7less) protein kinase and most
of the yeast protein kinases were identified through molecular
genetics. All of the yeast protein kinases identified to date fall within
the serine/threonine-specific class, despite directed attempts to
identify protein-tyrosine kinases in yeast (5). This observation,
together with the fact that many of the protein-tyrosine kinase
catalytic domains are components of growth factor receptor mole-
cules, suggests that tyrosine specificity may have been a recent
development in catalytic domain evolution, arising in conjunction
with the acquisition of multicellularity
and serving a role in cell-cell
communication.
Table 2. Protein-tyrosine
kinase
family
members.
* I _ i i i I l i i i i_
A. Src
subfamily
Src: cellular
homolog
of oncogene
product
from
Rous avian
sarcoma
virus
*-human
fetal liver
genomic
DNA (77)
-mouse brain
cDNA; neuronal
alternate
splice
form (78)
-chicken
genomic
DNA (79)
-Xenopus
laevis
ovary
cDNA (partial)
(80)
Yes:
cellular
homolog
of oncogene
product
from
Yamaguchi
73 avian
sarcoma virus
*-human
embryo
fibroblast
cDNA (81) D
Fgr:
cellular
homolog
of oncogene
product
from Gardner-Rasheed
feline
sarcoma
virus
*-human
genomic
DNA (82)
-human
B lymphocyte
cell line cDNA (amino terminus)
(83)
FYN:
putative
protein-tyrosine
kinase
related
to Fgr and
Yes
*-human
fibroblast
cDNA (84)
LYN:
putative
protein-tyrosine
kinase
related
to LCK and Yes
*-human
placenta
cDNA (85)
LCK:
lymphoid
cell protein-tyrosine
kinase
*-human
(JURKAT)
Tcell leukemia
line cDNA (86)
-mouse
(LSTRA)
T cell lymphoma
line cDNA (87)
HCK: hematopoietic
cell putative
protein-tyrosine
kinase
*-human
placenta
and peripheral
leukocyte
cDNAs (88)
Dsrc64:
Drosophila
gene product
related to Src;
polytene
locus
64B
*-D. melanogaster genomic DNA (89, 90)
Dsrc28:
Drosophila
gene product
related
to Src;
polytene
locus 28C
*-D. melanogaster
adult female cDNA (91)
B. Abl subfamily
Abl:
cellular
homolog
of oncogene
product
from Abelson
murine E
leukemia virus
*-human
fetal liver
cDNA (92)
ARG:
putative
protein-tyrosine
kinase
related
to Abl
-human
genomic
DNA (partial)
(93)
Dash:
Drosophila
gene product
related
to Abl
*-D. melanogaster
genomic DNA (90)
Nabl:
nematode
gene product
related
to Abl
*-Caenorhabditis
elegans
genomic DNA (94)
Fes/Fps:
cellular
homolog
of oncogene products
from Gardner-
Arnstein and Snyder-Theilen
feline
sarcoma
viruses and
Fujinami
and PRCII
avian
sarcoma
viruses
*-human
genomic
DNA (95) F
-feline
genomic
DNA (96)
-chicken
genomic
DNA (97)
C. Epidermal
growth
factor
receptor
subfamily
EGFR:
epidermal
growth
factor
receptor;
cellular
homolog
of
*Protein kinases
that have
catalytic
domains
included
in the amino
acid
sequence
alignment.
44
oncogene product
(v-Erb-B)
from
AEV-H avian
erythroblastosis
virus
*-human
placenta
and A431 cell line cDNAs (98)
NEU: cellular
oncogene
product
activated
in induced
rat
neuroblastomas
(also
called ERB-B2
or HER2)
*-human
placenta
and
gastric
cancer
cell line cDNAs (99)
-rat neuroblastoma
cell line cDNA (100)
DER:
Drosophila
gene product
related
to EGFR
*-D. melanogaster
genomic DNA (101)
Insulin
receptor
subfamily
INS.R: insulin
receptor
*-human
placenta
cDNA (102)
IGF1R:
insulin-like
growth
factor 1 receptor
*-humran
placenta
cDNA (103)
DILR:
Drosophila
gene product
related
to INS.R
*-D. melanogaster
embryo cDNA (104)
Ros: cellular
homolog
of oncogene
product
from UR2 avian
sarcoma virus
*-human
placenta genomic
DNA (105)
-chicken
genomic
DNA (106), chicken
kidney
cDNA (107)
7less:
Drosophila
sevenless
gene product
essential
for R7
photoreceptor
cell development
*-D. melanogaster
eye imaginal disc cDNA (108)
TRK: colon carcinoma
oncogene
product
activated
by genetic
recombination
*-human
tumor
cell cDNA (109)
MET:
N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG)-induced
oncogene
product
*-human
HOS cell line cDNA (110)
Platelet-derived
growth
factor
receptor
subfamily
PDGFR:
platelet-derived growth
factor
receptor
*-mouse
NR6 fibroblast cell line cDNA (111)
CSF1R:
colony-stimulating
factor-type
1 receptor;
cellular
homolog
of oncogene
product
(v-Fms)
from
McDonough
feline sarcoma
virus
*-human
placenta
cDNA (112)
Kit:
cellular
homolog
of oncogene product
from
Hardy-Zuckerman
4
feline sarcoma
virus
*-human
placenta
cDNA (113)
RET: cellular
oncogene product
activated
by recombination
*-human
T cell lymphoma
cDNA (114)
Other
receptor-like protein-tyrosine
kinases
TKR11:
putative protein-tyrosine
kinase
-chicken
genomic
DNA (partial)
(115)
TKR16: putative
protein-tyrosine
kinase
-chicken
genomic
DNA (partial)
(115)
Illllll Illll I I 'l I I Illlllllll Ill II IIII Illlllll I I I [~~
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
SCIENCE, VOL. 241
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Conserved Features of the Catalytic Domains
To compare primary
structures,
we have aligned catalytic
domains
from the 65 protein kinases marked by an asterisk
in Tables 1 and 2
(Fig. 1). The 65 sequences represent each of the separate entries in
the Tables except for six family members that are not included
because their catalytic domain sequences have been only partially
determined. The alignment was made by eye and is parsimonious in
nature; the amount of gapping introduced into the sequences in
order to optimize positional similarities was kept to a minimum.
The alignment clearly demonstrates the overall similarity among the
catalytic
domains. The catalytic domains are not conserved uniform-
ly but, rather, consist of alternating regions of high and low
conservation. Eleven major conserved subdomains are evident (Fig.
1, I to XI), separated by regions of lower conservation wherein fall
the larger gaps or inserts. Very large inserts (in excess of 60 residues)
occur in CDC7 between subdomains VII and VIII and between
subdomains X and XI, and in PDGFR, CSF1R, and Kit between
subdomains V and VI. A similarity profile of the aligned catalytic
domains provides a ready visualization of the subdomain structure
(Fig. 2). Such an arrangement
of alternating regions of high and low
conservation is a common feature of homologous globular proteins
(10) and gives some clues to higher order structure. The conserved
subdomains must be important for catalytic function, either directly
as components of the active site or indirectly by contributing to the
formation of the active site through constraints imposed on second-
ary structure. The nonconserved regions, on the other hand, are
likely to occur in loop structures, where folding allows the essential
conserved regions to come together.
Highly conserved individual amino acids within the catalytic
domains are expected to play important roles in catalysis. We will
refer to amino acid positions using the residue numbering for
bovine adenosine 3',5'-monophosphate (cAMP)-dependent pro-
tein kinase catalytic subunit, (x form (cAPK-(x, Fig. 1). Nine
positions in the alignment contain the identical amino acid residue
in each of the 65 sequences. These invariant residues correspond to
cAPK-I(: Gly52, Lys72,
Glu91,
Asp166,
Asn'71, Asp'84, Gly186,
Glu208,
and Arg280.
An additional five positions contain the identical amino
acid in all but one of the sequences: Gly50, Val'17,
Phe'85, Asp220
and Gly225. Many of these most highly conserved residues directly
participate
in adenosine triphosphate (ATP) binding and phospho-
transfer.
The consensus Gly-X-Gly-X-X-Gly, found in many nucleotide
binding proteins in addition to the protein kinases (11), is found in
subdomain I, very near the catalytic domain amino terminus. The
invariant or nearly invariant residues corresponding to cAPK-(x
Gly5?
and Gly52
fall within this consensus. Only two positions on the
amino-terminal side of this consensus show conservation through-
out the protein kinase family; hydrophobic residues occupy posi-
tions one and seven upstream from the first glycine in the consensus.
The amino terminus of some catalytic domain polypeptides lies as
close as ten residues from the first conserved glycine. A model for
the ATP-binding site of v-Src (12), based on the three-dimensional
structures
from other nucleotide binding proteins, shows the Gly-X-
Gly-X-X-Gly
residues forming an elbow around the nucleotide, with
the first glycine in contact with the ribose moiety and the second
glycine lying near the terminal pyrophosphate. A nearly invariant
valine residue lies within subdomain I, located just two positions on
the carboxyl-terminal side of the Gly-X-Gly-X-X-Gly consensus
(Val57
for cAPK-(x) and may contribute to the positioning of the
conserved glycines.
In subdomain II lies an invariant lysine, corresponding to cAPK-
c( Lys72, that is certainly the best characterized catalytic domain
residue. This lysine appears to be directly involved in the phospho-
I JULY 1988
transfer
reaction, possibly mediating proton transfer (13). In cAPK-
ot (14), v-Src (15), and EGFR (16), Lys72
or its equivalent reacts
with the ATP analog p-fluorosulfonyl 5'-benzoyl adenosine, thereby
inhibiting enzyme activity. Site-directed mutagenesis techniques
have been used to substitute alternate amino acids at this position in
v-Src (13, 17), v-Mos (18), v-Fps (19), EGFR (20), INS.R (21), and
PDGFR (22). All substitutions, including arginine, result in loss of
protein kinase activity. In all but three of the aligned sequences, an
alanine is present two positions on the amino-terminal side of the
invariant
lysine in subdomain II. The invariant lysine lies 14 to 23
residues downstream of the last conserved glycine in subdomain I,
but no mutations have been made to test whether this spacing is
critical.
The central core of the catalytic domain, the region with greatest
frequency of highly conserved residues, consists of subdomains VI
through IX. The invariant
or nearly invariant residues in subdomain
VI (corresponding to Asp'66 and Asn'71) and subdomain VII
(corresponding to Asp'84, Phes,85 and Gly'86) also have been
implicated in ATP binding. These residues are part of a feature
found in a number of bacterial
phosphotransferases
that use ATP as
phosphate donor (23). The aspartic acid residues corresponding to
cAPK-at
Asp'66 and Asp'84 may interact with the phosphate groups
of ATP through Mg2+ salt bridges (23). The triplet corresponding
to Asp'84-Phel85-Gly'86
in subdomain VII is of further interest in
that it represents the most highly conserved short stretch in the
catalytic domains. It is flanked for two positions on either side by
hydrophobic or near-neutral
residues.
Subdomain VIII contains the consensus triplet Ala-Pro-Glu, a
conserved feature often mentioned as a key protein kinase catalytic
domain indicator (1). The invariant residue corresponding to
cAPK-(x
Glu208
contributes to the Ala-Pro-Glu consensus. In addi-
tion to the conservation of these residues, several other lines of
evidence implicate this region as important in catalysis. Mutagenesis
studies have shown that each residue in the Ala-Pro-Glu consensus is
required for activity of v-Src (24). Other studies have provided
evidence that this consensus lies very near the catalytic site. An
affinity
peptide substrate analog reacts with cAPK-(x
Cys'99, thereby
inhibiting enzyme activity (25). Also, sites of autophosphorylation
found in many protein-tyrosine kinases (1) as well as cAMP-
dependent protein kinase [Thr'97 (26)] lie within 20 residues
upstream of the Ala-Pro-Glu consensus. The role of this autophos-
phorylation site is not entirely settled, but for several protein-
tyrosine kinases there is evidence that phosphorylation of this site
leads to increased catalytic activity (27). Autophosphorylation may
result in a conformational change that allows better access of
exogenous substrates to the active site.
Subdomains VI and VIII are of additional interest in that they
contain residues that are specifically
conserved in either the protein-
serine/threonine or the protein-tyrosine kinases and, as such, may
play a role in recognition of the correct hydroxyamino acid. The
most striking indicator of amino acid specificity is found in subdo-
main VI, lying between the invariant residues corresponding to
cAPK-(x
Asp'66 and Asn'71; two of the residues implicated in ATP
binding. The consensus Asp-Leu-Lys-Pro-Glu-Asn in this region is
a strong indicator of serine/threonine specificity, whereas the pro-
tein-tyrosine kinase consensus is either Asp-Leu-Arg-Ala-Ala-Asn
(for the vertebrate members of the Src subfamily) or Asp-Leu-Ala-
Ala-Arg-Asn (for all others). Another such region is found in
subdomain VIII and lies immediately on the amino-terminal side of
the Ala-Pro-Glu consensus. This region is highly conserved among
the protein-tyrosine kinases with a more limited conservation
among the protein-serine/threonine kinases. The protein-tyrosine
kinase consensus through this region is Pro-Ile/Val-Lys/Arg-Trp-
Thr/Met-Ala-Pro-Glu while the protein-serine/threonine kinase
ARTICLES 45
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consensus is Gly-Thr/Ser-X-X-Tyr/Phe-X-Ala-Pro-Glu.
These re-
gions in subdomains VI and VIII that indicate substrate specificity
have been targeted for the design of degenerate oligonucleotide
probes for use in screening cDNA libraries to identify novel
members of both the protein-serine/threonine (4) and protein-
tyrosine (28) kinase families.
To date, no evidence has been reported concerning the possible
functions of residues in conserved subdomains III, IV, V, IX, X, and
XI. Subdomain IX contains a very well conserved short stretch that
includes the nearly invariant residues corresponding to Asp220
and
Gly225.
Subdomains III and XI each contain an invariant residue,
corresponding to Glu91
and Arg280.
The latter or its equivalent must
lie very near the catalytic domain carboxyl terminus. Arginine
residues occupying this position reside just 16 residues upstream
from both the CDC28 and HSVK polypeptide carboxyl termini,
and just 19 residues upstream from both the Mos and Fes carboxyl
termini. Deletion analysis of v-Src places the carboxyl terminus of
the catalytic domain of the protein-tyrosine kinases at a conserved
hydrophobic residue ten residues downstream of this arginine (8).
The point mutation conferring temperature
sensitivity in some cdc28
mutants replaces this conserved arginine with glutamine (29).
A leap in our understanding of the functional roles of the
conserved catalytic domain residues will come with the solution of a
crystal
structure
for one of the protein kinase catalytic
domains. The
similarities in primary
strucure should carry
over to the higher order
structure and catalytic mechanism as well. Other investigators have
been making progress toward the solution of the three-dimensional
structure of cAPK-o (30).
Catalytic
Domain Phylogeny
Amino acid sequence alignments can be used to deduce phyloge-
netic relationships (31). We have used the alignment data from Fig.
1 to construct a phylogenetic tree of the protein kinase catalytic
domains (Fig. 3). All 65 of the sequences in the alignment are
included in the tree. They derive from both vertebrate and inverte-
brate sources and, in some cases, presumed functional homologs
from both vertebrate and invertebrate sources are represented. The
tree, therefore, reflects catalytic domain evolution stemming from
gene duplication events (for example, when the vertebrate, mostly
human, sequences are compared), speciation events (when verte-
brate and invertebrate
functional homologs are compared), or both.
The tree reveals a relation between catalytic domain sequence and
certain biochemical properties; catalytic domains from protein
kinases having similar modes of regulation or substrate specificities
tend also to have similar primary structures and cluster together
within the tree. Five major branch clusters are present in the tree: (i)
protein-tyrosine kinases, (ii) cyclic nucleotide- and calcium-phos-
pholipid-dependent protein kinases, (iii) calcium-calmodulin-de-
pendent protein kinases, (iv) protein kinases closely related to
SNF1, and (v) protein kinases closely related to CDC28. These
major
clusters account for all but 12 of the 65 sequences included in
the tree. Generally, a sequence found within one of these clusters
shares in excess of 35% identical amino acids with each of the other
sequences in the cluster, whereas the catalytic
domain sequences that
do not map within the same cluster have identities in the range of
20 to 25%.
The most highly populated cluster contains all 27 confirmed or
putative protein-tyrosine kinases. The large number of protein-
tyrosine kinases probably reflects the intense research
effort devoted
to this group, rather than a true indication of their abundance
relative
to the protein-serine/threonine kinases. Branches leading to
the Src subfamily and to each of the three receptor subfamilies
46
diverge from the main line at about the same point. In light of the
oncogenic potential of many of the protein-tyrosine kinases, it is of
interest that the protein-serine/threonine kinases having the least
divergence from this group include Raf and Mos, cellular
homologs
of retroviral oncogene products. However, another potentially
oncogenic protein-serine/threonine kinase, PIM-1, is not closely
related to the protein-tyrosine kinases.
The next most populous cluster in the tree includes two separate
subfamilies that can be classified according to their mode of
regulation: the cyclic nucleotide-dependent protein kinases and the
calcium-phospholipid-dependent protein kinases.
The similarities in
the mode of regulation of the members of these two subfamilies,
namely, activation by "second messengers" released in response to
ligand binding at the cell surface, may be a reflection of their recent
evolutionary divergence.
The third major catalytic
domain cluster contains the subfamily of
protein kinases that have activities regulated by calmodulin. The
calmodulin-dependent cluster falls near the cyclic nucleotide- and
calcium-phospholipid-dependent cluster. All members of the cal-
modulin-dependent subfamily have a calmodulin binding domain,
characterized
by a high proportion of basic amino acid residues and
having a propensity for formation of an amphiphilic a helix, residing
outside the catalytic domain. (Note that the calmodulin binding
domain sequences were not included in the phylogenetic analysis.)
The different
protein kinases thus far described as being regulated by
calmodulin, therefore, appear to have diverged from a common
ancestor after acquisition of the calmodulin binding domain. The
mapping of the putative protein kinase PSK-H1 within this cluster
predicts that this enzyme will also prove to be regulated by
calmodulin.
Also mapping near the cyclic nucleotide- and calcium-phospho-
lipid-dependent protein kinases is a small cluster composed of four
protein kinases recently identified in the budding or fission yeasts;
SNF1, niml+, KIN1, and KIN2. Whether these protein kinases
Fig. 1. Multiple
amino
acid
sequence alignment
of 65 protein
kinase
catalytic
domains. The first 38 sequences
derive from protein-serine/threonine
kinases
(indicated
by asterisks
in Table
1) and
the remaining
27 sequences
in
the alignment
are from protein-tyrosine
kinases
(indicated
by asterisks in
Table
2). cAPK-a
and Src have been chosen
as prototype
protein-serine/
threonine and protein-tyrosine
kinases,
respectively;
their
catalytic
domain
sequences
are
numbered to indicate residue
position
from the polypeptide
amino
terminus.
(Although
the human Src
sequence
is shown,
the number-
ing is actually
taken
from
the chicken Src
sequence
to maintain
established
convention).
The number
of additional
amino- and
carboxyl-terminal
flank-
ing residues
lying
outside
the catalytic
domains
are shown at the beginning
and
end, respectively,
of each
sequence.
In several
cases the sequences
have
not been
determined
through
to the polypeptide
amino
or carboxyl
termini;
for
these,
the
number
of determined
residues
is given
followed
by a plus
(+)
sign.
An asterisk
(*) at the beginning
or end of a sequence
indicates
that no
additional
flanking
residues are
contained
in the polypeptide.
Gaps, repre-
sented by dashes, were introduced
into the sequences
to optimize the
alignment.
In six cases,
long insert
segments
have been excluded
from the
alignment
to shorten the figure.
The positions
and
lengths
of the excluded
inserts
within the alignment
are indicated
by numbers
within braces
(for
example,
{-48-}); the excluded
gap positions
in the other sequences
that
correspond
to these
long inserts are
shown as double
slashes
(//). Residues
conserved in 62 or more of the 65 sequences
are
shown as white letters in
black boxes.
Positions
where
residues of similar
structure
are
conserved
in 63
or more
sequences
are
shown in shaded
boxes.
Structurally
similar
groupings
used for this purpose
are nonpolar
chain
R groups
(M, L, I, V, and C);
aromatic
or ring-containing
R groups;
(F, Y, W, and H); small R groups
with
near neutral
polarity
(A, G, S, T, and
P); acidic
and
uncharged
polar
R
groups
(D, E, N, and Q); and basic
polar
R groups (K, R, and H). The
single-letter
amino acid code is used (A, alanine;
C, cysteine;
D, aspartic
acid; E, glutamic acid; F, phenylalanine;
G, glycine; H, histidine; I,
isoleucine;
K, lysine;
L, leucine;
M, methionine;
N, asparagine;
P, proline;
Q, glutamine;
R, arginine;
S, serine;
T, threonine;
V, valine;
W, tryptophan;
and Y, tyrosine).
Roman numerals
at bottom indicate
conserved
subdo-
mains.
SCIENCE, VOL. 241
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A Al
1 3
9 1 S-9 A A V A 3 A I 1 1 d 9-- 0 A S 3 V 9 A 1 A I
1 J N 1 1 0-9 A 3 A I d -- 9 I 3 VA 1 1 N A
1 J N 1 1 O- A 3 3
1A
3 1I A A d -----9 H 1 3 V9 1 1 N A
1 A a A 1 a-3 A 3 A 3 1 I I A I d -----9 1 3 V 9 1 1 N A
I j N 1 0-9 H A W
A d 1 A A 1 d S 9----3S A 1 3 I 9 1 1 S 1
1 jA N 1 A-9 H A W
A 3 A A W
1 1 d A-----9 3 1 3 A 9 j j 8 A
1 A S 1 1 a-9 V 3 W
H 3 W
I 1S I S 3-----1 a A 3 I 9 A A
d A
1 A i 1 -9 9 3 W
1 3 1 I I A & d 3-----N 1 1 3 A 9 1 b A 1
1 S 1
J-A
S >- W
1 3 W A A V d ----- S 3 A 9 1 A
1 A S a 1 0-9 a I W
1 3 W
I A 1 1 d -----9 A A 9 1 1 A
I A S A
1 A-9 H V W 3 W A
A 1 d ----- A A A 9 1 1 A
A A a 1 1 3-9 1 d W
1 b I A W W
b ------ s w 3 A V 1 1 N
A H a 1 1 3-9 A d W
1 A
A S------ 1 1 I 9 1 1
A A a 1 1 3-9 J d W
1 I A S------
1 1
A I 9 1 1 d 3
1 1 1 J -9 9A A A 1 3 A I A I d ----- a 1 3 A 9 I 1
3 A
1 A 3 1 1 N-9 N 3 W 3 1 I I A d V----- H 1 3 AA
91 1 d A
1 J 1 1 N-9 H S W J 3 1 I I A d d-----3 3 A
A 9 I 1 A
1 A a 1 1 N-9 A I W J 3 1 I I A d d-----3 1 3 A 9 1 1 A
1 A N 1 S-9 H N W
A 3 1 A I A I -----H 1 3 A
1 J 1 1 S-9 S W
A 3 O A I AId 3-----3 S 3 A
J a 1 S-9 N V J 3 1 I I A I d 3------ A A
1 J a A 1 S-9 N 3 W
A 3 1 I IA I 3------ A A
1 J a 11 S-9 N V W A 3 1 I I A Id 3-----3 A A
1 J a l S-9 ) N W A 31A IAI d ------ 3 S A A
l J a S-9 H 3 W
A 3 A IAI 3------ 3 S A A
1 J a S-9 S W 3 1 A IAI d ------ 3 S A A
1 a 1 S-9 A S W A
IAIA Id 3------ 3 S A
s0? Ot? 0??
9A 1 A
VA1V A
VA 1 A
VAIbA
VA 1 A
A 1d A
I
I
I
I
I
H d H N------A IN
1 AN
N W
H N-----91 A S 1 A
N 3 H A-----91 H S W I ) 1
N I H d-----91 H S W
I N 1
N d H S------A a A W I I 9
H a H A------I WH
1 113 A
H A I a------ 3 ) A V
H H 3 i------J 9 A W A S V
N A H 3-----3 S V I A V
A d S ------A 9 V A A
H d N a------A S V W AA V
N d H S------A a I A) V
N A H H------1 a A w I V V
N d H )------W 3 )t W IV V
N d H A------I 3 w A
AV
N d H
N H
I a H
'd H
X a H
X a H
X O H
X a H
A 3 H
0Z?
ANH
AAH
AAH
b------ 1 I W
A
------J A A W I V V
------ 1
i W A N V
O------1 & w
W
1 N V
O------ I1
i W
1 N V
1------1 >
X W
I a v
------1 1 1 W A a v
------ 1 N W I A
a------1 N W A a
III II
S W 3 3
SW1V3A3
SWHVOA3
SdSVN3 s
J1HVSdAl
a VH V S
SSaViSN
1 S A 3----------------------------9 I a I d N
J 0 0 8 V
u v----------------------------- s 3 S v 3 >
a 1 3 V J 3----------------------------- - S V 9
s d
) 1 A 3 I ) 3----------------------------? a IS 9 >) >
S 1 J N I d 3----------------------------A a I V N 3 N
N 1 J 3 I V. 3----------------------------? W S V V 3 N
N 1 3 I 3---------------------------- S S 3
d 1 j 3 3 S S----------------------------3 V 9 IS X 1
a 1 I 3 N V---------------------------- d S 1 N 3 d
a I I 3 X N V----------------------------? d S i V 3 d
a1 J V I----------------------------a d d 1 3
V 1 J 3 H1H d----------------------------- - W V a 3 >
31 )
j a v ?
v----------------------------- - w i a 3 )I
) 1 J 3 3 A 3----------------------------- - W 1 a 3 )
3 I J a a 3 s----------------------------- - W 1i9 3 )
b1 V V i s----------------------------- - w 1i 9 3
V 1 J V 3 A S----------------------------- - W S 9 d N
V d 1 b 9 V S----------------------------- - W S 9 a )
3 1 A V a A S----------------------------- - W 1 9 d >
3 1
A S 3 d S----------------------------- - W 1 9 d >
3 1 J V > d S----------------------------- - W 1 3 d A
a1 J V 3 d W----------------------------- - W 1 9 d >
1 3 d S----------------------------- - W 1 9 d
00?
A I i A 9---------AVAA1H J V 1 AV A
A W
V ---------aS)I A V I 3 A
A ) I A V---------a03N199 V i V 3 A
A N W
I V---------SHS19 H V V3 A
3 H I )-----N------aa 1 1 H A
A 1 W a--------- 3dllN H 33V l J
A d 3----------3S031 1 9 3 A
A I 3 9--------S9A9I A V 1 9 3 A
3 3 a A---------9NddJS 1 I 9 3 A
A l1 3 d
--------- 3 AA9 V A 9 3 A
A d 1 3 ---------39IIA N 3 A
A d I A----------N393d A M
A 9 'A
A d I A----------N390d I M I 9 N A
A d I A---------- 393d I M
1 9 N A
A 1 1 N a--------------A l 1 A 9 s
I 1 3 a H--------------d . M A 93 A
A 1 N 9 A-------------- M A 3 A
A S A-------------- M
A 3 A
I I ---------------9
A a I S ---------------N
A A 1 H ---------------
A A I 1 -------------9
A >1
IS ---------------N
A > I N ---------------9
A IS ---------------9
A N I1 ---------------
A l 1 --------------9
06Z
J 3
V
J v
Vl
AH
S
V
V
AS
J N
A a
A
A
d
b M
N 9
A3 HA I J N
N A I V W A
33 A
N AA 9 WM 3 3
N M
1 9 W M 3
N M
1 9 1 M a 3
J
N M
1 9 W
M 31 3 3
N M 1 9 W 1313 3
j
I A 9 1 A 1 N A/SAS
)I 9 J S 1 'A
N/98S
N 9
3J 1 N N/08S
1 A 9 I A 1 0 a/s9s
A 3 N J H A I 1/?601
3 M
N 1 A I a /AOES
A 1 1 1 b0
S/Sb9T
1 1 A 1 1 1 N 3/+ESI
Ad V 1 I I N 3/+ZIL
A3 S W I I A 3/996
3 A 1 1 1 I 3/020I
A 9 9 d 1I 3 V/+S89
A A A A
A 1 3 l/LIL
A I
N A 3 1/589
3 91 A 10 3/+I9S
A N H W
I I 3 S/+6SI
A H
A W
I a 1/+ZI
A H A W
I I 1/6?E
3 3 W 1 W 1 a IILZE
A A 1 1 0 I 3 S/18Z
A
) 3 1 A 1 S 3/8EZ
A 3 A 1 A 1i 3/E1 Z
d A A 1 > I S 3
/AZ
A n
I 1 ) 1 s 3/89Z
A A 3 1 1 I S S/09Z
A A 3 1 d 1 S 3/ALZ
A 3
A1 1 S 3/Z9Z
e>z
I A 0 H 1 I-A N 9 9 J 3 W
I I 1 9 1 SNS9Vdl d I S V V A A A
A
1 H H A 1 S-S 9 3 A
M I I V A 9 d------ d I W 3W J d 1
1 H H A S-S 9 3 3 M I I I 9 d------
Ad 1 J 3 W 3 1 1
1 H A 1 S-S 9 3 M
a I A I 1 N a------ I
I W
A 9 W
31 1
I J a aOA
d 3 d
A 3 1 I 1 A S -----d d 3 J MA 1 1 d I
I A 1 A 1 0-9 N d 3 A a 1 A A A I V 0-----3 1 3 1A a H 1 1 I
1 A I A 1 a-- V a A > d 1 A 1 3 1 A 9-----S A A H i a 1 1 d 1
1 J A a 1 S-9 N 3 9 1 3 A a W A 1 9-----9 H 3 M S a W 1 3 A
A I X a 1 S-9 9 a W A 3 W 3 W
A A V A-----3 I A A V 9 A J a A
1 I A a 1 S-9 3 a s A 3 W
1 I I I 3 N--NIHO
N A A V 9 A 1 I
A J I A J 3-3 H d A A d 1 A V I A a a-----A A d > V a 3 1 d V
A 1 A A a
-I N N A H 3
A I 1A d I d------ -S A d a > A
AA
I A
A3 1 a- Id
A J 3 1 A1 N
1 N
1 -----A V W J I a I 1 3 I
1 A I d 1 a-- b a A H 3 J A 1 1 A A I3AlIIS I V 3 A a w 1 l A
1 A I a-- W S 1 A 3 A I 1 A 8l S-----a 0 w A a 0 1 s A
W
A A 1
Ia-- W
a J 3 A 1 A 1 S-----3 V H
1 I ia 1 1 A
W
A d > 1 a-- 1 a 1 J 3 J A 1 A 1
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This content downloaded from 132.239.1.231 on Sun, 21 Apr 2013 19:08:54 PM
All use subject to JSTOR Terms and Conditions

230 240 250
LIYEMAA-GYTPFY F ?-? DQ?
S Q ITYEKIT
L I Y E M A A - G Y P P F F A-----------D Q P I Q I Y E K I V
L I Y E M
A A - G Y P P F F A-----------D Q P I Q I Y E K I V
L I Y E M L A - G Y T P F Y D-----------S N T M K T Y E K I L
L I Y E M L A - G Y T P F Y D-----------S N T M K T Y E K I L
L I Y E M L A - G Y T P F Y D-----------T TT P MKT Y E K I L
LIYEMLA-GYTPFY N-----------S N TMKT YENIL
LMYELLT-GSPPFS G-----------P DPMKTYN IIL
260
S G - K-V R F P S H
S G - K-V R F P S H
N A - E-L R F P P F
..N A - E-L R F P P F
Q G - K-V V Y P PY
N A - E-L K F P P F
R G I DMI
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- -------------//-----------------F S S D L K D L
- - - - - - -------------//-----------------F S S D L K D L
- - - ------//-----------------FN E D V K D L
- - -------------//-----------------F N E D VK D L
- - --- -//-----------------FQ P D V V D
- - - -------------//------------- F H P D A Q D L
_ _ _ -------------//---------------I A K N A A.N L
LL Y E M L A - Q P P F D G-----------E D E D E LF Q S I M E H - N-V KS - - - - - - -------------//-----------------L S K E A V S I
LL Y E M LA-G Q A P F E G --- E D E DEL F Q S I M E H - N-V A Y P K S - - - - - - -------------//-----------------M SK E A V A I
LY E M L A - Q P P F D -----------E D E E E L F Q A I M E Q - T-V T Y P K S - - - - - - -------------//-----------------L S R E A V A I
L L YE M L V - Q P P F D G-----------E D E E E L F A A I TD H- N-V S K
S - - - - - - -------------//-----------------L S K E A K E A
I L Y I L V - YP P F W
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D T-- ------------//-----------------V T P E A K D L
I L Y IL V - Y P P F W D-----------E DQH K L Y Q Q I K A G- A-Y D F P S P E W D T - - -------------//-----------------V T P E A K N L
IMYTLLA-G S P P F W
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I T Y M L L S - L S P F G-----------D DD T E T L N N V L S - N-W Y F D E E F E A - - ------------------------------V S D E A K D F
I C Y I L V S - LS P F M
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I A Y I S - M P FE D-----------D N R R L Y R Q I L RG - K-Y S Y S G E P W P S -- ------------//-----------------V S N LA K D F
I Y V M C - R P F D D-----------E S I P V L F K N I S N - V-Y T P K F - --------------//--------------- L S P G A A
I Y S S - N K P F G G-----------Q N T DV I Y N K I R H - A-Y D L P S - - - - - - -------------//-----------------I SS A A Q D L
V L F V L V C - G K V P F D D-----------E N S S V L H EK I K Q G - K-V E Y P Q H - - - - - - -------------//-----------------L S I E V I S L
V LYV V C - K V P F D D-----------E N S I L H E K I K K - K-V D Y P S H - - - - - - -------------//-----------------L S I E V I S
I F A E M
C N - R K P I F S GDSEI-------D Q I F K I F R V L G T P N E-A I W P D I V Y L P D F KPS---------- //--FPQWRRKDLSQVVPSL
D P R G I D L
I F A E M I R - R S P L F P GDSEI-------D E I F K I F Q V L G T P N E-E V W P G V T L L Q D Y KST---------- //--FPR WKRMDLHKVVPNG
E E D A I E L
I F A E L A T - K K P L F H GDSEI-------D Q L F R I F R A L G T P N N-E V W P E V E S L Q D Y KNT---------- //-- FPKWKPGSLASHVKNL
D E N G L D L
'I F A E M F R - R K P L F C GNSEA-------D Q L G K I F D L I G L P P E-D D W P R D V S L P R G A------------ //--FPPRGPRPVQSVVPEM
E E S G A Q L
I F A E L M L - R I P Y LP GQNDV-------D Q M E V T F R A T P D-R D W P E V S S F M T YNKLQI--------//YPPSRDELRKRFIAA S E Y A L D F
M AS M I F - R K E P F F HGHDNY------DQ V R I AK V GT E E L-Y AY D K Y N ID L PRFHDILQRHS--//--RKRWERFVHSDNQHLV
S P E ALD F
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H Y W C F Q V
MI I E L V T - G E F P L G GHN---------D T P D G I L D L L Q R I V N-E P S P R L P K D R I - -------------//-----------------Y S K E M T D F
S I LE M
A L - GR Y P Y P P-----------E T Y D N I F S Q L S A I V D-G P P P R L P S D K - - -------------//-----------------F S S D A Q D -F
T V FE A A A - N I V P D NG----------QS W Q K R S D L S D AP-R S S T D N S S T SSSRETPANSII-// -----------------G Q L D R
V I FE T A V - HN A S L F SAPRGPKRGPCDS
Q I T R I I R Q A Q V H V D-E F S P H P E S R L T S RYRSRAAGNN---//--RPPYTRPAWTRYYK-M
D ID V E Y L
I L I N L C C - K R N P W K RAC---------S Q T D G T Y R S Y V H N P S-T L L S I L P - - - - - -- -----------//-----------------I S R E L N S L
L YD M V C - D I P F - ------------E H D E E I I K G Q V F F R Q-T -- //-----------------V S S E C Q H
V L YE L M - G E L P Y S HI----------N N R DQ I I F M V G R G Y A-S P.D L S K L Y K N - - -------------//-----------------C P K A M K R L
V L YE L M T - G S L P Y S HI----------G C. R D Q I I F M V G R G Y L-S P D L S K I S S N - - -------------//-----------------C P K A M R R L
V L Y E LM T - G S P Y S HI----------G C R D Q I I F M
V G R Y L-S P D S K I S S N - - -------------//-----------------C P K AM R R L
T L W Q M T T - K Q A P Y S - E R Q H I LY A VV A Y D L-R P SL S A A V F E D S L------------//-----------------P G Q R L D V
LLT ELT
L Q T E L V
L L T ELI
L L T E L V
LL Y E I V
L L TE IV
L L M E I V
LL M ELF
L MW
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460 470 480 490
T K G R V P Y P G-----------M VN R E V L D Q V E R G Y R-M
P C P P E - - - - - - -------------//-----------------C P E S L H D L
T K G R V P Y P G-----------M V N R E V L E Q V E R G Y R-M
P C P Q G - - - - - - -------------//-----------------C P E S L H E L
T K G R I P Y P G-----------M N K R E V L E Q V E Q G Y H-M P C P P G - - - - - - ------------//-----------------C P A S L Y E A
T K G R V P Y P G-----------M N N R E V L E Q V E R G Y R-M
P C P Q D - - - - - - -------------//-----------------C P I S L H E L
T Y G K I P Y P G-----------R T N A D V MT A L S Q G Y R-M P R V E N - - - - - - ------------//-----------------C P D E L Y D I
T H G R I P Y P G-----------M T N P E V I Q N L E R G Y R-M
V R P D N - - - - - - -------------//-----------------C P E E L Y Q L
T Y G R I P Y P G-----------M S N P E V I R A L E R G Y R-M P R P E N - - - - - - -------------//-----------------C P E E L Y N I
T Y G Q V-P Y P G-----------M H S R E V I E N I E R G F R-M P K P T N H Y - - - - -------------//-----------------F P D N I Y Q L
T C'G K'M
PEY
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L L W E I A T Y G M
S P Y P G-----------I D P.S Q V Y E L L E K D Y R-M
K R P E G - - - - - - -------------//-----------------C P E K V Y E L
L L W E I A T Y G M S P Y P A-----------I D L T D V Y H K L D K G Y R-M
E R P P G - - - - - - ------------------------------C P P E V Y D L
L L W E I A T Y G M A P Y P G-----------V E L S N V Y.G,L L E N.G F R-M
D G P Q G - - - - - - -------------//-----------------C P P S V Y R L
L L W E T F S L G A S P Y P N----------L S N Q Q T R E F V E K G G R-L P C P E L - - - - - - -------------//-----------------C P D A V F R L
T V W E L M T F G S K P Y D G-----------I P A S E I S S I L E K G E R-L P Q P P I - - - - - - -------------//-----------------C T I D V Y M I
T VW E L M TF G A K P Y D G-----------I P A R E I P D L L E K G E R-LP Q P P I - - - - - - -------------//-----------------C I D V Y M I
T I W E L L T F G Q R P H E N-----------I P A K D I P D L I E V G L K-L E Q P E I - - - - - - -------------//-----------------C S L D I Y C T
V L W E I T S L A E Q PY Q G-----------L S N E Q V L K F V M D G G Y-L D Q P D N - - - - - - -------------//-----------------C P E R V i D L
V L W E I A T L A E Q P Y Q G-----------L S N E Q V L R F V M E G G L-L D K P D N - - - - - - -------------//-----------------C P D M L F E L
V L W E M A TL A A Q P Y Q G-----------L S N E Q V L R Y V ID G G V-M
E R P E N - - - - - - -------------//-----------------C P D F L H K L
L I W E I L T L G H Q P Y P A-----------H S N L D V L N Y V Q T G G R-L E P P R N - - - - - - -------------//-----------------C P D D L W N L
L C W E I L T L G Q Q P Y A A-----------R N N F E V L A H V K E G G R-L Q Q p
P M - - - - - - ------------//-----------------C T E K L Y S L
V L W E I F T Y G K Q P W Y Q-----------L S N T E A I D C I T Q G R E-L E R P R A - - - - - - -------------//-----------------C P P E V Y A I
V L W E L M T R G A P P Y P D-----------V N T F D I T V Y L L Q G R R-L L Q P E Y - - - - - - -------------//-----------------C P D P L Y E V
L L W
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A Q P A H - - - - - - -------------//-----------------A S D E I Y I
L L W E I F S L G L N P Y P GI----------L V N S K F Y K L V K D G Y Q-M A Q P A F - - - - - -------------//-----------------A P K N I Y S I
F L WE L F S L G S S P Y P GM----------P V D S K F Y K M I K E G F R-M
L S P E H - - - - - - -------------//-----------------A P A E M Y D I
L L.W
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QNG T EDV K N H P W F KE V/53
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Q S G S R D I K A H P W F S E V/53
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LG C GP EGER DI K E H A F F R Y 1/70
L G SGPDG E P T I R A H G F F R W /80
L G C G S S G E E D V R L H P F S R R 1/37
I T A A E A L K - - - - - H P W I S H R/203
I T A H E A L K - - - - - H P W
V C Q R/258
Y T A E EA L A----- H P F F QQ Y/96
M S A A Q C L A-----H P W L N N L/51
L N C T Q C L Q - - - - - H P W L Q K D/27
M T ALQALR -- - - - H P W
VV S M/139
I S I H E I M
Q - - - D D W F K V D/324
I T I P 'E F F S - - - - - H P F L MG C/109
A T L K Q V V E - - - - - H H W
M
V R G/663
A T L K NV V E- - ---H P W
MN R G/773
I S A K R A L Q - - - Q N Y L R D F H
ISARRAAI-----HPYFQES*
ISAKRALQ-----QNYLRD FH*
I S G K M A L N- ----H P Y F N D L/7
I.S A F R A L Q- - - - H S Y L H K D/5
WTAVQCLE------S DYFKEL/13
L T A R E A M
A - - - - - HP Y F L P I/11
S S A E D L L K-----T P F F N E L/35
S S I H E LL H----- D
HD L I M K Y/46
P T Y A ALT E - - - - - H P W L V K Y/84
P T ID I LA T D - - - E V C WV E M/32
P S AAELLC----- L F Q Q K *
I T L P E L S T - - - - - L V S N C K N/170
P S F E E I R N - - - - - H P W
M
Q G-D/20
P L F P Q I L S S I - - -E L L Q H S L/35
P L F P Q I L A T I - - - E L L Q R S L/32
PLFPQI LATI ---ELLQRSL/15
PSARLLLVDL---TSLKAELG*
510
PTFEYLQAFL ---ED
PTFEY I QSFL - - - ED
PTFEYLQSFL - - - ED
PTFEYLQSFL - - - ED
PTFDYLQSVL - - -DD
PTFDYLRSVL - - - ED
PTFEYIQSVL- - -DD
PTFEFLNHY F - - - ES
PAFRVLMDQL---AL
520
Y F T S T/10
Y F T A T/10
Y F T S A/10
Y F T A T/10
F Y T A T/8
F F T A T/8
F Y T A T/8
F S V T S/9
V A Q T L/2
P S F AE I H Q A F - - - E T M F Q ES/630
PTFKSIHHAL - - - H M F Q V
QG/9+
P R F R D I H F N L - - - E N L I SS N/137+
PS FS TI YQ EL - - - QS I R KRHR
P K F REL II E F - - - S K M
A R D P/235
P R F RE L V S E F - - - S R M A R DP/272
PT F K Q L T TV F - - A E F A R D P/183
PT F LE I V N L L - - - K D LH PS/85
PSFLEIISS I - - - K M E PG/94
P S F LD I I A Y L - - - E P Q C P NS/95
P T F HR I Q D Q L - - - Q L F R N FF/38
PSFRR C Y N TL - - - A I S
DHAISTD L/66
HS I KDV H A R L - - - Q AA A P/8
P S FS E L V S R I - - -S A I F S T F/46
P P F SQ LV LLL- - - ER LLGE G/134
P T F Q Q I C S F L - - - Q E Q A Q E D/55
P T F K Q I V Q L I - - - E K Q I S E S/45
PVFADISKDL---EKMMVKR/60 00
_
220
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cAPK-p W
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This content downloaded from 132.239.1.231 on Sun, 21 Apr 2013 19:08:54 PM
All use subject to JSTOR Terms and Conditions

Fig. 2. Similarity profile of protein kinase catalytic domains. For each 90
position in the alignment shown in Fig. 1, a relative similarity score was
determined based on the "structure-genetic" scoring matrix (116) for amino
acid similarities. Similarity scores were calculated as the sum of all possible a 80 -
pairwise comparisons between the individual amino acids at each position 8
and expressed as the percentage of the highest possible score (that is, the
score obtained when an identical residue occupies the position in all 65
aligned sequences). To smooth out the curve, a 9-position running average E 70-
of the relative scores was determined, and every third position was plotted. a
Positions that contain gaps for ten or more of the sequences were not ,
included in the profile; however, the locations of the major gap sites are 60 -
indicated by breaks
in the curve. The mean relative score for all the positions
included in the profile is 66 with a standard deviation of 14.9. Relative
similarity scores obtained when the catalytic domain sequences were ran-
domly scrambled had a mean of 47 and standard deviation of 1.85. Roman 50
numerals indicate conserved subdomains.
.o.
Raf
A?Raf
STE7 -
Fig. 3. Deduced phylogeny of
protein kinase catalytic do-
mains. The phylogenetic tree
was constructed from the multi-
ple alignment shown in Fig. 1. I i
The tree-building concept of
Fitch and Margoliash (117) was
used as implemented by Feng and Doolittle (118). Briefly, similarity
scores were obtained
for all possible pairwise comparisons and transformed
into a difference
matrix from which
branch order and length were determined. Programs were run on a VAX-785 computer
equipped with 40 megabytes physical memory under virtual memory operating system
(VMS). Systems limitations required that the branch lengths for the protein-serine/
threonine and protein-tyrosine kinases be calculated
separately,
and the tree shown is thus
a composite of these two determinations. The position of the protcin-tyrosine kinase
cluster was determined by including two protein-tyrosine kinases (Src and EGFR) in the
protein-serine/threonine kinase tree construction. The individual sequences are indicated
by the abbreviated names in Tables 1 and 2. The protein-tyrosine kinases arc not labeled
in (A), but are shown in the cluster enlargement in (B). The tree is shown "unrooted"
in
(A) as the branches are all measured relative to one another with no outside reference
point. The scale bars represent
a branch length corresponding to a relative
difference
score
of 25. The tree depicted is likely to underestimate distances between the least related
members of the family, particularly since the alignment used in its construction is
parsimonious.
50
Amino acid position (cAPK-a)
-cGPK
SCIENCE, VOL. 24I
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have similar modes of regulation remains to be determined. KIN1
and KIN2 were identified through screening a Saccharomyces
cerevisi-
ae DNA library with probes designed to recognize sequences
characteristic
of protein-tyrosine kinases and, as such, have been
suggested to represent "structural
mosaics" with some features of
catalytic domain structure more indicative of the protein-tyrosine
kinases than the protein-serine/threonine kinases (5). The deduced
phylogeny of KIN1 and KIN2, however, does not suggest a close
evolutionary relationship with protein-tyrosine kinases. In fact, the
probe target used to identify KIN1 and KIN2 encodes the stretch of
amino acids corresponding to cAPK-a Asp220-Gly225
in conserved
subdomain IX, a region of high conservation in all of the catalytic
domains regardless of substrate specificity.
The subfamily related to CDC28 includes functional homologs
from three widely divergent species: CDC28 from the budding
yeast S. cerevisiae,
cdc2+ from the fission yeast Schizosaccharomyces
pombe, and human CDC2Hs. Functional homology was demon-
strated by heterologous complementation of conditional mutants
defective in cell cycle progression (32, 33). The other two sequences
mapping within this cluster are putative protein kinases identified in
Saccharomyces
cerevisiae
(KIN28) and human HeLa cells (PSK-J3).
The members of this cluster are also distinguished by the small sizes
of the catalytic domain-containing polypeptides, suggesting their
activities may be regulated through association with other polypep-
tides in a holoenzyme complex. Indeed, support for this notion has
been obtained for cdc2+ (34).
Perspectives
The tremendous diversity of the protein kinase family is just now
beginning to be appreciated.
Most of the catalytic
domain sequences
referenced
in Tables 1 and 2 were reported within the past 2 years.
With continued characterizations of regulatory mutants in inverte-
brates, along with the recent development of new hybridization
approaches
for the identification of DNA clones that encode novel
protein kinase catalytic
domains, it is likely that the rate of discovery
will continue to accelerate through the next several years. The
difficult tasks will be to confirm protein kinase activities for the
newly identified family members and to elucidate their functional
roles. Clues to function may come through an analysis of catalytic
domain primary
structure and subsequent phylogenetic mapping. A
catalytic domain that has only limited divergence from another,
better characterized,
member of the family can be expected to play a
similar role in cellular physiology. Further clues are likely to come
from an inspection of amino acid sequences lying outside the
catalytic
domain where residues involved in enzyme regulation may
be found.
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oncogene
product
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from
I JULY 1988
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group
are
effectively
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closely
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cellular
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by
grant
GM38793
from the NIH (SKH).
SCIENCE, VOL. 241
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