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(Received for publication, June
23, 1995; and in revised form, November 8, 1995) From the
The cDNA of a novel, ubiquitously expressed protein kinase
(Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid
sequence (763 amino acids) contains a catalytic domain that is only
distantly related to that of other mammalian protein kinases. Its
closest relative is the protein kinase Mnb of Drosophila,
which is presumably involved in postembryonic neurogenesis (85%
identical amino acids within the catalytic domain). Outside the
catalytic domain, the sequence comprises several striking structural
features: a bipartite nuclear translocation signal, a tyrosine-rich
hydrophilic motif flanking the nuclear localization signal, a PEST
region, a repeat of 13 histidines, a repeat of 17 serine/threonine
residues, and an alternatively spliced insertion of nine codons. A
recombinant glutathione S-transferase-Dyrk fusion protein
catalyzed autophosphorylation and histone phosphorylation on tyrosine
and serine/threonine residues with an apparent K Reversible phosphorylation of proteins represents the main
mechanism of signal transduction in cells (Edelman et al.,
1987; Cohen, 1992; Hunter, 1991). It is catalyzed by a large family of
protein kinases that share structural similarities (11 subdomains)
within a catalytic domain of about 300 amino acids (Hanks et
al., 1988). Protein kinases appear to represent the largest family
of enzymes (Hunter, 1987), and to date more than 100 mammalian protein
kinases have been identified by molecular cloning and/or functional
characterization. This large number of homologous proteins provides the
basis of a complex signaling network that transmits and coordinates the
response to extracellular stimuli. Two major subgroups of protein
kinases, the protein tyrosine kinases and the protein serine/threonine
kinases, have been distinguished on the basis of functional but also of
structural parameters (Hanks et al., 1988; Hunter, 1991).
Initially it was assumed that the members of these subfamilies are
specific for phosphorylation of either tyrosine or serine/threonine
residues. More recently, however, several kinases have been identified
that catalyze their autophosphorylation on both tyrosine and
serine/threonine residues when isolated as recombinant proteins from Escherichia coli (Lindberg et al., 1992). In
addition, two other kinases have dual specificity toward a specific
substrate in vivo, i.e. in the intact cell. The dual
specificity kinase MEK1 phosphorylates MAP ( In this paper, we report the
cloning and characterization of a novel dual specificity protein kinase
with unique structural features. The activity of this kinase appears to
be regulated by tyrosine phosphorylation in the presumed activation
loop between subdomains VII and VIII. Its sequence comprises a nuclear
targeting motif, a PEST region, and two striking repeats of unknown
function. It is suggested that the enzyme is part of a signaling
pathway that controls nuclear functions.
Three
hybridizing cDNA clones (A, B, and D) of different lengths were
isolated from a rat brain cDNA library and characterized by restriction
mapping and/or sequencing. One of these clones (clone B, 5 kilobase
pairs) contained a full reading frame and a poly(A) tail. Clone A
contained a poly(A) tail but lacked 2.3 kilobase pairs in the
3`-untranslated region; clone D had a deletion of 27 bp within the open
reading frame (see below).
Figure 1:
Structural characteristics of the dual
specificity protein kinase Dyrk. A, nucleotide and deduced
amino acid sequence of the cDNA of rat Dyrk. The deduced amino acid
sequence of rat Dyrk is given in single-letter code above the
respective codons. The catalytic domain is boxed, and the PEST
region (dashed underline), a histidine repeat (underline), and a serine/threonine repeat (double
underline) are indicated. The shaded box depicts an
alternatively spliced segment of the sequence. A putative bipartite
nuclear targeting signal is marked by + symbols above the
respective residues, and amino acid residues that were later exchanged
by site-directed mutagenesis (Lys-188, Tyr-219, Tyr-319, and Tyr-321)
are depicted by asterisks. B, schematic presentation
of the structure of Dyrk. Boxes depict the position of the
different structural motifs and domains in the sequence of Dyrk. The
amino acid sequences of the spliced domain, the nuclear targeting
signal, and some of the kinase subdomains are given in single-letter
code. Roman numerals designate the kinase subdomains according
to the nomenclature of Hanks (Hanks and Quinn,
1991).
The deduced amino acid sequence (763 amino acids)
contains all conserved regions of the catalytic domain (amino acids
159-479) according to the classification of Hanks (Hanks and
Quinn, 1991) (subdomains I-XI, Fig. 1B). In
addition, the sequence exhibits several striking characteristics (Fig. 1B). First, the sequence harbors a bipartite
nuclear targeting sequence (Robbins et al., 1991; Dingwall and
Laskey, 1991) flanking the N-terminal side of the catalytic domain.
Second, the amino acid sequence of Dyrk comprises several tyrosine
residues that may represent phosphorylation sites. Two tyrosines
located between the subdomains VII and VIII are potential
phosphorylation sites regulating the activity of the catalytic domain,
in analogy to other protein kinases, e.g. ERK/MAPK and
GSK3
Figure 7:
Sequence comparison of protein kinases
that are regulated by phosphorylation in the presumed activation loop
between domains VII and VIII. Amino acids are given in single-letter
code; residues identical with the corresponding amino acids in Dyrk are
designated by periods. The subdomains VII and VIII are boxed. Asterisks above tyrosines or threonines
indicate residues that have been identified as activating
phosphorylation sites.
As a third unique
structural feature of Dyrk, the large domain flanking the C terminus of
the catalytic domain contains several striking motifs. Immediately
following the catalytic domain, a serine/threonine-rich region fulfills
the requirements of a PEST region. These motifs represent domains
abundant in proline, glutamic acid, serine, and threonine and are
believed to initiate a rapid degradation of the protein (Rogers et
al., 1986). Furthermore, the C terminus of Dyrk harbors a stretch
of 13 consecutive histidine residues (amino acids 607-619), and a
domain containing an unusually high portion of serine and threonine
residues (46 serines out of a total of 284 amino acids). Within the
serine/threonine-rich segment is a stretch (amino acids 659-672)
of 17 subsequent serine/threonine residues. Data base searches
turned up several partial human and mouse cDNA sequences (expressed
sequence tags) with high similarity to the nucleotide sequence of Dyrk.
Two of these sequence tags (mouse, accession no. Z31282; human,
accession no. L25452) comprise the histidine repeat, indicating that
the amino acid sequence of this repeat is fully conserved. The
chromosomal localization of the latter sequence tag has been determined
(Cheng et al., 1994) by hybridization to a set of mapped
YAC's derived from chromosome 21. Based on the high similarity of
this sequence tag with Dyrk (95% identical nucleotides), it appears
safe to conclude that the chromosomal localization of the human
homologue of Dyrk is 21q22.2.
Figure 2:
Comparison of the catalytic kinase domain
of Dyrk with that of other protein kinases. A, dendrogram of
an alignment of the catalytic domains of Dyrk and other protein
serine/threonine kinases. The dendrogram was constructed with the
PILEUP program. Data base accession numbers (Protein Identification
Resource) are as follows: Yak1, A32582; CDC2, A29539; CDK4
(cyclin-dependent kinase 4), JN0460; CDK5, A46365; ERK2/MAP-kinase,
S16444; GSK3b (glycogen synthase kinase 3
Figure 3:
Expression of Dyrk as an active protein
kinase in E. coli. A, Coomassie stain of the
recombinant proteins partially purified by affinity absorption on
glutathione-Sepharose and separated on SDS-PAGE (10% gel). GST, transformation of E. coli with vector encoding
glutathione S-transferase alone; GST-Dyrk,
transformation with vector comprising the coding regions of GST and
Dyrk in a single reading frame. B, protein kinase activity of
a 90-kDa fragment of Dyrk. The recombinant proteins were separated on
SDS-PAGE (10%) and were transferred onto PVDF membranes, renatured, and
subjected to an in situ kinase assay. C,
phosphorylation of exogenous substrates by Dyrk. The in vitro kinase assay was carried out with partially purified GST-Dyrk in
the absence (GST-Dyrk) or presence of the indicated
substrates (histone, casein, poly-Glu/Tyr). The reaction products were
separated by SDS-PAGE (14%) and subjected to
autoradiography.
Fig. 3C illustrates a series
of experiments designed to further characterize the protein kinase
activity of Dyrk. In vitro protein kinase assays with
recombinant Dyrk in the presence of magnesium, manganese, and
[
Figure 5:
Dual specificity protein kinase activity
of Dyrk: autophosphorylation and histone phosphorylation on tyrosine
and serine/threonine residues. A, tyrosine autophosphorylation
of the 90-kDa fragment of Dyrk as detected with anti-phosphotyrosine
antibody. Partially purified GST-Dyrk was separated by SDS-PAGE (10%
gel) and transferred onto a nitrocellulose membrane, and
phosphotyrosine was detected immunochemically with specific antiserum. B, detection of phosphotyrosine in hydrolysates of
phosphorylated GST-Dyrk. Partially purified GST-Dyrk was subjected to
an in vitro autophosphorylation in the presence of
[
Figure 4:
Expression of Dyrk in COS-7 cells. COS-7
cells were transiently transfected with a Dyrk construct (HA-Dyrk) or blank vector (Control) as
described. A, cells were boiled with SDS, and lysates were
separated by SDS-PAGE (10%), transferred onto nitrocellulose, and
probed with anti-HA antiserum. B, cells were lysed with
Nonidet P-40, and HA-Dyrk was isolated by immunoprecipitation. The
immunocomplexes were subjected to a kinase assay in the presence of
histone and separated by SDS-PAGE, and the incorporated
It
was reported previously that the serine kinase phosphorylase kinase
exhibited a tyrosine kinase activity toward angiotensin II in the
presence of manganese (Yuan et al., 1993). Thus, we studied
the dependence of both total and tyrosine phosphorylating activity of
Dyrk on bivalent ions (Fig. 5C). Magnesium alone was
sufficient for the total protein kinase activity but was much less
effective than manganese; EDTA fully suppressed the kinase activity of
Dyrk. The tyrosine kinase activity of Dyrk does not appear to require
the presence of manganese, since the ratio of phosphotyrosine to
phosphoserine/threonine was identical with both bivalent ions (Fig. 5D). In order to further characterize the
protein kinase activity of Dyrk, a preliminary kinetic analysis of the
histone phosphorylation was performed (Fig. 6). The K
Figure 6:
Kinetic analysis of the phosphorylation of
histone by Dyrk. Samples of recombinant Dyrk (10 ng/10 µl) were
incubated at room temperature for 10 min in phosphorylation buffer
containing the indicated concentrations of histone and
[
Figure 8:
Autophosphorylation and protein kinase
activities of Dyrk-mutants of tyrosines 219 and 319/321. Tyrosine
residues Tyr-219 and Tyr-319/Tyr-321 were substituted for
phenylalanine; Lys-188 was exchanged for arginine as described under
``Materials and Methods.'' A, the partially purified
GST-fusion proteins of Dyrk-wild type and mutants were separated on
SDS-PAGE (10% gels), transferred onto nitrocellulose membranes, and
phosphotyrosine was detected immunochemically with specific antibody.
In parallel experiments, it was ascertained that equal amounts of the
90-kDa fragment were present in each lane. B, partially
purified GST-fusion proteins of Dyrk-wild type and mutants were
phosphorylated in vitro as described in the presence of
histone, and reaction products were separated by SDS-PAGE (14% gels).
The gel was dried and autoradiographed for 18 h. Equal amounts of the
90-kDa fragments were present in each lane.
The present data indicate that Dyrk is a dual specificity
protein kinase that catalyzes its autophosphorylation on both
serine/threonine and tyrosine residues. Moreover, the data give a first
insight into the regulation of Dyrk. Its kinase activity depends on the
presence of tyrosine residues between subdomains VII and VIII
(activation loop, Fig. 7). Thus, in analogy to the
serine/threonine kinases ERK2 (Payne et al., 1991; Rossomando et al., 1992), GSK3 Dual specificity
protein kinases represent a class of the protein kinase superfamily
that is defined solely by their ability to phosphorylate both
serine/threonine and tyrosine. The so far identified dual specificity
kinases (Clk1, ERK2, GSK3 Dyrk exhibits
several striking structural characteristics of potential functional
relevance. The sequence harbors a bipartite nuclear targeting motif at
amino acids 117-134 that consists of 2 basic amino acids, a
spacer of 10 amino acids, and 4 additional basic amino acids. Similar
motifs have previously been found in a number of nuclear proteins, e.g. steroid hormone receptors, transcription factors, and
enzymes and proteins involved in transcription or mitosis (Dingwall and
Laskey, 1991). This motif appears to be a reliable indicator of nuclear
localization since it is found in about 50% of nuclear proteins but in
less than 5% of nonnuclear proteins. The region of Dyrk flanking the
C terminus of its catalytic domain contains an unusual portion of
uncharged hydrophilic amino acids (31 threonines and 46 serines in a
total of 284 amino acids). A computerized search revealed that a
portion of this region immediately following the catalytic domain
fulfilled the requirement of a PEST region (Rogers et al.,
1986). Because PEST regions are typical of rapidly metabolized
proteins, they are believed to signal their degradation (Rogers et
al., 1986). The PEST region in Dyrk showed a score that would rank
second among those listed (Rogers et al., 1986). In addition
to the PEST region, the C-terminal portion of Dyrk harbors a repeat of
13 histidines (amino acids 607-619) and a stretch of 13
subsequent serine/threonine residues (amino acids 659-672). A
data base search revealed that similar histidine repeats have
previously been found in other proteins, e.g. a protein kinase
from yeast (SNF1, Celenza and Carlson, 1986) and several transcription
factors (e.g. accession no. P39020, P15463, and P25490), but
their exact function is as yet unclear. At present, we can only
speculate on the possible function of Dyrk on the basis of its
structural features and its autophosphorylation on tyrosine residues.
The closest relative of Dyrk is the product of the mnb gene
from Drosophila with 85% identical amino acids within the catalytic
domain (Tejedor et al., 1995). It appears reasonable to assume
that Dyrk is the mammalian homologue of this gene, although it
exhibited several striking differences outside the catalytic domains. mnb appears to be involved in postembryonic neurogenesis
because the gene was found disrupted in the minibrain mutant
(Tejedor et al., 1995). Another relative of Dyrk, Yak1, has
been suggested to arrest cell growth in yeast because its inactivation
allowed growth deficient strains to proliferate (Garrett and Broach,
1989; Garrett et al., 1991). Based on the unproven assumption
that the structural similarities reflect a functional relationship, it
might be speculated that Dyrk is involved in cell cycle control. All
other kinases that are regulated by tyrosine phosphorylation between
subdomains VII and VIII, e.g. MAPK/ERK, JNK, and GSK3 The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
X79769[GenBank].
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3488-3495
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
of approximately 3.4 µM. Exchange of two
tyrosine residues in the ``activation loop'' between
subdomains VII and VIII for phenylalanine almost completely suppressed
the activity and tyrosine autophosphorylation of Dyrk. Tyrosine
autophosphorylation was also reduced by exchange of the tyrosine
(Tyr-219) in a tyrosine phosphorylation consensus motif. The data
suggest that Dyrk is a dual specificity protein kinase that is
regulated by tyrosine phosphorylation in the activation loop and might
be a component of a signaling pathway regulating nuclear functions.
)kinase/ERK2 on
both threonine 183 and tyrosine 185 (Payne et al., 1991),
thereby activating the kinase and presumably inducing its translocation
to the nucleus. Similarly, the dual specificity kinase Wee1 of Schizosaccharomyces pombe appears to phosphorylate and inhibit
the serine/threonine kinase Cdc2 by phosphorylation on tyrosine 15
(Lundgren et al., 1991).
RNA Preparation and cDNA Synthesis
3T3-L1 cells
(Green and Kehinde, 1974) were differentiated as described previously
(Weiland et al., 1991), washed twice with phosphate-buffered
saline, frozen in liquid nitrogen, and lysed in a solution of 4 M guanidine thiocyanate and 7% mercaptoethanol. Rat tissues were
homogenized with a Polytron homogenizer in 4 M guanidine
thiocyanate supplemented with 7% mercaptoethanol. The lysates were
layered on a cesium chloride cushion (5.88 M) and centrifuged
at 28,000 rpm (rotor SW 40) for 29 h at 20 °C. Pelleted RNA was
dissolved with 300 µl of 0.1 M sodium acetate/Tris buffer
(pH 9.0) and was neutralized by the addition of 50 µl of 2 M potassium acetate (pH 5.5). First strand cDNA was synthesized with
reverse transcriptase (First-strand cDNA synthesis kit, Pharmacia
Biotech Inc.) by oligo(dT) priming.PCR Cloning of Protein Serine/Threonine
Kinases
Highly degenerate oligonucleotide primers were designed
from the conserved regions VIb and IX (according to Hanks and
Quinn(1991)) of the catalytic domain of protein serine/threonine
kinases. Forward primer,
5`-A(C/T)(A/C)GIGA(C/T)(A/C/T)TIAA(A/G)(C/T)CI(C/G)A(A/G)AA-3`; reverse
primer,
5`-A(A/G/T)(A/C/G)A(C/T)ICC(A/C/G)A(A/G)I(A/G)(A/C/T)CCAI(A/C)(A/C/T)(A/G)TC-3`.
PCR amplification, separation of the products, and cloning was carried
out as described previously (Becker et al., 1994). Plasmid DNA
was isolated and characterized by sequencing or Southern blotting.Library Screening and DNA Sequencing
Cloned PCR
products were isolated with restriction enzymes and used as probes to
screen a rat fat cell gt11 library (Clontech Laboratories, Palo
Alto, CA, catalog no. RL 10011b) and a rat brain
-zap cDNA library
(Stratagene, La Jolla, CA, catalog no. 937502). Deletions were
generated by exonuclease digestion and by sonication (Branson sonifier
450, cup horn, 1 min at maximum setting) and were sequenced by the
method of Sanger (T7-sequencing-kit, Pharmacia). Sequences were
determined with overlapping fragments from both directions.
Northern Blot Analysis
Samples of total RNA (20
µg) dissolved in a denaturing solution containing formaldehyde
(6.5%) and formamide (50%) and incubated at 65 °C for 15 min were
separated by electrophoresis through 1% agarose gels containing 6.5%
formaldehyde. Gels were stained with ethidium bromide before transfer
onto nylon membranes (Hybond, Amersham-Buchler, Braunschweig, Germany)
in order to ascertain that equal amounts of total RNA had been
separated. Membranes were hybridized at 42 °C with partial cDNA
probes labeled with [P]dCTP by random
oligonucleotide priming (Feinberg and Vogelstein, 1983). The blots were
washed twice at 55 °C in 0.12 M NaCl, 0.012 M sodium citrate, 0.1% SDS and once with 0.015 M NaCl,
0.0015 M sodium citrate, 0.1% SDS.
Preparation of Recombinant GST-Dyrk
A PCR fragment
(bp 112-745) of the Dyrk cDNA (clone B) comprising an EcoRI site before the initiation codon was prepared and used
for construction of an expression vector pGEX-2TK (Pharmacia) harboring
the cDNA of Dyrk (bp 119-2414) fused with that of GST in a single open
reading frame. Due to the cloning strategy, the two amino acids before
the stop codon were exchanged for a short linker-derived segment. PCR
product and fusion domain were verified by sequencing. Site-directed
mutagenesis with single-stranded DNA as template was performed as
described previously (Kunkel et al., 1987). Mutants were
confirmed by sequencing and subcloned into pGEX. Transformants in E. coli DH5
were isolated, induced with
isopropyl-1-thio-
-D-galactopyranoside for 2 h at 37
°C, and lysed by sonication. The recombinant fusion protein was
purified by affinity adsorption to glutathione-Sepharose and eluted
with glutathione as described by the manufacturer (Pharmacia). The
concentration of active recombinant kinase was determined by laser
densitometry of the Coomassie-stained 90-kDa product in comparison with
bovine serum albumin standards.Expression of Dyrk in COS-7 Cells
The cDNA of Dyrk
was subcloned into a derivative of the mammalian expression vector
pSVL, which contains a translation start and a short tag sequence for
immunochemical detection (HA1 protein of influenza virus). In addition,
the C terminus was extended by a short linker-derived segment by the
cloning strategy. Transfection of COS-7 cells was performed as
described previously (Wandel et al., 1994). For immunochemical
detection, cells were lysed by boiling in 1% SDS in 10 mM Tris
buffer (pH 7.4). For immunoprecipitation, cells were lysed in buffer
containing 50 mM Hepes, 150 mM NaCl, 2 mM EDTA, 20 mM NaF, 2 mM sodium pyrophosphate, 1
mM phenylmethylsulfonyl fluoride, 1 mM sodium
vanadate, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml
pepstatin, and 1% Nonidet P-40. Antiserum against the HA epitope was
from BabCo, Richmond, CA. Immunocomplexes were isolated with protein
A-Sepharose, and a kinase assay (see below) was carried with the
adsorbed material in the presence of histone. The products were
separated by SDS-PAGE and detected by autoradiography.Autophosphorylation of Recombinant GST-Dyrk Transferred
to PVDF Membranes
Samples of the fusion protein were separated
by SDS-PAGE and transferred onto PVDF membranes (Immobilon-P,
Millipore, United Kingdom). Proteins were denatured and renatured as
described previously (Ferrell and Martin, 1991), and the membranes were
incubated in 2 ml of phosphorylation buffer consisting of 33 mM Hepes, 6.6 mM manganese chloride, 6.6 mM magnesium chloride, 0.7 mM dithiothreitol, and
[P]ATP (40 µCi). Membranes were washed as
described by Ferrell and Martin(1991) and autoradiographed for 16 h.
Assay of Protein Kinase Activity
Samples of the
fusion proteins were incubated in phosphorylation buffer containing
[P]ATP (2 µCi, tracer only or final
concentration of 10-100 µM as indicated) in a total
volume of 12 µl for 30 min at room temperature. Histone (type IIS,
catalog no. H6005, Sigma),
-casein (catalog no. C8032), or
poly-Glu/Tyr 4:1 (catalog no. P0275) were added to a final
concentration of 0.66 µg/µl or as indicated. Thereafter, the
samples were boiled with Laemmli's sample buffer (Laemmli, 1970)
and separated on 10 or 14% polyacrylamide gels. The gels were dried and
autoradiographed for 1-4 h.Assay of Tyrosine Phosphorylation of
GST-Dyrk
Samples of the fusion protein GST-Dyrk were separated
on 10% polyacrylamide gels, transferred onto nitrocellulose membranes,
and probed with anti-phosphotyrosine antibodies (Transduction
laboratories, Nottingham, UK).Phosphoamino Acid Analysis
Autophosphorylation of
recombinant GST-Dyrk was carried out as described above. Reaction
products were precipitated with 10% trichloroacetic acid, and
hydrolyzed in 5.8 M hydrochloric acid for 2 h at 110 °C.
The lysates were dried under nitrogen, washed with water, and dissolved
in 10 µl of water supplemented with carrier phosphoamino acids. The
phosphoamino acids were separated by thin-layer chromatography (silica
gel, 25% ammonium hydroxide, 96% ethanol, 1.6:3.5). In a separate
experimental series, phosphorylation was carried out in the presence of
histone. The reaction products were separated by SDS-PAGE and
transferred onto PVDF membranes. The phosphorylated histone was
identified by autoradiography, cut, and hydrolyzed as described above.
Cloning of Dyrk
Degenerate oligonucleotide
primers matching the regions VIb and IX according to the nomenclature
of Hanks (Hanks and Quinn, 1991) were designed to amplify a domain of
about 150 bp, including the activation loop of protein serine/threonine
kinases. PCR products were cloned, and 70 clones encoding 13 different
sequences were identified as protein kinases by a data base search or
on the basis of structural criteria. One of them (PSK47, later
designated Dyrk) was chosen for further characterization.Structural Characteristics of Dyrk
The amino acid
sequence as depicted in Fig. 1A was deduced from an open
reading frame (clone B) in which the translation start was assigned to
the first AUG codon after a stop codon. The sequence of clone D
differed from that of clone B by a deletion of 27 bp within the open
reading frame (codons 69-78, shaded box). In order to
confirm that two different RNA species were present in rat brain, the
corresponding domain was amplified by PCR with cDNA from rat brain as
the template. Indeed, two bands of the expected size were identified
(data not shown). Thus, it appears reasonable to conclude that the mRNA
of Dyrk is alternatively spliced, generating two products that differ
by 27 nucleotides.
(see Fig. 7) (Rossomando et al., 1992; Hughes et al., 1993). Dyrk then contains two other putative consensus
motifs of tyrosine phosphorylation (Tyr-112 and Tyr-219), which are
preceded by lysine and glutamic acid, 7 or 3 residues to the N-terminal
side, respectively (Cooper et al., 1984). In addition, a
tyrosine-rich, highly charged motif that contains 4 tyrosines and 5
aspartic acid residues is located between the nuclear targeting domain
and the N terminus of the catalytic domain.
Relationship of Dyrk with Other Kinases
Data bases
were searched (SWISS-PROT, the Protein Identification Resource, EMBL
data base) in order to find relatives of Dyrk by a comparison of its
catalytic domain with that of known protein serine/threonine kinases,
and a dendrogram of a selected number of protein kinases was
constructed (Fig. 2A). The dendrogram demonstrates that
the structure of the catalytic domain of Dyrk differs considerably from
that of most other kinases. Its closest relative (85% identical amino
acids within the catalytic domain) is encoded by a gene from Drosophila (mnb), which has recently been found disrupted in a mutant
with abnormal neurogenesis (minibrain; Tejedor et
al.(1995)). The second closest relative (46% identical amino
acids) is a partial human sequence (PSK-H2) that was published without
further information (Hanks and Quinn, 1991). In addition, a third
relative appears to be the 89-kDa protein serine/threonine kinase
(Yak1) from yeast (39% identical amino acids), which is believed to
inhibit mitosis (Garrett et al., 1991). The dendrogram
indicates that Dyrk is located on the same branch of the protein kinase
superfamily as Mnb, PSK-H2, and Yak1, but is only distantly related to
the other subfamilies. Fig. 2B depicts an alignment of
Mnb, PSK-H2, and Yak1 with Dyrk, demonstrating that the identities
within this subfamily are mainly restricted to the subdomains I, II,
III, V, VIb, VII, VIII, IX, and XI of the catalytic domain. There is no
similarity outside the catalytic domains (comparison not shown) except
for 70 amino acids flanking the N terminus of the catalytic domain of
Mnb. Thus, Dyrk is a novel member of a small subfamily of protein
kinases with a unique structure of both the catalytic domain and the
flanking N- and C-terminal regions.
), S14708; MEK1
(MAPK/ERK-kinase), S29863; PKA (cAMP-dependent protein kinase), S21640;
PKC, A26037; TIK, A40813; ESK, A44439. GenBank accession numbers were
as follows: Mnb, X70794; Clk1, L29219; Clk2, L29218; Clk3, L29217; P38,
L35253; JNK1 (jun-kinase 1), L26318. Asterisks indicate known
dual specificity kinases. B, sequence alignment of the
catalytic domains of Dyrk, Mnb, PSK-H2, and Yak1. The deduced amino
acid sequences of the catalytic domains were aligned with the aid of
the CLUSTAL program (gap penalty 5, open gap cost 10, unit gap cost
10). Hyphens represent gaps introduced for optimal alignment,
{ } indicates a portion of the sequence of PSK-H2 that was
not shown in the source (Hanks and Quinn, 1991). The kinase subdomains
according to the nomenclature of Hanks (Hanks and Quinn, 1991) are
depicted on the top of the alignment by Roman numerals.
Residues identical with Dyrk are boxed. Asterisks indicate amino acids identical in all compared sequences; periods designate conservative substitutions. References for
the kinases are as follows: Mnb (Tejedor et al., 1995); PSK-H2
(Hanks and Quinn, 1991); Yak1 (Garrett and Broach,
1989).
Expression of Dyrk mRNA in Various Rat
Tissues
Northern blot analysis of total RNA from a series of
different rat tissues detected two transcripts (2.8 and 5.4 kb) in all
tissues examined (brain, heart, skeletal muscle, lung, intestine, fat
cells, adrenal gland, testis, thymus, kidney, liver, and spleen; data
not shown). These sizes correspond reasonably well with those derived
from the isolated clones. Although mRNA levels appeared somewhat
different in the various tissues, the data indicate a ubiquitous
expression of Dyrk mRNA.Protein Kinase Activity of Dyrk
In order to
demonstrate the protein kinase activity of Dyrk, a recombinant GST-Dyrk
fusion protein was expressed in E. coli DH5 and was
purified by affinity adsorption on glutathione-Sepharose. Analysis of
the partially purified fusion protein by SDS-PAGE showed a major
product of an apparent molecular mass of 90 kDa and several other bands
of higher mobility (60, 41, and 30 kDa; Fig. 3A). All
bands appear to be derived from the recombinant GST-Dyrk fusion protein
because they were not present in control preparations of GST alone. As
judged from the calculated molecular weight of GST-Dyrk (112 kDa), only
fragments of the protein kinase were isolated. Variation of the
conditions of expression and isolation (temperature, duration of
induction, addition of protease inhibitors) failed to prevent the
partial degradation of the fusion protein or allow the isolation of a
112-kDa protein. However, the truncated 90-kDa GST-Dyrk exhibited a
marked protein kinase activity that was detected after renaturing of
the protein on PVDF membranes (Fig. 3B). As is also
illustrated in Fig. 3B, the smaller fragments lacked a
detectable protein kinase activity. Preliminary experiments were
carried out in order to find out which domain(s) were removed from the
90-kDa fragment. Thrombin cleavage of the 90 kDa GST-Dyrk, presumably
at the cleavage site engineered in the linker between GST and Dyrk,
generated an active kinase of approximately 60 kDa (data not shown).
Furthermore, the 90-kDa fragment appeared to cross-react with an
antibody against GST. Finally, taking advantage of the histidine
repeat, we isolated an active protein kinase by affinity adsorption on
nickel-chelating agarose (data not shown). Thus, it is tentatively
concluded that the 90-kDa band represents a fragment that comprises
both GST-domain and histidine repeat but lacks the remaining part of
the C terminus of Dyrk.
P]ATP indicated that the kinase, in addition to
autophosphorylating the 90-kDa fragment, stimulated a marked
P incorporation into its smaller fragments (Fig. 3C, first lane). Furthermore, the fusion
protein catalyzed the
P incorporation into histone and
casein but failed to phosphorylate the tyrosine kinase substrate
poly-Glu/Tyr. A quantitative assessment of the stoichiometry of
autophosphorylation of the 90 kDa band (Fig. 3C, first lane) indicated that 1 mol of phosphate was incorporated
per 8 mol of recombinant protein kinase, suggesting that the protein
was already phosphorylated in E. coli (see Fig. 5A).
P]ATP. The reaction products were hydrolyzed
and separated by thin-layer chromatography as described under
``Materials and Methods.'' The positions of carrier
phosphotyrosine (PY) and phosphoserine/phosphothreonine (PS/PT) as determined by ninhydrine staining are
marked. C and D, tyrosine phosphorylation of histone
by Dyrk. Histone was phosphorylated by partially purified Dyrk in the
presence of the indicated bivalent ions or EDTA. The reaction products
were separated by SDS-PAGE and subjected to autoradiography (C). D, additional samples prepared by the same
procedure were transferred onto PVDF membranes (14% gel, right
panel). The phosphorylated 13 kDa band was cut out of the membrane
and hydrolyzed. Aliquots adjusted for their content of radioactivity
were separated by thin-layer chromatography and subjected to
autoradiography.
Expression of Dyrk in COS-7 Cells
COS-7 cells were
transiently transfected with a construct comprising the Dyrk cDNA fused
to the HA tag for immunochemical detection. Western blotting of total
cell lysate (Fig. 4A) identified an immunoreactive
product of 105 kDa, which was absent in controls transfected with blank
vector. As is illustrated in Fig. 4B, the
immunoprecipitated product of the transfection catalyzed its
autophosphorylation as well as phosphorylation of histone. It should be
noted that the calculated molecular weight of the HA-Dyrk construct
(90.5 kDa) is somewhat lower than the observed molecular mass.
P
was detected by autoradiography.
Dual Specificity Protein Kinase Activity of
Dyrk
Since the sequence of Dyrk harbors two consensus motifs for
tyrosine phosphorylation, we studied the possibility that Dyrk was
autophosphorylated on tyrosine residues or catalyzed the tyrosine
phosphorylation of histone. The GST-Dyrk preparation was partially
purified, separated by SDS-PAGE, and transferred onto nitrocellulose
membranes. Probing with anti-phosphotyrosine antibody (Fig. 5A) revealed that the 90-kDa fragment indeed contained
phosphotyrosine. In addition, a second, much weaker immunoreactive band
was detected at approximately 41 kDa. Since E. coli is devoid of
tyrosine kinase activities (Kornbluth et al., 1988), the
tyrosine phosphorylation of GST-Dyrk must result from its
autophosphorylating activity. In order to confirm the identity of the
phosphotyrosine by an analysis of the phosphorylated amino acids,
GST-Dyrk was in vitro phosphorylated by
[P]ATP and hydrolyzed, and the hydrolysate was
separated by thin-layer chromatography (Fig. 5B). As
anticipated, phosphotyrosine as well as phosphoserine/phosphothreonine
were detected. In addition, hydrolysis and amino acid analysis of
histone (11-15 kDa), which was phosphorylated by Dyrk revealed
that the kinase catalyzed a marked tyrosine phosphorylation of the
exogenous substrate (Fig. 5, C and D).
value of the total phosphorylation reaction
obtained in the presence of 40 µM ATP was 0.044
µg/µl or 3.4 µM (slope of the Lineweaver-Burke
plot as determined by linear regression: 2.01 ± 0.14 (min
µg)/(pmol µl)). Since mainly one component of
the histone preparation was phosphorylated, the true K for phosphorylation of this isotype is even lower and was
estimated to be less than 2 µM. V
was 2.2 nmol/(min mg) (ordinate intercept, 45.9 ±
8.8 min/pmol). At all tested histone concentrations, more
phosphotyrosine was detected than phosphoserine (Fig. 6, inset). Moreover, the ratio of phosphotyrosine to
phosphoserine was not altered at different histone concentrations,
indicating similar K values of both reactions.
Thus, the data warrant the conclusion that Dyrk is a dual specificity
kinase, phosphorylating both tyrosine and serine/threonine residues in
its sequence and in exogenous substrates.
-
P]ATP (final concentration, 40
µM). The samples were separated by SDS-PAGE, and the
radioactivity incorporated was determined by cutting and scintillation
counting of the histone band. Linear regression analysis of the
Lineweaver-Burke plot yielded a slope of 2.01 ± 0.14 (min
µg)/(pmol µl)) and an ordinate intercept of
45.9 ± 8.8 min/pmol from which K = 0.044 µg/µl and V
= 2.2 nmol/(min mg) were calculated, respectively.
Aliquots of the phosphorylation were subjected to phosphoamino acid
analysis (inset).
Regulation of the Protein Kinase Activity of Dyrk by
Tyrosine Phosphorylation in the Putative Activation Domain
Dyrk
contains 2 tyrosine residues between the subdomains VII and VIII (Fig. 1A and Fig. 7). Similar residues are
present in other kinases (Fig. 7) (Rossomando et al.,
1992; Han et al., 1994; Derijard et al., 1994; Hughes et al., 1993), which have recently been shown to be
phosphorylated, giving rise to activation of the enzymes. Consequently,
the domain is believed to be crucial for the regulation of these
kinases (Marshall, 1994). Since we suspected a similar pattern of
regulation of Dyrk, constructs were generated encoding mutants of Dyrk,
and the resulting recombinant proteins were tested for their kinase
activities. As is illustrated in Fig. 8, exchange of the two
tyrosines in the activation loop (GST-Dyrk-Y319/321F in lane
4) suppressed the autophosphorylation of Dyrk on tyrosine (panel A) as well as its kinase activity (tyrosine and
serine/threonine) toward histone (panel B). Exchange of
Tyr-219 for phenylalanine largely reduced the autophosphorylation of
Dyrk on tyrosine, as detected with anti-phosphotyrosine antibody, but
failed to significantly reduce the histone phosphorylation. As an
additional control, a kinase-negative mutant (Dyrk-K188R) was
constructed by exchange of the conserved lysine in the ATP-binding
motif. Like that of Dyrk-Y319F/Y321F, the kinase activity of the
resulting recombinant protein was considerably lower than that of Dyrk.
Longer exposure (data not shown) revealed that both Dyrk-Y319F/Y321F
and Dyrk-K188R retained a minute kinase activity toward histone. A low
residual kinase activity of constructs similar to Dyrk-K188R has
previously been reported for several other kinases (Zhou and Elledge,
1993; Brill et al., 1994; Bowdish et al., 1994).
These data indicate that Tyr-219 and Tyr-319/Tyr-321 are
autophosphorylated, and that the tyrosines in the activation loop
regulate the protein kinase activity of Dyrk.
(Hughes et al., 1993), JNK1
(Derijard et al., 1994), and p38 (Han et al., 1994),
the conclusion appears to be justified in that the activation of Dyrk
depends on the phosphorylation of one or both of these tyrosines. It
should be noted that the activation motif in Dyrk (YQY, see Fig. 7) is somewhat different from that in the other
tyrosine-regulated serine kinases, which contain a single tyrosine
residue or a tyrosine and a threonine. C-terminal to the YQY motif are
three residues (RFY), which are conserved in all other tyrosine
phosphorylation-regulated kinases (Fig. 7) and might therefore
belong to the activation motif. The YQY motif resembles that present in
Mnb (YHY), Yak1 and PSK-H2 (YTY), and we anticipate on the basis of
this structural similarity that these kinases are also activated by
tyrosine phosphorylation in the activation loop. It remains to be
elucidated whether the activation of Dyrk is regulated by a protein
kinase or by an activator of autophosphorylation., MEK1, TIK, ESK; see Fig. 2A) share no easily recognizable structural
similarities other than the domains common for all serine/threonine
kinases (Lindberg et al., 1992). Some of these kinases harbor
the activation motif as depicted in Fig. 7, others (TIK, ESK,
Clk) lack this motif. Thus, at present there are no structural
similarities predicting dual specificity. Accordingly, the catalytic
domain of Dyrk is only distantly related to those of the other dual
specificity kinases. The so far characterized dual specificity kinases
show different functional characteristics (Lindberg et al.,
1992). Some can use poly-Glu/Tyr as a substrate, others do not. Some
dual specificity kinases catalyze tyrosine phosphorylation exclusively
as autophosphorylation (ERK2, Wu et al.(1991); GSK3, Wang et al.(1994)), others can stimulate both autophosphorylation
and phosphorylation of exogenous substrates on tyrosine (e.g. MEK, Zheng and Guan(1993)). Dyrk appears unique in that it
catalyzed both autophosphorylation and histone phosphorylation on
tyrosine, but it failed to phosphorylate poly-Glu/Tyr. Thus, the
substrates of Dyrk appear to require specific consensus motifs, which
may not be identical with those of other kinases.,
are components of signaling pathways that transduce receptor-initiated
signals to a nuclear phosphorylation of transcription factors (Hill and
Treisman, 1995; Woodgett, 1991). By analogy, we speculate that Dyrk is
a component of a similar signaling pathway, possibly mediating the
specific phosphorylation of transcription factors within the nucleus.
)
We thank B. Feulner for skillful technical assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
