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Structural and Functional Characteristics of Dyrk, a Novel Subfamily of Protein Kinases with Dual Specificity
Structural and Functional Characteristics of Dyrk, a Novel Subfamily of Protein Kinases with Dual Specificity WALTER BECKERAND HANS-GEORG JOOST Institutfur Phurmnkologie und
Toxikologie 0-52057 Aachen, Germany I. Introduction.
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XI. Identificationand Nomenclature of the Dyrk/Minibrain Family ......
111. Structural Comparison of the Dyrk Family Members and D e f ~ t i o n of Common Characteristics ..................................... A. Comparison of the Catalytic Domain .......................... B. Comparison of N and C Termini .............................. C. The DH-Box @DDNxDY) ................................... D. Evolutionary Comparison and Relation of the Dyrk Family to Other Kinase Subfamilies ......................................... n! Tissue Distribution, Alternative Splicing, and Polyadenylation of Dyrk mRNA ....................................................... V. Genomic Localization of the Dyrk Kinases ........................ VI. Enzymatic Characteristics of the Dyrk Family ..................... A. Dual-SpecificityProtein Kinase Activity ........................ B. Presumed Activation of Dyrk Protein Kinase Activity by Tyrosine Phosphorylation ............................................ VII. Potential Cellular Functions of the Dyrk Family of Protein Kinases VIII. Futureperspectives ............................................ Note Added in Proof ...................... ................ References ...................................................
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Dyrk-related kinases represent a novel subfamily of protein kinases with unique structural and enzymatic features. Its members have been identified in distantly related organisms. The yeast kinase, Yakl,has been characterized as a negative regulator of growth. Mnb from Drosophiia is encoded by the minibraingene, whose mutation results in specific defecb in neurogenesis. Its mammahn ’ homolog, DyrklA, is activated by tyrosine phosphorylation in the activation Imp between subdomains VII and VIII of the catalytic domain. The human gene for DyrklA is located in the “Down syndrome critical region” of chromosome 21 and is therefore a candidate gene for mental retardation in Down syndrome. More recently, six additional mammalian Dyrk-relatedb a s e s have been identified (DyrklB, DyrklC, Dyrk2, Dyrk3, Dyrk4A, and Dyrk4B). All members of the Dyrk family contain in the activation Imp Progress in Nudeic Acid Reseaich and Molecular Biology Vol 62
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Copyngfit 0 1999 by Academic Press
AU nghts of reproducbon in any form reserved
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WALTER BECKER AND HANS-GEORG JOOST
the tyrosines that are essential for the full activity of DyrklA. Outside their catalytic domains, Dyrk kinases exhibit little sequence similarity except for a small segment immediately precedingthe catalytic domain (DH-box,Dyrk homology box). An unusual enzymatic property of Dyrk-related kinases is their ability to catalyze tyrosine-directed autophosphorylation as well as phosphorylation of serine/threonine residues in exogenous substrates. The exact cellular function of the Dyrk kinases is yet unknown.However, it appears reasonable to assume that they are involved in the regulation of cellular growth and/or development. B 1999 Academic Press
I. Introduction Protein phosphorylation and dephosphorylation are the predominant mechanisms of signal transduction in eukaryotic cells. The enzymes catalyzing phosphorylation of serine, threonine, or tyrosine residues of proteins provide a high degree of versatility and substrate specificity, and represent one of the largest gene families. On the basis of the numbers of genes encoding protein kinases in the genomes of yeast (113 of a total of 6121 genes) and Caenorhabditis elegans (270 of the approximately 15,000 sequenced genes), the total number of human protein kinases might be estimated to exceed 1000, a number already suggested by Hunter more than 10 years ago (1,2). This superfamily of protein kinases can be further divided into phylogenetically related subfamilies,which in most cases have related functions (e.g.,cell cycle kinases, receptor tyrosine kinases) (3).This article provides a summary of the knowledge on a new protein kinase family, Dyrk, whose prototype has been identified by polymerase chain reaction cloning and by positional cloning. This enzyme, and its yeast and Drosophila homologs, showed striking structural, enzymatic, and functional characteristics. Furthermore, several related mammalian genes have been identified, defining a novel subfamily of protein kinases with unique structural, enzymatic, and probably also functional features that will be summarized herein.
II. Identification and Nomenclature of the DyrklMinibrain Family Dyrkl has been identified independently by three different experimental strategies. First, its Drosophila homolog (Mnb) has been identified by positional cloning of the minibrain mutations (mnb),which exhibit specific behavioral defects and a reduced number of neurons in distinct areas of the brain (optic lobes and central brain hemispheres) (4).Four mutant alleles resulting in reduced expression of minibrain have been found, and it was suggested that the expression of minibrain is required for proliferation of distinct neuroblasts during larval development.
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STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
Second, rat Dyrkl was identified in a polymerase chain reaction (PCR) cloning approach as a dual-specificity protein kinase that autophosphorylates as well as catalyzes the phosphorylation of histone on serinelthreonine and on tyrosine residues (5).Subsequently, six additional mammalian Dyrk-related kinases were identified, namely DyrklB, DyrklC, Dyrk2, Dyrk3, Dyrk4A, and Dyrk4B, defining a distinct family of protein kinases with common structural and functional characteristics (accession nos. Y13493, Y12735, Y09306, and U49952; 54.The acronym “Dyrk (dualspecificity Yak-related ljnase) refers to the unusual ability of these kinases to phosphorylate serinelthreonine and tyrosine residues, and to the significant sequence similarity with the protein kinase Yak1 from Saccharomyces cerevisiae. YAK1 was identified as a functional antagonist of the Ras/PKA pathway, and has been characterized as a negative regulator of growth (6, 7). Like Dyrkl, Dyrk3, and Mnb, recombinant Yak1 is autophosphorylated on tyrosine residues (see below), and is thus considered the yeast homolog of the Dyrk family. Based on the proposed role of the conserved tyrosine residues in the activation loop (see below), “Dyrk can alternatively be read as “dual-specificity tyrosine-phosphorylation regulated ljnase.” Due to the high sequence similarity of the rat Dyrkl cDNA with a human expressed sequence tag (EST)that had been mapped to chromosome 2 1,the gene of a human homolog was localized to 21q22.2 (5). By correlation of phenotype with genotype in patients with partial trisomy 2 1,this region has been defined as the “Down syndrome critical region” (8)(Fig. 1);its triplication appears to be responsible for many features of Down syndrome, including mental retardation (9-11). Indeed, several groups independently identi-
I I
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230E8
141z-L?&6 i 152F7tl
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.+; 1
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c”Down Syndrome Critical Region”+’
FIG.1. L o c i a t i o n of the human DyrklA gene in the Down syndrome critical region of chromosome 21. The Down syndrome critical region spans about 3 Mb of chromosome 21 at 21q22.2 andis definedbythe markers D21S17 (centromericborder),D21S55, and ERG (telomeric border) (8). The position of selected YACs in this region is indicated (230E8,141G6,152F7, 336(311,28SE6). D YRKl A maps to YACs 152F7 and 336G11(12,14,34).Smith et d.(15)identified the DYRKlA gene on a 180-kb telomeric fragment of 1S2F7 (tel).
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WALTER BECKER A N D HANS-GEORG JOOST
fied the human Dyrkl gene by screening the Down syndrome critical region for genes whose triplication may cause defects in neurogenesis (12-14). Because of its high degree of similarity to Mnb, the DYRK1 gene was an obvious candidate for phenotypic manifestations of Down syndrome. This hypothesis was supported by the finding that mice transgenic with a 180-kb fragment of human chromosome 21 including the DYRK1 gene (Fig. 1) showed defects in learning tasks (15).
111. Structural Comparison of the Dyrk Family Members and Definition of Common Characteristics
A. Comparison of the Catalytic Domain 1. CONSERVED RESIDUES AND MOTIFS
To date, sequences of 14 Dyrk-related kinases (Table I> are available for sequence comparisons, including several sequences that were identified in genome sequencing projects of fission yeast (Schixosaccharomycespornbe), the roundworm Caenorhabditis elegans, and Drosophila. A comparison of these sequences with the CLUSTAL W program (16)allows a definition of the common structural features of the Dyrk family (Fig. 2), and of its differences with regard to other protein kinase subfamilies. In addition to the sequences compared in Fig. 2, two cDNA fragments with high similarity to Dyrkl, and one fragment with high similarity to Dyrk4, have been identified (5a).These sequences were designated DyrklB, DyrklC, and Dyrk4B. They will not further be discussed here. However, Dyrkl will be referred to as DyrklA throughout. All Dyrk-related kinases contain a canonical kinase domain of 297-335 amino acids with the conserved regions as defined by Hanks and Quinn (17) (subdomains I-XI in Fig. 2).In addition, the sequences exhibit a number of features that are not (or rarely) found in other kinases and are therefore characteristic for the Dyrk family (indicated by boxes in the sequence alignment). Three specific sequence motifs are found in the vicinity of the catalyhc cleft (subdomains VIB-IX). First, in all kinases of the Dyrk family, the conserved DFG motif of subdomain VII is followed by the amino acids serine, serine, and cysteine (SSC). Second, the arginine residue in subdomain VIB, which is highly conserved in all other kinases, is substituted for cysteine in the Dyrk family. Third, the YxY motif between subdomains VII and VIII (YxYIQ) is specific for the Dyrk family. In addition, there are several other conserved amino acids (boxed in Fig. 2) that are characteristic for the Dyrk family: a cysteine between subdomains IV and V, the NLY motif in subdomain V, and a glycine between subdomains VIII and IX. Finally, Dyrk-related kinases
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STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
TABLE I COMPILATION OF SEQUENCES ENCODINC DYRK-RELATED K~NASES Gene product
Species
DyrklA DyrklA DyrklA DyrklA DyrklA DyrklA Dyrk2 Dyrk3 Dyrk4 AD-1 AD-1 Mnb 34dc6z
Mouse Human Human Human Human Human Drosophila Drosophila
T04c10 F4 9 9e 11 KAB7 KA23 Yak1
Caenorhabditis eleguns Cuenurhubditis elegans Schizosaccharomyces pombe Schizosucchuromycespombe Sacchumyces ceruisim
Human Human Human Human Rat
Accession no. U52373 D85759 U58497" D86550 X79769 U58796 Y13493 Y12735 YO9305 YO9306 U57894 X70794 L43478 L43480 269885 270308 254354 250142 X16056
Type
Ref.
cDNA cDNA cDNA cDNA cDNA cDNA cDNA cDNA cDNAb CDNA~ CDNA" Gene Geneb,' Geneh." Gene" Gene" Gene' Gene' Gene
12 13 14
5 14 -
"This database entry represents a fusion of the muline and the human sequences. "The nucleotide sequence encodes a partial open reading frame. <:Thesequence has been determined in a genome sequencing project.
contain an unusual insertion of six amino acids between subdomains I11 and lV. The protein kinase AD-1 was included in the comparison in order to further illustrate the defining features of the Dyrk family. AD-1 is a close relative of Dyrkl but is not considered a member of the Dyrk family, because three of the above described conserved motifs are not found in its sequence: the cysteine in VIB, the SSC in VII, and the YxY motif. However, it shares several other characteristics of the Dyrk family, e.g., a glutamine in the sequence before subdomain VIII (YIQSRFY), where most other kinases possess a s m d , nonpolar residue (3). Song et d.(14) have suggested that a motif of four leucine residues (Leu274, Leu-281, Leu-288, and Leu-295) might form aleucine zipper structure. They tested the proposed ability to mediate proteinjprotein interactions with the help of the two-hybrid system, and demonstrated dimerization of DyrklA (18). However, the respective leucine residues are conserved in a large pro-
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WALTER BECKER AND HANS-GEORG JOOST
FIG.2. Sequence alignment of the catalytic domains of Dyrk kinases. The deduced amino acid sequences of the catalytic domains were aligned with the help of the CLUSTAL W program (26). Hyphens represent gaps introduced for optimal alignment. The kinase subdomains according to the nomenclature of Hanks and Quinn (17) are indicated by Roman numerals at the top of the alignment. Residues conserved in all or nearly all protein kinases [according to Hanks and Hunter (3)]are in black boxes. Amino acids that are conserved in the Dyrk family but are very rare at this position in other kinases are highlighted by shaded boxes. Asterisks indicate amino acids identical in all compared sequences; periods designate conservative substitutions. Arrows point to s i g d c a n t differences between AD-1 and the Dyrk family. Sequence sources (EM, EMBLGenBank; others, SwissProt database):DyrklA, rat, EM:X79769; Dyrk2, Dyrk3, and Dyrk4, human, EM:Y13493, EM:Y12735, and EM:Y09305; Mnb and 34dc6z, Drosophila, P49657 and EM:L43478/L43480; T04c10 and F49el1, Caenmhabditis elegans, EM:Z69885 and 270308; KAB7 and KA23, Schizosaccharomyces pombe, Q09815 and Q09690; Yakl, Saccharornyces cerevisiae, P14680; and AD-1, human, EM:AF004849.
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STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
Dyrkl Mnb TO4clO Dyrk2 Dyrk3 F49ell 34dc6z Dyrk4 KA23
KAB7
Yakl AD-1
Dyrkl Mnb T04c10 Dyrk2 Dyrk3 F49ell 34dc6z Dyrk4 KA23 KAB7
Yakl AD-1
Dyrkl Mnb T04c10 Dyrk2 Dyrk3 F49ell 34dc6z Dyrk4 KA23
- - - - - - - - HTVADY LKFKDLILRMLDYD
XI
.--HSVSDYLKFKDLILRMLDFD - - - - - - - - HSVEDYSKFKDLIKRMLQFD - --- --.- - -- -- LKGCDDPLFLDFLKQCLEWD -LKGCDJYLFIEFLKXCLSXD - - - - - - - - LKNMGDELFVDFLKRCLDWD
- - --- - --
KAB7
- -- - - - - -- --- - - -
AD-1
DLEGSDLLAEKADRREFVSLLKKMLLIDADI&TP~~ETLNHPFV
Yakl
.
* .* * .
*. .
FIG.2 (Continued).
portion of all protein kinases (3).Furthermore, the definition of the leucine zipper motif requires that the leucine residues are located within a single ahelix (19,20).Based on the tertiary structure of other kinases, the four leucine residues are located within different structural elements (&-helixE, P-strand 7, and the catalytic loop) (24. Thus, the putative dimerization of DyrklA is probably not mediated by the proposed leucine zipper. 2. THEYxY MOTIFIN THE ACTIVATION LOOP The position of the conserved YxY motif between subdomains VII and VIII corresponds exactly with that of the TxY motif in the MAP kinases, which are activated by phosphorylation of these residues (22). Activating phosphorylation of serine, threonine, and/or tyrosine residues in this so-
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WALTER BECKER AND HANS-GEORG JOOST
called “activation loop’’ is a common mechanism in many protein kinases (23) (Fig. 6). Indeed, exchange of these tyrosines for phenylalanine almost completely suppressed the activity of recombinant DyrklA (5). Based on the structure of phosphorylated CAMP-dependentkinase (PKA) (21),a general mechanism of the activation of protein kinases by phosphorylation was proposed by Johnson d al. (23).In their model, the binding of the phosphate in the activation loop to specific positively charged residues stabilizes an active conformation of the enzyme. These arginines, i.e., an arginine in subdomain VIB (YRDLKPEN in PKA) and a lysine in subdomain VII (DFGFAKin PKA),are conserved in many kinases that are activated by phosphorylation (23).Strikingly, the Dyrk family kinases contain the characteristic cysteine residues at the equivalent positions (see above). Thus, the role of the phosphotyrosine must differ from that of the phosphothreonine in PKA.
B. Comparison of N and C Termini In all kinases of the Dyrk family, the catalytic domain is located between N-terminal and C-terminal regions of variable length (99-699 and 22-424 amino acids) (Fig. 3). Outside the catalytic domain, kinases of the Dyrk family show no extended regions of sequence similarity to any other known protein, and the noncatalytic regions of these kinases are not generally related within the Dyrk family. Sequence similarity is apparent only in closely related members of the family, such as Dyrk2 and Dyrk3 (Fig. 3), and in species homologs, e.g., mammalian Dyrkl A, Drosophila Mnb, and Caenorhabdicis T04c10.
FIG.3. Schematic presentation of the structures of the known mammalian Dyrk kinases. Outside the catalytic domain (cat), black bars indicate sequence regions without similarity to other proteins. Homologous regions in Dyrk2 and Dyrk3 are depicted as open bars. A small segment of the N-terminal region that is conserved in all kinases of the Dyrk family is symbolized by an open box in front of the catalytic domain. The positions of different structural motifs in the sequence of DyrklA are given (0,nuclear localization signal, NLS; PEST region; H, histidine repeat; S, serineithreonine repeat). Only partial sequence information is available for Dyrk4.
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STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
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FIG.4. The DH-box (DDDNxDY): a conserved sequence motif preceding the catalytic domain in the Dyrk family and related kinases. Related sequences were identified in a protein database search performed with residues 135-180 of DyrklA (accession no. X79769), which include the DH-box and subdomain I of the catalytic domain. Conservation of amino acids is highlighted by shaded boxes. Invariable amino acids are shown in boldface. The number of amino acids between the last residue of the alignment and the first residue of the catalytic domain is given in brackets. In the consensus sequence, amino acids that are given are found at this position in at least six of the nine sequences. Periods in the consensus sequences designate nonconserved residues. The consensus sequence for the C k family has been derived from seven different kinases (24-26); the consensus for Prp4 has been deduced from three homologs [mouse, 5’. pombe, and C. eleguns (27),and database entries U48737 and 2712621.
C. The DH-Box (DDDNxDY) A short stretch of amino acids immediately preceding the catalytic domain appears to be conserved in all kinases of the Dyrk family. Because of its potential importance (see below), we designated this motif DH-box @yrk homology box). As is illustrated in Fig. 4, the core of this stretch appears particularly well conserved within the family; its consensus sequence is DDDNxDY. This observation prompted us to analyze other kinases for the presence of related motifs. Indeed, a similar motif was identified in the kinases of the Clk family (also called LAMMER kinases, because of the conserved signature motif EHLAMMERILG in subdomain X, including mammalian Clkl-3, Doa from Drosophila, and AFC1-3 from Arabidopsis) (24-26) and in Prp4 (27, 28). The consensus sequences of the motif differ somewhat among the three groups of kinases (Fig. 4). However, two aspartic acid residues followed by a third acidic residue are conserved in the Clk family and in Prp4. Furthermore, a tyrosine and an acidic residue (boxed in Fig.
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WALTER BECKER AND HANS-GEORG JOOST
4) appear conserved. Interestingly, mutations of one or both of the aspartic acid residues in Prp4 cause the functional inactivation of this gene in S. pombe (28).However, the DH-box is not essential for the enzymatic activity of recombinant DyrklA because an N-terminally truncated construct exhibits full enzymatic activity (W, Becker and H.-G. Joost, unpublished data). In the absence of further functional evidence, it can only be speculated that the DH-box represents a site of interaction with the substrates or with other proteins, and thereby regulates the kinase activity. In addition to the DH-box, some kinases of the Dyrk family harbor striking sequence motifs in their noncatalytic regions. Putative nuclear localization signals have been identified in the amino-terminal regions of Mnb and DyrklA (4,5).Indeed, a construct of the N-terminal region of DyrklA fused to green fluorescent protein is detected in the nucleus of transfected mammalian cells (Sa, 18). Immediately following the catalytic domain, DyrklA contains a region rich in proline, glutamic acid, serine, and threonine (PEST) residues (Fig. 3). Such PEST regions are believed to represent signals for a rapid turnover of the protein (29).A region rich in glycine, alanine, and serine (GAS domain) was identified in the carboxy-terminaldomains of Mnb (4).A GAS sequence has also been detected in the Drosophila sgg protein kinase. Several members of the Dyrk family harbor striking repeat structures in their noncatalyhc domains. DyrklA contains stretches of 13 consecutive histidines and 17 subsequent serine/threonine residues in its carboxy-terminal regions. Yak1 and T04c10 harbor repeats of 12 and 9 glutamine residues, respectively. Although histidine and glutamine repeats are frequently found in transcription factors, their exact function has not yet been elucidated.
D. Evolutionary Comparison and Relation of the Dyrk Family to Other Kinase Subfamilies The relationship of the Dyrk family to other kinases can be deduced from a sequence comparison of their catalytic domains. In Fig. 5, the results of such a sequence alignment are illustrated as a phylogenetic tree constructed with the TREE program (30). According to the classification of Hanks and Hunter (3),the Dyrk family belongs to the CMGC group of protein kinases, which includes the families of the cyclin-dependent kinases, the MAP kinases, the GSK3 family, and the Clk family (also called LAMMER kinases) (25). The closest relatives of the Dyrk family are the Clk kinases and Prp4; these kinases appear to be involved in the regulation of RNA synthesis and splicing (25,27,31). Interestingly,Clkl/STY have also been shown to exhibit dual specificity when expressed in Escherichia coli. There are several kinases in the CMGC group that contain a tyrosine residue in the activation loop (marked by asterisks in Fig. 5 ). However, activating phosphorylation of this
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
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Dyrk 1 Mnb*(D.m.) T04cl0* (C.O.) AD- 1 F20b6+ (C.O.) Yakl*(S.c.) Prp4* Clk2
I
I
Cdk4 Kklalro' Make Gsk3@*
FIG.5. Relationship between the Dyrk family and other protein kinases. The dendrograrn was constructed with the TREE program (30) on the basis of a sequence alignment of the catalytic domains. Source organisms are indicated for nonmammalian sequences (Ce., Caenarhab&.s elegans; Dm., Drosophila melanogaster; S.C.,Saccharomyces cerevisiae; S.P., Schizosaccharomyces pornbe).Asterisks indicate kinases with a tyrosine residue in the activation loop.
tyrosine has so far been demonstrated only for the MAP kinases (Erkl12,Jnk, and p38) and GSK3 (22,32). The tree in Fig. 5 also illustrates the relationship of the kinases within the Dyrk family. The branching pattern suggests that DyrklA, Mnb, and T04c10 are species homologs of mammals, Drosophila, and Caenorhabditis, respectively. The other main branch of the tree also contains kinases from invertebrates and mammals (Dyrk2, Dyrk3, F49el1, and 34dc6z). Hence, the last common ancestor of Drosophila, Caenorhabditis, and mammals possessed at least two genes for Dyrk-related kinases. In contrast, Yak1 is the only member of the Dyrk family in the yeast Saccharomyces cerevisiae. Furthermore, it is noteworthy that this gene is not essential for viability (6).
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WALTER BECKER AND HANS-GEORG JOOST
As a result of sequencing projects, partial sequences of two different Dyrk-relatedkinases of Arabidopsis thaliana were published in the EST databases (accession nos. N65563 and R84192). The presence of Dyrk-related kinases in plants, fungi, and animals suggests that these enzymes regulate a fundamental function of the eukaryotic cell.
IV. Tissue Distribution, Alternative Splicing, and Polyadenylationof Dyrk mRNA mRNA of DyrklA has been found in all human, murine, and rat tissues studied ( 5 1 2 - 1 4 ) . The distribution of DyrklA mRNA in brain has been studied by Northern blot analysis using human brain (13) and by in situ hybridization using embryonic mouse brain (14) and adult mouse brain (12). The data obtained from these studies differ considerably but indicate that DyrklA is widely expressed in most regions of the brain. In striking contrast to DyrklA, the other members of the family (DyrklB, Dyrk2-4) and the related AD-1A and AD-1B were found to be predominantly expressed in rat testis. However, their transcripts are not absolutely testis specific, because cDNA clones for Dyrk2, Dyrk3, and Dyrk4 ( 5 4 have been isolated from a human fetal brain cDNA library. Furthermore, the EST database (33) contains a considerable number of expressed sequence tags for Dyrk2-4 and AD-1 that were derived from organs other than testis. Particularly, fetal tissues (brain, liver/spleen) were the sources of many of these ESTs. Thus, it seems reasonable to assume that the kinases of the Dyrk family may also be expressed during embryonal development. Alternative splicing of DyrklA mRNA has been observed in rat tissues (5).Two cDNA clones that were identified differed by an in-frame insertion of 2 7 nucleotides, and mRNA corresponding to both splicing products has been detected by PCR in several tissues. Both transcripts appear to be generated also in human tissue (longer form, U52373; short form, D85759). Analysis of the human gene reveals that the inserted segment is indeed located at an exon/intron border (13).It should be noted that the conservation of the insertion in rat and human mRNA points to a functional role of the two splicing variants. In rodents, DyrklA is encoded by two alternatively polyadenylated mRNAs of approximately 3 and 6 kb (5,12).Human tissues contain only the larger transcript (12, 13; W. Becker and H.-G Joost, unpublished), probably due to a difference of a single base in the sequence of the putative polyadenylation signal of the rate mRNA (AATAAA vs. AGTAAA; nucleotide 2759 in the rat cDNA sequence, accession no. X79769). Alternative splicing and polyadenylation were also shown for Dyrk2. A
STRUCTURE AND FUNCTION O F DYRK PROTEIN KINASES
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transcript that was isolated contained a 149-bp insertion in the 5' flanking region of the open reading frame; this insertion appeared to extend the coding region by 73 codons. Furthermore, the human gene generates at least three differently polyadenylated transcripts: one with only a 184-bp 3' untranslated region (UTR), one with an additional 1461-bp 3' UTR, and one with an even longer 3' UTR as predicted from the sequences of several expressed sequence tags (accession nos. R63 190, R63622, R25341, and 225301) ( 5 4 . Expression of the mnb gene during development of Drosophila has been studied by immunocytochemistry (4). In the larval brain (third instar larva), protein levels are highest in areas where neuronal progeny is generated during postembryonal development (outer proliferation centers of the optic lobes, mushroom body neuropils). Highest expression in adult flies was observed in mushroom bodies and retinal pigment cells, whereas expression in the optic lobes was low. Thus, the embryonal pattern of expression correlates with the defects of the mutant flies (reduced number of neurons in the optic lobes), but the sites of expression in the adult flies are not visibly affected by mnb mutations.
V. Genomic Localization of the Dyrk Kinases As was discussed earlier,human DYRKlA is located on 21q22.2 at the STS marker D21S70 (YAC clones 152F7 and 336G11) (12,14,34). The most precise localization is given by Smith et aZ. (15), who identifed the gene on a 180-kb telomeric fragment of the YAC 152F7. Murine DyrklA was mapped to chromosome 16 between markers D16Mit6 and D16Mit71, a region homologous to human chromosome 21 (18).A human EST corresponding to DYRK2 (accession no. 225301) has been mapped to chromosome 12 (35).The chromosomal localization of DYRK3 is lq32, as determined by fluorescencebased in situ hybridization with a corresponding EST clone (accession no. R38268) (36). The gene responsible for a dominant craniofacial disorder (Van der Woude's syndrome)has been mapped to this region on chromosome 1 (37).However, DYRK3 has been excluded as a candidate (B. Schutte and J. C. Murray, personal communication).
VI. Enzymatic Characteristics of the Dyrk Family
A. Dual-Specificity Protein Kinase Activity Recombinant DyrklA is autophosphorylated and exhibits protein kinase activity toward exogenous substrates, e.g., histone and casein (5).Both the
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WALTER BECKER AND HANS-GEORG JOOST
full-length protein (obtained by transfection of COS-7 cells) and a Cterminally truncated product (from E. coli) were active. Because both serinejthreonine and tyrosine residues are phosphorylated in autophosphorylation reactions, DyrklA was classified as a dual-specificityprotein kinase. A number of protein kinases that do not belong to the family of tyrosine kinases, e.g., members of the LAMMER kinase family, have been found to contain phosphotyrosine when expressed in E. coli (38, 39). So far, no defining sequence motifs for this biochemical feature have been identified, and dualspecificity protein kinases are found in diverse branches of the phylogenetic tree of protein kinases (38, 40). However, dual-specificityprotein kinase activity appears to be a common feature of the Dyrk family because recombinant Mnb, Dyrk2, Dyrk3, and Yakl also contain phosphotyrosine (54.The evolutionary conservation of this feature strongly suggests that dual specificity is important for the natural function of these kinases.
B. Presumed Activation of Dyrk Protein Kinase Activity by Tyrosine Phosphorylation In analogy to other protein kinases (Fig. 6), it appeared possible that the activity of Dyrk is regulated by phosphorylation of the YxY motif. Indeed, mutation of these residues essentialIy inactivated recombinant DyrklA (5). However, attempts to demonstrate phosphorylation or dephosphorylation of DyrklA in transfected COS-7 cells have failed so far (W. Becker, unpublished results). The yeast homolog Yakl has been shown to be activated 50-fold by growth arrest in Go (7). In the same study Yakl was also shown to be phosphorylated by an unidentified kinase.
Growth Factors
Stress (Heat shock, UV) Cytoklnes
1
1
I
1
I [: i% , )
1
Differentiation Prollferation
Stress response
Insulin EGF
?
1
1
1
1
? Glycogen Synthesls Difhrsntiation
FIG.6. Function and signal transduction of protein kinases regulated by phosphorylation in the activationloop. The figure depicts a simplified scheme of known signal transduction pathways, which include a tyrosine-phosphorylated protein kinase (Erk, Jnk, p38, Gsk3). Phosphorylated amino acids are given in one-letter code.
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
15
VII. Potential Cellular Functions of the Dyrk Family of Protein Kinases The biochemical analysis of four prototypes (DyrklA, Dyrk3, Mnb, and Yakl) revealed that members of the Dyrk family catalyze autophosphorylation on tyrosine residues. Thus, in view of the large evolutionary distance between DyrklA and Yakl, the capability for tyrosine autophosphorylation appears to be a general features of the Dyrk family. Furthermore, it has been suggested previously that the activity of Dyrk is modified by phosphorylation of a tyrosine in the activation loop (5).The Dyrk kinases therefore appear to be components of yet unknown signal transduction pathways with activating tyrosine kinases upstream of Dyrk, similar to the ERK/MAP kinase pathway. The exact functional role of the Dyrk-controlled signaling pathway has yet to be defined. However, there are several clues pointing to a role of Dyrk in cell replication and/or differentiation. First, the involvement of the rninibrain mutation in neurogenesis appears to be well established. Furthermore, overexpression of human Dyrk in mice produces learning defects, supporting the hypothesis that the gene for DyrklA is responsible for mental retardation in Down syndrome. It remains to be established, however, why both reduced (in the fly) and enhanced expression (in Down syndrome) cause a “retarded phenotype. Second, the predominant expression of Dyrk2-4 in testis points to a role of these isoforms in spermatogenesis. It should be noted that DyrklA may be involved in spermatogenesis as well, because male Down syndrome patients are infertile. Third, the yeast homolog Yakl has been shown to be a negative regulator of the cell cycle, because its disruption induces growth in a strain arrested by disruption of PKA. Note that there is only one Yak isoform in S. cmeoisiae, and that the Dyrk family has considerably expanded during evolution to at least seven isofoims in humans. Taken together, these pieces of evidence strongly suggest that Dyrk kinases are involved in the control of the cell cycle in differentiating cell or tissues.
VIII. Future Perspectives All lines of available evidence suggest that DyrklA is involved in neuronal development. Thus, future research will start from this hypothesis and will concentrate on its expansion. With respect to the physiologic function of Dyrk, transgenic mice overexpressing a Dyrk isoform as well as null mutants will be the most important models. Second, it is clear that Dyrk is involved in a novel regulatory pathway that resembles others, e.g., the MAP kinase pathway. Elucidation of the key upstream and downstream players in this pathway will be attempted using strategies suitable for identification of in-
16
WALTER BECKER AND HANS-GEORG JOOST
teracting proteins (e.g., two-hybrid screens), and by analysis of cells transfected with the kinases. Third, it will be important to study the molecular basis of the remarkable dual specificity of Dyrk, and the role of its typical structural characteristics, e.g., the DH-box. Here, the experimental approach is a mutational analysis of Dyrk expressed in E. coli or COS-7 cells. Findy, the elucidation of the physiologic functions of Dyrk and its isoforms may help to define their potential involvement in diseases. NOTEADDEDIN PROOF:The kinase termed AD-1 in this review is identical to PKY, a human protein kinase with increased expression in mdtidrug-resistant cells [D. A. Begley, M. B. Berkenpas, K. E. Sampson, and I. Abraham, Gene 200,35-43 (1998)l.
REFERENCES 1. T. Hunter, Cell 50,823 (1987). 2. T.Hunter and G. D. Plowman, Trends Biochem. Sci. 22,18 (1997). 3. S. K. Hanks and T. Hunter, FASEB]. 9,576 (1995). 4. F. Tejedor, X. R. Zhu, E. Kaltenbach, A. Ackermann, A. Baumann, I. Canal, M. Heisenberg, K. F. Fischbach, and 0. Pongs, Neuron 14,287 (1995). 5. H. Kentrup, W. Becker, J. Heukelbach, A. Wilmes, A. Schiirmann, C. Huppertz, H. Kainulainen, and H.-G. Joost,]. Biol. Chem. 271,3488 (1996). 5u. W. Becker, Y.Weber, K. Wetzel, K. Eirmbter, F.J. Tejedor, and H.-G. Joost,]. Biol. Chem., in press (1998). 6. S. Garrett and J. Broach, Genes Deu. 3,1336 (1989). 7. S. Garrett, M. M. Menold, and J. Broach, Mol. Cell. Biol. 11,4045 (1991). 8. P. M. Sinet, D. Theophile, Z. Rahmani, Z. Chettouh, J. L. Blouin, M. Prieur, B. Noel, and J. M. Delabar, Biomed. Phumother. 48,247 (1994). 9. M. K. McCormick, A. Schinzel, M. B. Petersen, G. Stetten, D. J. Driscoll, E. S. Cantu, L. Tranebjaerg, M. Mikkelsen, P. C. Watkins, and S. E. Antonarakis, Genmics 5,325 (1989). 10. Z. Rahmani, J. L. Blouin, N. CrBau-Goldberg,P. C. Watkins, J. F. Mattei, M. Poissonier, M. Priew, Z. Chettouh, A. Nicole, A. Aurias, P. M. Sinet, and J. M. Delabar, A-oc. Natl. Acad. Sci. U.S.A. 86,5958 (1989). 11. J. M. Delabar, D. Thkophile, Z. Rahmani, Z. Chettouh, J. L. Blouin, M. Prieur, B. Noel, and P. M. Sinet, Eur. 1.Hum. Genet. 1, 114 (1993). 12. J. Guimeri, C. Casas, C. Pucharcos, A. Solans, A. Domenech, A. M. Planas, J. Ashley, M. Lovett, X. Estivill, and M. A. Pritchard, Hum. Mol. Genet. 5,1305 (1996). 13. N. Shindoh, J. Kudoh, H. Maeda, A. Yamaki, S. Minoshima, Y. Shimizu, and N. Shimizu, Biochem. Biuphys. Res. Commun. 225,92 (1996). 14. W.-J. Song, L. R. Stemberg, C. Kasten-Sport&,M. L. van Keuren, S.-H. Chung, A. C. Slack, D. E. Miller, T. W. Glover, P.-W. Chiang, L. Lou, and D. M. Kurnit, Genomics 38,331 (1996). 15. D. J. Smith, M. E. Stevens, S. P. Sudanagunta, R. T. Bronson, M. Makhinson, A. M. Watabe, T. J. ODell, J. Fung, H.4. G. Weier,J.-F. Cheng, andE. M. Rubin, Nature Genet. 16,28 (1997). 16. J. D. Thompson, D. G. Higgins, and T. J. Gibson, Nucleic Acids Res. 22,4673 (1994). 17. S. K. Hanks and A. M. Quinn, Methods Enzymol. 200,38 (1991). 18. W.-J. Song, S.-H. Chung, and D. M. Kurnit, Biochem. Biuphys. Res. Commun. 231,640 (1997).
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
17
19. W. H. Landschulz, P. F. Johnson, and S. L. McKnight, Science 243,1681 (1989). 20. T. E. Ellenberger, C. J. Brand, K. Struhl, and S. C. Harrison, Cell 71,1223 (1992). 21. D. R. Knighton, J. H. Zheng, L. F. Eyck, V. A. Ashford, N. H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253,407 (1991). 22. C.-J. Marshall, Cell 80, 179 (1995). 23. L. N. Johnson, M. E. M. Noble, and D. J. Owen, Cell 85,149 (1996). 24. J. Hanes, H. von der Kammer, J. Klaudiny, and K. H. Scheit,]. Mol. Bwl. 244,665 (1994). 25. B. Yun, R. Farkas, K. Lee, and L. Rabinow, Genes Den 8,1160 (1994). 26. J. Bender and G. R. Fink, Proc. Natl. Acud. Sci. U.S.A. 91,12105 (1994). 27. S. K. Alahari, H. Schmidt, and N. F. Kaufer, Nucleic Acids Res. 21,4079 (1993). 28. T. Gross, M. Lutzelberger, H. Wiegmann, A. Klingenhoff, S. Shenoy, and N. F. Kaufer, Nucleic Acids Res. 25,1028 (1997). 29. S. Rogers, R. Wells, and M. Rechsteiner, Science 234,364 (1986). 30. D. I? Feng and R. F. Doolittle,J. MoZ. Euol. 25,351 (1987). 31. K. CoIwill, T. Pawson, B. Andrews, J. Prasad, J. L. Manley, J. C. Bell, and P. I. Duncan, EMBO ]. 15,265 (1996). 32. K. Hughes, E. Nikolakaki, S. E. Plyte, N. F. Totty, and J. R. Woodgett, EMBO]. 12,803 (1993). 33. M. S. Boguski, T. M. Lowe, and C . M. Tolstoshev, Nature G a e t . 4,332 (1993). 34. J. F. Cheng, V. Boyartchuk, and Y. Zhu, Genomics 23,75 (1994). 35. R. Houlgatte, R. Mariage-Samson, S. Duprat, A. Tessier, S. Bentolila, B. Lamy, and C. Auffray, Genome Res. 5,272 (1996). 36. S. Banfi, G. Borsani, E. Rossi, L. Bernard, A. Guffanti, F. Rubboli, A. Marchitiello, S. Gigho, E. Coluccia, M. Zollo, 0. Zuffardi, and A. Ballabio, Nature Genet. 13,167 (1996). 37. B. Schutte, A. Sander, M. Malik, and J. C. Murray, Genomics 36,507 (1996). 38. R. A. Lindberg, A. M. Quinn, and T. Hunter, Trends Biochem. Sci. 17,114 (1992). 39. K. Lee, C. Du, M. Horn, and L. Rabinow,]. Bwl. Chem. 271, 27299 (1996). 40. E. M. J. Douville, D. E. H. Afar,B. W. Howell, K. Lehvin, L. Tannock, Y. Ben-David, T. Pawson, and J. C . Bell, Mol. Cell. Biol. 12, 2681 (1992).
Toxikologie 0-52057 Aachen, Germany I. Introduction.
.................................................
XI. Identificationand Nomenclature of the Dyrk/Minibrain Family ......
111. Structural Comparison of the Dyrk Family Members and D e f ~ t i o n of Common Characteristics ..................................... A. Comparison of the Catalytic Domain .......................... B. Comparison of N and C Termini .............................. C. The DH-Box @DDNxDY) ................................... D. Evolutionary Comparison and Relation of the Dyrk Family to Other Kinase Subfamilies ......................................... n! Tissue Distribution, Alternative Splicing, and Polyadenylation of Dyrk mRNA ....................................................... V. Genomic Localization of the Dyrk Kinases ........................ VI. Enzymatic Characteristics of the Dyrk Family ..................... A. Dual-SpecificityProtein Kinase Activity ........................ B. Presumed Activation of Dyrk Protein Kinase Activity by Tyrosine Phosphorylation ............................................ VII. Potential Cellular Functions of the Dyrk Family of Protein Kinases VIII. Futureperspectives ............................................ Note Added in Proof ...................... ................ References ...................................................
2 2
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12 13 13 13
14 15 15
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Dyrk-related kinases represent a novel subfamily of protein kinases with unique structural and enzymatic features. Its members have been identified in distantly related organisms. The yeast kinase, Yakl,has been characterized as a negative regulator of growth. Mnb from Drosophiia is encoded by the minibraingene, whose mutation results in specific defecb in neurogenesis. Its mammahn ’ homolog, DyrklA, is activated by tyrosine phosphorylation in the activation Imp between subdomains VII and VIII of the catalytic domain. The human gene for DyrklA is located in the “Down syndrome critical region” of chromosome 21 and is therefore a candidate gene for mental retardation in Down syndrome. More recently, six additional mammalian Dyrk-relatedb a s e s have been identified (DyrklB, DyrklC, Dyrk2, Dyrk3, Dyrk4A, and Dyrk4B). All members of the Dyrk family contain in the activation Imp Progress in Nudeic Acid Reseaich and Molecular Biology Vol 62
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Copyngfit 0 1999 by Academic Press
AU nghts of reproducbon in any form reserved
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WALTER BECKER AND HANS-GEORG JOOST
the tyrosines that are essential for the full activity of DyrklA. Outside their catalytic domains, Dyrk kinases exhibit little sequence similarity except for a small segment immediately precedingthe catalytic domain (DH-box,Dyrk homology box). An unusual enzymatic property of Dyrk-related kinases is their ability to catalyze tyrosine-directed autophosphorylation as well as phosphorylation of serine/threonine residues in exogenous substrates. The exact cellular function of the Dyrk kinases is yet unknown.However, it appears reasonable to assume that they are involved in the regulation of cellular growth and/or development. B 1999 Academic Press
I. Introduction Protein phosphorylation and dephosphorylation are the predominant mechanisms of signal transduction in eukaryotic cells. The enzymes catalyzing phosphorylation of serine, threonine, or tyrosine residues of proteins provide a high degree of versatility and substrate specificity, and represent one of the largest gene families. On the basis of the numbers of genes encoding protein kinases in the genomes of yeast (113 of a total of 6121 genes) and Caenorhabditis elegans (270 of the approximately 15,000 sequenced genes), the total number of human protein kinases might be estimated to exceed 1000, a number already suggested by Hunter more than 10 years ago (1,2). This superfamily of protein kinases can be further divided into phylogenetically related subfamilies,which in most cases have related functions (e.g.,cell cycle kinases, receptor tyrosine kinases) (3).This article provides a summary of the knowledge on a new protein kinase family, Dyrk, whose prototype has been identified by polymerase chain reaction cloning and by positional cloning. This enzyme, and its yeast and Drosophila homologs, showed striking structural, enzymatic, and functional characteristics. Furthermore, several related mammalian genes have been identified, defining a novel subfamily of protein kinases with unique structural, enzymatic, and probably also functional features that will be summarized herein.
II. Identification and Nomenclature of the DyrklMinibrain Family Dyrkl has been identified independently by three different experimental strategies. First, its Drosophila homolog (Mnb) has been identified by positional cloning of the minibrain mutations (mnb),which exhibit specific behavioral defects and a reduced number of neurons in distinct areas of the brain (optic lobes and central brain hemispheres) (4).Four mutant alleles resulting in reduced expression of minibrain have been found, and it was suggested that the expression of minibrain is required for proliferation of distinct neuroblasts during larval development.
3
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
Second, rat Dyrkl was identified in a polymerase chain reaction (PCR) cloning approach as a dual-specificity protein kinase that autophosphorylates as well as catalyzes the phosphorylation of histone on serinelthreonine and on tyrosine residues (5).Subsequently, six additional mammalian Dyrk-related kinases were identified, namely DyrklB, DyrklC, Dyrk2, Dyrk3, Dyrk4A, and Dyrk4B, defining a distinct family of protein kinases with common structural and functional characteristics (accession nos. Y13493, Y12735, Y09306, and U49952; 54.The acronym “Dyrk (dualspecificity Yak-related ljnase) refers to the unusual ability of these kinases to phosphorylate serinelthreonine and tyrosine residues, and to the significant sequence similarity with the protein kinase Yak1 from Saccharomyces cerevisiae. YAK1 was identified as a functional antagonist of the Ras/PKA pathway, and has been characterized as a negative regulator of growth (6, 7). Like Dyrkl, Dyrk3, and Mnb, recombinant Yak1 is autophosphorylated on tyrosine residues (see below), and is thus considered the yeast homolog of the Dyrk family. Based on the proposed role of the conserved tyrosine residues in the activation loop (see below), “Dyrk can alternatively be read as “dual-specificity tyrosine-phosphorylation regulated ljnase.” Due to the high sequence similarity of the rat Dyrkl cDNA with a human expressed sequence tag (EST)that had been mapped to chromosome 2 1,the gene of a human homolog was localized to 21q22.2 (5). By correlation of phenotype with genotype in patients with partial trisomy 2 1,this region has been defined as the “Down syndrome critical region” (8)(Fig. 1);its triplication appears to be responsible for many features of Down syndrome, including mental retardation (9-11). Indeed, several groups independently identi-
I I
II I II
I
230E8
141z-L?&6 i 152F7tl
I
’
.+; 1
-
1
1
I
c”Down Syndrome Critical Region”+’
FIG.1. L o c i a t i o n of the human DyrklA gene in the Down syndrome critical region of chromosome 21. The Down syndrome critical region spans about 3 Mb of chromosome 21 at 21q22.2 andis definedbythe markers D21S17 (centromericborder),D21S55, and ERG (telomeric border) (8). The position of selected YACs in this region is indicated (230E8,141G6,152F7, 336(311,28SE6). D YRKl A maps to YACs 152F7 and 336G11(12,14,34).Smith et d.(15)identified the DYRKlA gene on a 180-kb telomeric fragment of 1S2F7 (tel).
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WALTER BECKER A N D HANS-GEORG JOOST
fied the human Dyrkl gene by screening the Down syndrome critical region for genes whose triplication may cause defects in neurogenesis (12-14). Because of its high degree of similarity to Mnb, the DYRK1 gene was an obvious candidate for phenotypic manifestations of Down syndrome. This hypothesis was supported by the finding that mice transgenic with a 180-kb fragment of human chromosome 21 including the DYRK1 gene (Fig. 1) showed defects in learning tasks (15).
111. Structural Comparison of the Dyrk Family Members and Definition of Common Characteristics
A. Comparison of the Catalytic Domain 1. CONSERVED RESIDUES AND MOTIFS
To date, sequences of 14 Dyrk-related kinases (Table I> are available for sequence comparisons, including several sequences that were identified in genome sequencing projects of fission yeast (Schixosaccharomycespornbe), the roundworm Caenorhabditis elegans, and Drosophila. A comparison of these sequences with the CLUSTAL W program (16)allows a definition of the common structural features of the Dyrk family (Fig. 2), and of its differences with regard to other protein kinase subfamilies. In addition to the sequences compared in Fig. 2, two cDNA fragments with high similarity to Dyrkl, and one fragment with high similarity to Dyrk4, have been identified (5a).These sequences were designated DyrklB, DyrklC, and Dyrk4B. They will not further be discussed here. However, Dyrkl will be referred to as DyrklA throughout. All Dyrk-related kinases contain a canonical kinase domain of 297-335 amino acids with the conserved regions as defined by Hanks and Quinn (17) (subdomains I-XI in Fig. 2).In addition, the sequences exhibit a number of features that are not (or rarely) found in other kinases and are therefore characteristic for the Dyrk family (indicated by boxes in the sequence alignment). Three specific sequence motifs are found in the vicinity of the catalyhc cleft (subdomains VIB-IX). First, in all kinases of the Dyrk family, the conserved DFG motif of subdomain VII is followed by the amino acids serine, serine, and cysteine (SSC). Second, the arginine residue in subdomain VIB, which is highly conserved in all other kinases, is substituted for cysteine in the Dyrk family. Third, the YxY motif between subdomains VII and VIII (YxYIQ) is specific for the Dyrk family. In addition, there are several other conserved amino acids (boxed in Fig. 2) that are characteristic for the Dyrk family: a cysteine between subdomains IV and V, the NLY motif in subdomain V, and a glycine between subdomains VIII and IX. Finally, Dyrk-related kinases
5
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
TABLE I COMPILATION OF SEQUENCES ENCODINC DYRK-RELATED K~NASES Gene product
Species
DyrklA DyrklA DyrklA DyrklA DyrklA DyrklA Dyrk2 Dyrk3 Dyrk4 AD-1 AD-1 Mnb 34dc6z
Mouse Human Human Human Human Human Drosophila Drosophila
T04c10 F4 9 9e 11 KAB7 KA23 Yak1
Caenorhabditis eleguns Cuenurhubditis elegans Schizosaccharomyces pombe Schizosucchuromycespombe Sacchumyces ceruisim
Human Human Human Human Rat
Accession no. U52373 D85759 U58497" D86550 X79769 U58796 Y13493 Y12735 YO9305 YO9306 U57894 X70794 L43478 L43480 269885 270308 254354 250142 X16056
Type
Ref.
cDNA cDNA cDNA cDNA cDNA cDNA cDNA cDNA cDNAb CDNA~ CDNA" Gene Geneb,' Geneh." Gene" Gene" Gene' Gene' Gene
12 13 14
5 14 -
"This database entry represents a fusion of the muline and the human sequences. "The nucleotide sequence encodes a partial open reading frame. <:Thesequence has been determined in a genome sequencing project.
contain an unusual insertion of six amino acids between subdomains I11 and lV. The protein kinase AD-1 was included in the comparison in order to further illustrate the defining features of the Dyrk family. AD-1 is a close relative of Dyrkl but is not considered a member of the Dyrk family, because three of the above described conserved motifs are not found in its sequence: the cysteine in VIB, the SSC in VII, and the YxY motif. However, it shares several other characteristics of the Dyrk family, e.g., a glutamine in the sequence before subdomain VIII (YIQSRFY), where most other kinases possess a s m d , nonpolar residue (3). Song et d.(14) have suggested that a motif of four leucine residues (Leu274, Leu-281, Leu-288, and Leu-295) might form aleucine zipper structure. They tested the proposed ability to mediate proteinjprotein interactions with the help of the two-hybrid system, and demonstrated dimerization of DyrklA (18). However, the respective leucine residues are conserved in a large pro-
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WALTER BECKER AND HANS-GEORG JOOST
FIG.2. Sequence alignment of the catalytic domains of Dyrk kinases. The deduced amino acid sequences of the catalytic domains were aligned with the help of the CLUSTAL W program (26). Hyphens represent gaps introduced for optimal alignment. The kinase subdomains according to the nomenclature of Hanks and Quinn (17) are indicated by Roman numerals at the top of the alignment. Residues conserved in all or nearly all protein kinases [according to Hanks and Hunter (3)]are in black boxes. Amino acids that are conserved in the Dyrk family but are very rare at this position in other kinases are highlighted by shaded boxes. Asterisks indicate amino acids identical in all compared sequences; periods designate conservative substitutions. Arrows point to s i g d c a n t differences between AD-1 and the Dyrk family. Sequence sources (EM, EMBLGenBank; others, SwissProt database):DyrklA, rat, EM:X79769; Dyrk2, Dyrk3, and Dyrk4, human, EM:Y13493, EM:Y12735, and EM:Y09305; Mnb and 34dc6z, Drosophila, P49657 and EM:L43478/L43480; T04c10 and F49el1, Caenmhabditis elegans, EM:Z69885 and 270308; KAB7 and KA23, Schizosaccharomyces pombe, Q09815 and Q09690; Yakl, Saccharornyces cerevisiae, P14680; and AD-1, human, EM:AF004849.
7
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
Dyrkl Mnb TO4clO Dyrk2 Dyrk3 F49ell 34dc6z Dyrk4 KA23
KAB7
Yakl AD-1
Dyrkl Mnb T04c10 Dyrk2 Dyrk3 F49ell 34dc6z Dyrk4 KA23 KAB7
Yakl AD-1
Dyrkl Mnb T04c10 Dyrk2 Dyrk3 F49ell 34dc6z Dyrk4 KA23
- - - - - - - - HTVADY LKFKDLILRMLDYD
XI
.--HSVSDYLKFKDLILRMLDFD - - - - - - - - HSVEDYSKFKDLIKRMLQFD - --- --.- - -- -- LKGCDDPLFLDFLKQCLEWD -LKGCDJYLFIEFLKXCLSXD - - - - - - - - LKNMGDELFVDFLKRCLDWD
- - --- - --
KAB7
- -- - - - - -- --- - - -
AD-1
DLEGSDLLAEKADRREFVSLLKKMLLIDADI&TP~~ETLNHPFV
Yakl
.
* .* * .
*. .
FIG.2 (Continued).
portion of all protein kinases (3).Furthermore, the definition of the leucine zipper motif requires that the leucine residues are located within a single ahelix (19,20).Based on the tertiary structure of other kinases, the four leucine residues are located within different structural elements (&-helixE, P-strand 7, and the catalytic loop) (24. Thus, the putative dimerization of DyrklA is probably not mediated by the proposed leucine zipper. 2. THEYxY MOTIFIN THE ACTIVATION LOOP The position of the conserved YxY motif between subdomains VII and VIII corresponds exactly with that of the TxY motif in the MAP kinases, which are activated by phosphorylation of these residues (22). Activating phosphorylation of serine, threonine, and/or tyrosine residues in this so-
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WALTER BECKER AND HANS-GEORG JOOST
called “activation loop’’ is a common mechanism in many protein kinases (23) (Fig. 6). Indeed, exchange of these tyrosines for phenylalanine almost completely suppressed the activity of recombinant DyrklA (5). Based on the structure of phosphorylated CAMP-dependentkinase (PKA) (21),a general mechanism of the activation of protein kinases by phosphorylation was proposed by Johnson d al. (23).In their model, the binding of the phosphate in the activation loop to specific positively charged residues stabilizes an active conformation of the enzyme. These arginines, i.e., an arginine in subdomain VIB (YRDLKPEN in PKA) and a lysine in subdomain VII (DFGFAKin PKA),are conserved in many kinases that are activated by phosphorylation (23).Strikingly, the Dyrk family kinases contain the characteristic cysteine residues at the equivalent positions (see above). Thus, the role of the phosphotyrosine must differ from that of the phosphothreonine in PKA.
B. Comparison of N and C Termini In all kinases of the Dyrk family, the catalytic domain is located between N-terminal and C-terminal regions of variable length (99-699 and 22-424 amino acids) (Fig. 3). Outside the catalytic domain, kinases of the Dyrk family show no extended regions of sequence similarity to any other known protein, and the noncatalytic regions of these kinases are not generally related within the Dyrk family. Sequence similarity is apparent only in closely related members of the family, such as Dyrk2 and Dyrk3 (Fig. 3), and in species homologs, e.g., mammalian Dyrkl A, Drosophila Mnb, and Caenorhabdicis T04c10.
FIG.3. Schematic presentation of the structures of the known mammalian Dyrk kinases. Outside the catalytic domain (cat), black bars indicate sequence regions without similarity to other proteins. Homologous regions in Dyrk2 and Dyrk3 are depicted as open bars. A small segment of the N-terminal region that is conserved in all kinases of the Dyrk family is symbolized by an open box in front of the catalytic domain. The positions of different structural motifs in the sequence of DyrklA are given (0,nuclear localization signal, NLS; PEST region; H, histidine repeat; S, serineithreonine repeat). Only partial sequence information is available for Dyrk4.
+
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
9
FIG.4. The DH-box (DDDNxDY): a conserved sequence motif preceding the catalytic domain in the Dyrk family and related kinases. Related sequences were identified in a protein database search performed with residues 135-180 of DyrklA (accession no. X79769), which include the DH-box and subdomain I of the catalytic domain. Conservation of amino acids is highlighted by shaded boxes. Invariable amino acids are shown in boldface. The number of amino acids between the last residue of the alignment and the first residue of the catalytic domain is given in brackets. In the consensus sequence, amino acids that are given are found at this position in at least six of the nine sequences. Periods in the consensus sequences designate nonconserved residues. The consensus sequence for the C k family has been derived from seven different kinases (24-26); the consensus for Prp4 has been deduced from three homologs [mouse, 5’. pombe, and C. eleguns (27),and database entries U48737 and 2712621.
C. The DH-Box (DDDNxDY) A short stretch of amino acids immediately preceding the catalytic domain appears to be conserved in all kinases of the Dyrk family. Because of its potential importance (see below), we designated this motif DH-box @yrk homology box). As is illustrated in Fig. 4, the core of this stretch appears particularly well conserved within the family; its consensus sequence is DDDNxDY. This observation prompted us to analyze other kinases for the presence of related motifs. Indeed, a similar motif was identified in the kinases of the Clk family (also called LAMMER kinases, because of the conserved signature motif EHLAMMERILG in subdomain X, including mammalian Clkl-3, Doa from Drosophila, and AFC1-3 from Arabidopsis) (24-26) and in Prp4 (27, 28). The consensus sequences of the motif differ somewhat among the three groups of kinases (Fig. 4). However, two aspartic acid residues followed by a third acidic residue are conserved in the Clk family and in Prp4. Furthermore, a tyrosine and an acidic residue (boxed in Fig.
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WALTER BECKER AND HANS-GEORG JOOST
4) appear conserved. Interestingly, mutations of one or both of the aspartic acid residues in Prp4 cause the functional inactivation of this gene in S. pombe (28).However, the DH-box is not essential for the enzymatic activity of recombinant DyrklA because an N-terminally truncated construct exhibits full enzymatic activity (W, Becker and H.-G. Joost, unpublished data). In the absence of further functional evidence, it can only be speculated that the DH-box represents a site of interaction with the substrates or with other proteins, and thereby regulates the kinase activity. In addition to the DH-box, some kinases of the Dyrk family harbor striking sequence motifs in their noncatalytic regions. Putative nuclear localization signals have been identified in the amino-terminal regions of Mnb and DyrklA (4,5).Indeed, a construct of the N-terminal region of DyrklA fused to green fluorescent protein is detected in the nucleus of transfected mammalian cells (Sa, 18). Immediately following the catalytic domain, DyrklA contains a region rich in proline, glutamic acid, serine, and threonine (PEST) residues (Fig. 3). Such PEST regions are believed to represent signals for a rapid turnover of the protein (29).A region rich in glycine, alanine, and serine (GAS domain) was identified in the carboxy-terminaldomains of Mnb (4).A GAS sequence has also been detected in the Drosophila sgg protein kinase. Several members of the Dyrk family harbor striking repeat structures in their noncatalyhc domains. DyrklA contains stretches of 13 consecutive histidines and 17 subsequent serine/threonine residues in its carboxy-terminal regions. Yak1 and T04c10 harbor repeats of 12 and 9 glutamine residues, respectively. Although histidine and glutamine repeats are frequently found in transcription factors, their exact function has not yet been elucidated.
D. Evolutionary Comparison and Relation of the Dyrk Family to Other Kinase Subfamilies The relationship of the Dyrk family to other kinases can be deduced from a sequence comparison of their catalytic domains. In Fig. 5, the results of such a sequence alignment are illustrated as a phylogenetic tree constructed with the TREE program (30). According to the classification of Hanks and Hunter (3),the Dyrk family belongs to the CMGC group of protein kinases, which includes the families of the cyclin-dependent kinases, the MAP kinases, the GSK3 family, and the Clk family (also called LAMMER kinases) (25). The closest relatives of the Dyrk family are the Clk kinases and Prp4; these kinases appear to be involved in the regulation of RNA synthesis and splicing (25,27,31). Interestingly,Clkl/STY have also been shown to exhibit dual specificity when expressed in Escherichia coli. There are several kinases in the CMGC group that contain a tyrosine residue in the activation loop (marked by asterisks in Fig. 5 ). However, activating phosphorylation of this
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
11
Dyrk 1 Mnb*(D.m.) T04cl0* (C.O.) AD- 1 F20b6+ (C.O.) Yakl*(S.c.) Prp4* Clk2
I
I
Cdk4 Kklalro' Make Gsk3@*
FIG.5. Relationship between the Dyrk family and other protein kinases. The dendrograrn was constructed with the TREE program (30) on the basis of a sequence alignment of the catalytic domains. Source organisms are indicated for nonmammalian sequences (Ce., Caenarhab&.s elegans; Dm., Drosophila melanogaster; S.C.,Saccharomyces cerevisiae; S.P., Schizosaccharomyces pornbe).Asterisks indicate kinases with a tyrosine residue in the activation loop.
tyrosine has so far been demonstrated only for the MAP kinases (Erkl12,Jnk, and p38) and GSK3 (22,32). The tree in Fig. 5 also illustrates the relationship of the kinases within the Dyrk family. The branching pattern suggests that DyrklA, Mnb, and T04c10 are species homologs of mammals, Drosophila, and Caenorhabditis, respectively. The other main branch of the tree also contains kinases from invertebrates and mammals (Dyrk2, Dyrk3, F49el1, and 34dc6z). Hence, the last common ancestor of Drosophila, Caenorhabditis, and mammals possessed at least two genes for Dyrk-related kinases. In contrast, Yak1 is the only member of the Dyrk family in the yeast Saccharomyces cerevisiae. Furthermore, it is noteworthy that this gene is not essential for viability (6).
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WALTER BECKER AND HANS-GEORG JOOST
As a result of sequencing projects, partial sequences of two different Dyrk-relatedkinases of Arabidopsis thaliana were published in the EST databases (accession nos. N65563 and R84192). The presence of Dyrk-related kinases in plants, fungi, and animals suggests that these enzymes regulate a fundamental function of the eukaryotic cell.
IV. Tissue Distribution, Alternative Splicing, and Polyadenylationof Dyrk mRNA mRNA of DyrklA has been found in all human, murine, and rat tissues studied ( 5 1 2 - 1 4 ) . The distribution of DyrklA mRNA in brain has been studied by Northern blot analysis using human brain (13) and by in situ hybridization using embryonic mouse brain (14) and adult mouse brain (12). The data obtained from these studies differ considerably but indicate that DyrklA is widely expressed in most regions of the brain. In striking contrast to DyrklA, the other members of the family (DyrklB, Dyrk2-4) and the related AD-1A and AD-1B were found to be predominantly expressed in rat testis. However, their transcripts are not absolutely testis specific, because cDNA clones for Dyrk2, Dyrk3, and Dyrk4 ( 5 4 have been isolated from a human fetal brain cDNA library. Furthermore, the EST database (33) contains a considerable number of expressed sequence tags for Dyrk2-4 and AD-1 that were derived from organs other than testis. Particularly, fetal tissues (brain, liver/spleen) were the sources of many of these ESTs. Thus, it seems reasonable to assume that the kinases of the Dyrk family may also be expressed during embryonal development. Alternative splicing of DyrklA mRNA has been observed in rat tissues (5).Two cDNA clones that were identified differed by an in-frame insertion of 2 7 nucleotides, and mRNA corresponding to both splicing products has been detected by PCR in several tissues. Both transcripts appear to be generated also in human tissue (longer form, U52373; short form, D85759). Analysis of the human gene reveals that the inserted segment is indeed located at an exon/intron border (13).It should be noted that the conservation of the insertion in rat and human mRNA points to a functional role of the two splicing variants. In rodents, DyrklA is encoded by two alternatively polyadenylated mRNAs of approximately 3 and 6 kb (5,12).Human tissues contain only the larger transcript (12, 13; W. Becker and H.-G Joost, unpublished), probably due to a difference of a single base in the sequence of the putative polyadenylation signal of the rate mRNA (AATAAA vs. AGTAAA; nucleotide 2759 in the rat cDNA sequence, accession no. X79769). Alternative splicing and polyadenylation were also shown for Dyrk2. A
STRUCTURE AND FUNCTION O F DYRK PROTEIN KINASES
13
transcript that was isolated contained a 149-bp insertion in the 5' flanking region of the open reading frame; this insertion appeared to extend the coding region by 73 codons. Furthermore, the human gene generates at least three differently polyadenylated transcripts: one with only a 184-bp 3' untranslated region (UTR), one with an additional 1461-bp 3' UTR, and one with an even longer 3' UTR as predicted from the sequences of several expressed sequence tags (accession nos. R63 190, R63622, R25341, and 225301) ( 5 4 . Expression of the mnb gene during development of Drosophila has been studied by immunocytochemistry (4). In the larval brain (third instar larva), protein levels are highest in areas where neuronal progeny is generated during postembryonal development (outer proliferation centers of the optic lobes, mushroom body neuropils). Highest expression in adult flies was observed in mushroom bodies and retinal pigment cells, whereas expression in the optic lobes was low. Thus, the embryonal pattern of expression correlates with the defects of the mutant flies (reduced number of neurons in the optic lobes), but the sites of expression in the adult flies are not visibly affected by mnb mutations.
V. Genomic Localization of the Dyrk Kinases As was discussed earlier,human DYRKlA is located on 21q22.2 at the STS marker D21S70 (YAC clones 152F7 and 336G11) (12,14,34). The most precise localization is given by Smith et aZ. (15), who identifed the gene on a 180-kb telomeric fragment of the YAC 152F7. Murine DyrklA was mapped to chromosome 16 between markers D16Mit6 and D16Mit71, a region homologous to human chromosome 21 (18).A human EST corresponding to DYRK2 (accession no. 225301) has been mapped to chromosome 12 (35).The chromosomal localization of DYRK3 is lq32, as determined by fluorescencebased in situ hybridization with a corresponding EST clone (accession no. R38268) (36). The gene responsible for a dominant craniofacial disorder (Van der Woude's syndrome)has been mapped to this region on chromosome 1 (37).However, DYRK3 has been excluded as a candidate (B. Schutte and J. C. Murray, personal communication).
VI. Enzymatic Characteristics of the Dyrk Family
A. Dual-Specificity Protein Kinase Activity Recombinant DyrklA is autophosphorylated and exhibits protein kinase activity toward exogenous substrates, e.g., histone and casein (5).Both the
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WALTER BECKER AND HANS-GEORG JOOST
full-length protein (obtained by transfection of COS-7 cells) and a Cterminally truncated product (from E. coli) were active. Because both serinejthreonine and tyrosine residues are phosphorylated in autophosphorylation reactions, DyrklA was classified as a dual-specificityprotein kinase. A number of protein kinases that do not belong to the family of tyrosine kinases, e.g., members of the LAMMER kinase family, have been found to contain phosphotyrosine when expressed in E. coli (38, 39). So far, no defining sequence motifs for this biochemical feature have been identified, and dualspecificity protein kinases are found in diverse branches of the phylogenetic tree of protein kinases (38, 40). However, dual-specificityprotein kinase activity appears to be a common feature of the Dyrk family because recombinant Mnb, Dyrk2, Dyrk3, and Yakl also contain phosphotyrosine (54.The evolutionary conservation of this feature strongly suggests that dual specificity is important for the natural function of these kinases.
B. Presumed Activation of Dyrk Protein Kinase Activity by Tyrosine Phosphorylation In analogy to other protein kinases (Fig. 6), it appeared possible that the activity of Dyrk is regulated by phosphorylation of the YxY motif. Indeed, mutation of these residues essentialIy inactivated recombinant DyrklA (5). However, attempts to demonstrate phosphorylation or dephosphorylation of DyrklA in transfected COS-7 cells have failed so far (W. Becker, unpublished results). The yeast homolog Yakl has been shown to be activated 50-fold by growth arrest in Go (7). In the same study Yakl was also shown to be phosphorylated by an unidentified kinase.
Growth Factors
Stress (Heat shock, UV) Cytoklnes
1
1
I
1
I [: i% , )
1
Differentiation Prollferation
Stress response
Insulin EGF
?
1
1
1
1
? Glycogen Synthesls Difhrsntiation
FIG.6. Function and signal transduction of protein kinases regulated by phosphorylation in the activationloop. The figure depicts a simplified scheme of known signal transduction pathways, which include a tyrosine-phosphorylated protein kinase (Erk, Jnk, p38, Gsk3). Phosphorylated amino acids are given in one-letter code.
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
15
VII. Potential Cellular Functions of the Dyrk Family of Protein Kinases The biochemical analysis of four prototypes (DyrklA, Dyrk3, Mnb, and Yakl) revealed that members of the Dyrk family catalyze autophosphorylation on tyrosine residues. Thus, in view of the large evolutionary distance between DyrklA and Yakl, the capability for tyrosine autophosphorylation appears to be a general features of the Dyrk family. Furthermore, it has been suggested previously that the activity of Dyrk is modified by phosphorylation of a tyrosine in the activation loop (5).The Dyrk kinases therefore appear to be components of yet unknown signal transduction pathways with activating tyrosine kinases upstream of Dyrk, similar to the ERK/MAP kinase pathway. The exact functional role of the Dyrk-controlled signaling pathway has yet to be defined. However, there are several clues pointing to a role of Dyrk in cell replication and/or differentiation. First, the involvement of the rninibrain mutation in neurogenesis appears to be well established. Furthermore, overexpression of human Dyrk in mice produces learning defects, supporting the hypothesis that the gene for DyrklA is responsible for mental retardation in Down syndrome. It remains to be established, however, why both reduced (in the fly) and enhanced expression (in Down syndrome) cause a “retarded phenotype. Second, the predominant expression of Dyrk2-4 in testis points to a role of these isoforms in spermatogenesis. It should be noted that DyrklA may be involved in spermatogenesis as well, because male Down syndrome patients are infertile. Third, the yeast homolog Yakl has been shown to be a negative regulator of the cell cycle, because its disruption induces growth in a strain arrested by disruption of PKA. Note that there is only one Yak isoform in S. cmeoisiae, and that the Dyrk family has considerably expanded during evolution to at least seven isofoims in humans. Taken together, these pieces of evidence strongly suggest that Dyrk kinases are involved in the control of the cell cycle in differentiating cell or tissues.
VIII. Future Perspectives All lines of available evidence suggest that DyrklA is involved in neuronal development. Thus, future research will start from this hypothesis and will concentrate on its expansion. With respect to the physiologic function of Dyrk, transgenic mice overexpressing a Dyrk isoform as well as null mutants will be the most important models. Second, it is clear that Dyrk is involved in a novel regulatory pathway that resembles others, e.g., the MAP kinase pathway. Elucidation of the key upstream and downstream players in this pathway will be attempted using strategies suitable for identification of in-
16
WALTER BECKER AND HANS-GEORG JOOST
teracting proteins (e.g., two-hybrid screens), and by analysis of cells transfected with the kinases. Third, it will be important to study the molecular basis of the remarkable dual specificity of Dyrk, and the role of its typical structural characteristics, e.g., the DH-box. Here, the experimental approach is a mutational analysis of Dyrk expressed in E. coli or COS-7 cells. Findy, the elucidation of the physiologic functions of Dyrk and its isoforms may help to define their potential involvement in diseases. NOTEADDEDIN PROOF:The kinase termed AD-1 in this review is identical to PKY, a human protein kinase with increased expression in mdtidrug-resistant cells [D. A. Begley, M. B. Berkenpas, K. E. Sampson, and I. Abraham, Gene 200,35-43 (1998)l.
REFERENCES 1. T. Hunter, Cell 50,823 (1987). 2. T.Hunter and G. D. Plowman, Trends Biochem. Sci. 22,18 (1997). 3. S. K. Hanks and T. Hunter, FASEB]. 9,576 (1995). 4. F. Tejedor, X. R. Zhu, E. Kaltenbach, A. Ackermann, A. Baumann, I. Canal, M. Heisenberg, K. F. Fischbach, and 0. Pongs, Neuron 14,287 (1995). 5. H. Kentrup, W. Becker, J. Heukelbach, A. Wilmes, A. Schiirmann, C. Huppertz, H. Kainulainen, and H.-G. Joost,]. Biol. Chem. 271,3488 (1996). 5u. W. Becker, Y.Weber, K. Wetzel, K. Eirmbter, F.J. Tejedor, and H.-G. Joost,]. Biol. Chem., in press (1998). 6. S. Garrett and J. Broach, Genes Deu. 3,1336 (1989). 7. S. Garrett, M. M. Menold, and J. Broach, Mol. Cell. Biol. 11,4045 (1991). 8. P. M. Sinet, D. Theophile, Z. Rahmani, Z. Chettouh, J. L. Blouin, M. Prieur, B. Noel, and J. M. Delabar, Biomed. Phumother. 48,247 (1994). 9. M. K. McCormick, A. Schinzel, M. B. Petersen, G. Stetten, D. J. Driscoll, E. S. Cantu, L. Tranebjaerg, M. Mikkelsen, P. C. Watkins, and S. E. Antonarakis, Genmics 5,325 (1989). 10. Z. Rahmani, J. L. Blouin, N. CrBau-Goldberg,P. C. Watkins, J. F. Mattei, M. Poissonier, M. Priew, Z. Chettouh, A. Nicole, A. Aurias, P. M. Sinet, and J. M. Delabar, A-oc. Natl. Acad. Sci. U.S.A. 86,5958 (1989). 11. J. M. Delabar, D. Thkophile, Z. Rahmani, Z. Chettouh, J. L. Blouin, M. Prieur, B. Noel, and P. M. Sinet, Eur. 1.Hum. Genet. 1, 114 (1993). 12. J. Guimeri, C. Casas, C. Pucharcos, A. Solans, A. Domenech, A. M. Planas, J. Ashley, M. Lovett, X. Estivill, and M. A. Pritchard, Hum. Mol. Genet. 5,1305 (1996). 13. N. Shindoh, J. Kudoh, H. Maeda, A. Yamaki, S. Minoshima, Y. Shimizu, and N. Shimizu, Biochem. Biuphys. Res. Commun. 225,92 (1996). 14. W.-J. Song, L. R. Stemberg, C. Kasten-Sport&,M. L. van Keuren, S.-H. Chung, A. C. Slack, D. E. Miller, T. W. Glover, P.-W. Chiang, L. Lou, and D. M. Kurnit, Genomics 38,331 (1996). 15. D. J. Smith, M. E. Stevens, S. P. Sudanagunta, R. T. Bronson, M. Makhinson, A. M. Watabe, T. J. ODell, J. Fung, H.4. G. Weier,J.-F. Cheng, andE. M. Rubin, Nature Genet. 16,28 (1997). 16. J. D. Thompson, D. G. Higgins, and T. J. Gibson, Nucleic Acids Res. 22,4673 (1994). 17. S. K. Hanks and A. M. Quinn, Methods Enzymol. 200,38 (1991). 18. W.-J. Song, S.-H. Chung, and D. M. Kurnit, Biochem. Biuphys. Res. Commun. 231,640 (1997).
STRUCTURE AND FUNCTION OF DYRK PROTEIN KINASES
17
19. W. H. Landschulz, P. F. Johnson, and S. L. McKnight, Science 243,1681 (1989). 20. T. E. Ellenberger, C. J. Brand, K. Struhl, and S. C. Harrison, Cell 71,1223 (1992). 21. D. R. Knighton, J. H. Zheng, L. F. Eyck, V. A. Ashford, N. H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253,407 (1991). 22. C.-J. Marshall, Cell 80, 179 (1995). 23. L. N. Johnson, M. E. M. Noble, and D. J. Owen, Cell 85,149 (1996). 24. J. Hanes, H. von der Kammer, J. Klaudiny, and K. H. Scheit,]. Mol. Bwl. 244,665 (1994). 25. B. Yun, R. Farkas, K. Lee, and L. Rabinow, Genes Den 8,1160 (1994). 26. J. Bender and G. R. Fink, Proc. Natl. Acud. Sci. U.S.A. 91,12105 (1994). 27. S. K. Alahari, H. Schmidt, and N. F. Kaufer, Nucleic Acids Res. 21,4079 (1993). 28. T. Gross, M. Lutzelberger, H. Wiegmann, A. Klingenhoff, S. Shenoy, and N. F. Kaufer, Nucleic Acids Res. 25,1028 (1997). 29. S. Rogers, R. Wells, and M. Rechsteiner, Science 234,364 (1986). 30. D. I? Feng and R. F. Doolittle,J. MoZ. Euol. 25,351 (1987). 31. K. CoIwill, T. Pawson, B. Andrews, J. Prasad, J. L. Manley, J. C. Bell, and P. I. Duncan, EMBO ]. 15,265 (1996). 32. K. Hughes, E. Nikolakaki, S. E. Plyte, N. F. Totty, and J. R. Woodgett, EMBO]. 12,803 (1993). 33. M. S. Boguski, T. M. Lowe, and C . M. Tolstoshev, Nature G a e t . 4,332 (1993). 34. J. F. Cheng, V. Boyartchuk, and Y. Zhu, Genomics 23,75 (1994). 35. R. Houlgatte, R. Mariage-Samson, S. Duprat, A. Tessier, S. Bentolila, B. Lamy, and C. Auffray, Genome Res. 5,272 (1996). 36. S. Banfi, G. Borsani, E. Rossi, L. Bernard, A. Guffanti, F. Rubboli, A. Marchitiello, S. Gigho, E. Coluccia, M. Zollo, 0. Zuffardi, and A. Ballabio, Nature Genet. 13,167 (1996). 37. B. Schutte, A. Sander, M. Malik, and J. C. Murray, Genomics 36,507 (1996). 38. R. A. Lindberg, A. M. Quinn, and T. Hunter, Trends Biochem. Sci. 17,114 (1992). 39. K. Lee, C. Du, M. Horn, and L. Rabinow,]. Bwl. Chem. 271, 27299 (1996). 40. E. M. J. Douville, D. E. H. Afar,B. W. Howell, K. Lehvin, L. Tannock, Y. Ben-David, T. Pawson, and J. C . Bell, Mol. Cell. Biol. 12, 2681 (1992).