(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 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.
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 ()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).
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 (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.
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), 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).
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
[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).
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
[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.
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 P
was detected by autoradiography.
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 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.
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
[-
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).
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 (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.
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, 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.
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,
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X79769[GenBank].