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Laguna, A., de la Luna, S. DYRK family of protein kinases: evolutionary relationships, biochemical proper- ties, and functional roles. FASEB J. 25, 449–462 ...
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DYRK family of protein kinases: evolutionary relationships, biochemical properties, and functional roles Sergi Aranda,*,†,1 Ariadna Laguna,*,†,2 and Susana de la Luna*,†,‡,3 *Center for Genomic Regulation, University Pompeu Fabra, Barcelona, Spain; †Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras, Barcelona, Spain; and ‡Institucio´ Catalana de Recerca i Estudis Avanc¸ats, Barcelona, Spain Dual-specificity tyrosine-regulated kinases (DYRKs) comprise a family of protein kinases within the CMGC group of the eukaryotic kinome. Members of the DYRK family are found in 4 (animalia, plantae, fungi, and protista) of the 5 main taxa or kingdoms, and all DYRK proteins studied to date share common structural, biochemical, and functional properties with their ancestors in yeast. Recent work on DYRK proteins indicates that they participate in several signaling pathways critical for developmental processes and cell homeostasis. In this review, we focus on the DYRK family of proteins from an evolutionary, biochemical, and functional point of view and discuss the most recent, relevant, and controversial contributions to the study of these kinases.—Aranda, S., Laguna, A., de la Luna, S. DYRK family of protein kinases: evolutionary relationships, biochemical properties, and functional roles. FASEB J. 25, 449 – 462 (2011). www.fasebj.org

ABSTRACT

Key Words: cell differentiation 䡠 cell survival 䡠 endocytosis 䡠 phosphorylation

Protein phosphorylation has been extensively studied for the past 60 yr and is regarded as the most widespread post-translational modification in the proteome. Protein kinases are one of the largest superfamilies of proteins in eukaryotes, and given the effect of phosphorylation on protein function, they have historically emerged as key enzymes for the regulation of basic cellular processes (1). Human protein kinases are classified into a hierarchy of groups, families, and subfamilies based on the taxonomic classification of Hanks and Hunter (2). Dualspecificity tyrosine-regulated kinases, or dual-specificity yak-related kinases (DYRKs), belong to the CMGC group, which also includes cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinase-3 (GSK3), CDK-like kinases, serine/arginine-rich protein kinases, cdc2-like kinases, and RCK kinases. Homology within the kinase domain allows for the division of the DYRK family into 3 subfamilies: DYRK kinases, homeodomain-interacting protein kinases (HIPKs), and pre-mRNA processing 0892-6638/11/0025-0449 © FASEB

protein 4 kinases (PRP4s) (Fig. 1A). Within the DYRK subfamily, 3 groups are distinguished: the Yak group, with no members in animals, and the DYRK1 and DYRK2 groups (Fig. 1). For practical purposes, DYRK will herein refer to the subfamily of DYRK proteins. Since the discovery of Yak1p (3), the founding member of the DYRK family, in budding yeast some 20 yr ago, DYRK family members have been found in all eukaryotes (Fig. 1), and studies of their function have placed different members of this family as key players in a great variety of cellular processes. Here, we offer a review on mammalian DYRK kinases, highlighting both the commonalities within the family and the aspects that lead to biological differences among distinct members. We also present a short overview of the family members in lower eukaryotes, in the worm and in Drosophila, which may help in understanding the mammalian DYRK kinases from an evolutionary viewpoint.

DYRK IN LOWER EUKARYOTES Two DYRK proteins have been widely characterized in yeast, Yak1p in Saccharomyces cerevisiae and Pom1p in Schizosaccharomyces pombe. These two kinases belong to two distinct branches within the DYRK subfamily (Fig. 1A). A third DYRK family member studied in lower eukaryotes is Dictyostelium discoideum YakA, which clusters with S. cerevisiae Yak1p. In general, the roles of these kinases are related to the control of the cell cycle, cytokinesis, and cell differentiation. Yak1p is a negative regulator of proliferation under nutritional stress, antagonizing RAS/cAMP (3) and 1

Current address: Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden. 2 Current address: Ludwig Institute for Cancer Research Ltd., Stockholm Branch, SE-171 77 Stockholm, Sweden. 3 Correspondence: Center for Genomic Regulation-CRG, Dr. Aiguader, 88, 08003-Barcelona, Spain. E-mail: [email protected] doi: 10.1096/fj.10-165837 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 449

Figure 1. DYRK family of proteins. A) Unrooted phylogenetic tree showing the evolutionary divergence of the kinase domain in the different DYRK family members. The degree of conservation within the kinase domain allows the classification of the DYRK family members into 3 subfamilies: DYRK (green), HIPK (violet), and PRP4K (orange). Within the DYRK subfamily, 3 main branches are distinguished: one represented by the well-known yeast kinase Yak1p (red lines) and with no members in animals, and the other two, DYRK1 and DYRK2, with members from yeast to humans (blue and green lines). Three D. discoideum DYRK proteins are identified in databases: YakA, DdDyrk1, and DdDyrk2, belonging to one of each of the groups. In the nematode, insect, and vertebrate lineages, several members can be distinguished, likely due to gene duplication events during metazoan evolution. For the vertebrate lineage, only human sequences were included. B) DYRK subfamily members can be classified into 2 main groups: class I and class II. The percentage of conservation at the protein level between orthologues is indicated above the arrows and between 2 paralogues is indicated in parentheses within the boxes. Two members of the family have been reported in C. elegans (MBK-1 and MBK-2), whereas 33 members exist in Drosophila (minibrain, dDyrk2, and dDyrk3). While MBK-1 and minibrain are most closely related to mammalian DYRK1A and DYRK1B, dDyrk2 is related to mammalian DYRK4. Finally, MBK-2 and dDyrk3 are related to mammalian DYRK2 and DYRK3. Note that the phylogenetic classification correlates to the functional classification of the DYRK subfamily as class I and class II kinases (29). Protein sequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov); see Supplemental Table S3 for accession numbers. Only the kinase domain was used for comparison. The tree was generated by ClustalW2 (http:// www.ebi.ac.uk/Tools/clustalw2) and Phylodendron (http:// iubio.bio.indiana.edu/treeapp).

target of rapamycin (TOR) signaling (4), two of the major nutrient-sensing pathways that control eukaryotic cell growth. The subcellular localization of Yak1p 450

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acts as a regulatory mechanism for its function, as rapamycin treatment and glucose depletion induce its translocation to the nucleus (4, 5), where most of its substrates are located. Known Yak1p substrates include Msn2p and Hsf1p, two essential stress-responsive transcription factors downstream of the RAS/cAMP pathway; Pop2p, a component of the transcription factor complex CCR4 and of the deadenylase complex; Crf1p, a corepressor of the transcription of ribosomal protein genes downstream of TOR signaling; and the protein kinase A (PKA) regulatory subunit Bcy1p (for references, see Supplemental Table S1). All of these findings delineate Yak1p as a key element within the sensing mechanisms for nutritional or oxidative stresses that transduce these signals into the transcriptional control of the cell cycle machinery. Protein putative kinase 15 (Ppk15p) is the DYRK protein most related to Yak1p in S. pombe, but Pom1p is the most studied member of the DYRK family in fission yeast. Pom1p is necessary to provide positional information for both cell growth and cell division (6), and it appears to work by coupling cell size to cell cycle progression via regulation of the G2-M transition (7, 8). The activity of the kinase is regulated during the cell cycle, where it is high during bipolar growth and cell division and low after cell division (9). Similarly to Yak1p, subcellular location is a key determinant in regulating Pom1p activity. During interphase, Pom1p is located in the growing cell tips where it interacts with and regulates Rag4p, a GTPase-activating protein. This results in Cdc42p activation and the triggering of F-actin polarization, which is necessary for the growth of cell tips (10). Pom1p localization in growing tips forms a concentration gradient that, during G2, enables the proper function of cell cycle determinants, which are located at the medial cell cortex. These determinants are Mid1p, a determinant of the division plane position, and Cdr2p, an inhibitory kinase of Wee1 that ultimately controls Cdk1 mitotic activity (7, 8, 11, 12). YakA is one of the 3 Dictyostelium DYRKs that can be identified in databases (Fig. 1A). YakA regulates the transition between the vegetative growing phase and the developmental phase that is induced by nutrient starvation in D. discoideum, by regulating exit from the cell cycle (13). In addition, YakA controls PKA activity by inhibiting the expression of PfuA, a translational inhibitor of the PKA catalytic subunit (13). Similarly to Yak1p, YakA is involved in the regulation of growth rates and cell survival in response to several cellular stresses (nitrosative/oxidative and heat stress) (14).

DYRK IN NEMATODES Two DYRK genes have been identified in Caenorhabditis elegans: mbk-1 and mbk-2 (MiniBrain Kinase homologue-1 and -2). While little is known about the function of mbk-1, mbk-2 has recently been the subject of intense research. mbk-1 loss-of-function mutants are viable and do not show gross morphological alterations.

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However, extra copies of mbk-1 cause behavioral defects in chemotaxis toward volatile chemoattractants (15). In contrast, null mbk-2 mutants are lethal due to severe defects in cytokinesis during embryonic development (15). Work by several groups has shown that MBK-2 is required to promote the oocyte-to-embryo transition by acting at several different levels (see Supplemental Table S1 for a complete list of substrates and/or partners). MBK-2 not only regulates the proper localization of the mitotic spindle during asymmetric cell divisions, but also the asymmetric segregation of germ line determinants and the degradation of the maternal proteins OMA-1/OMA-2 and MEI-1, a homologue of the microtubule-severing protein katanin (16 –20). MBK-2 function relies on the relocation of the protein from the cortex to the egg cytoplasm during meiotic divisions (21), thereby granting MBK-2 access to its substrates, and also on its activation by the removal of a yet unknown repressor (22, 23). These control mechanisms are linked to the formation of protein complexes with the pseudophosphatases EGG-3, EGG-4, and EGG-5 and to MBK-2 phosphorylation by CDK-1 (21– 23). The activity of the polarity proteins MEX-5 and MEX-6 and the role of OMA-1 as a general transcriptional repressor during early embryonic development are also modulated by MBK-2 phosphorylation (24, 25).

DYRK IN MAMMALS The mammalian DYRK subfamily consists of 5 members: DYRK1A, DYRK1B (also named Mirk), DYRK2, DYRK3 (also named REDK), and DYRK4 (31) (Figs. 2 and 3). The analysis of the genomic structure and the degree of conservation of the kinase domain of these family members (Figs. 1 and 3) reveals that all of the DYRK genes arose from gene duplication events that occurred during the late periods of metazoan evolution. The pairs DYRK1A-DYRK1B and DYRK2-DYRK3 are paralogous genes with common orthologous genes in C. elegans (mbk-1 and mbk-2, respectively) and Drosophila (mnb and dDyrk3, respectively), whereas the closest ancestor of DYRK4 is Drosophila dDyrk2 (Fig. 1B). At the protein level, conservation among the different mammalian members is restricted to the kinase domain and to a sequence just upstream of the kinase domain named the DYRK homology (DH)-box, which is characteristic of the subfamily (31) (Fig. 2B). The paralogous pair DYRK1A-DYRK1B shares additional homology outside the kinase domain in a nuclear localization signal (NLS) and a PEST motif within the N-terminal and C-terminal regions, respectively. The other 3 DYRKs present a common functional domain in the N-terminal region known as the NAPA-domain, which can also be found in DYRKs from other species belonging to the DYRK2 branch (29) (Fig. 2B). DYRKs are dual-specificity protein kinases

DYRK IN DROSOPHILA Three DYRK subfamily members have been identified in Drosophila melanogaster: minibrain (mnb) (26), dDyrk2 (smell-impaired 35A, smi35A) (27), and a third member annotated in the Fly database as dDyrk3 (CG40478; http:// flybase.org) (Fig. 1). Although biochemical work on Drosophila DYRKs has led to the current model of how DYRKs become catalytically active (28, 29), very little is known on their substrates or targets (see Supplemental Table S1). Minibrain was named after the brain phenotype of hypomorphic mutant flies (26). Disruption of mnb causes an abnormal spacing of neuroblasts in the outer proliferation centers of the larval brain, and consequently, the adult brain shows a specific and marked reduction in the size of the optic lobes and central brain hemispheres, indicating that mnb plays an essential role during postembryonic neurogenesis. These morphological alterations are associated with specific behavioral abnormalities in learning, memory, and visual and olfactory tasks (26). dDyrk2, which displays the highest degree of homology with mammalian DYRK4 (Fig. 1B), is catalytically active during all stages of fly development, with increased levels during embryogenesis and pupation (27). Finally, dDyrk3 was identified as a hit in a genome-wide screen in Drosophila for regulators of the nuclear factor of activated T-cells (NFAT) transcription factor (30). DYRK FAMILY OF PROTEIN KINASES

DYRK proteins are defined as dual-specificity protein kinases because they have phosphorylation activity on tyrosine, serine, and threonine residues, although the tyrosine phosphorylation activity is restricted to autophosphorylation. Indeed, no phosphorylated tyrosines have been found in any DYRK substrate to date (a complete list is supplied in Supplemental Tables S1 and S2). As members of the CMGC group of kinases, DYRKs have CMGC-specific structural features, such as the CMGC-insert between subdomains X and XI, and DYRK-specific constraints such as the replacement of the HRD-arginine in the catalytic loop with cysteine (32) (Fig. 2C). The activation loop is characterized by a conserved YXY sequence whose tyrosine residues have been found phosphorylated in vivo (for Drosophila DYRKs, see ref. 28; for mammalian DYRKs, consult the Phosphosite database at http://www.phosphosite.org). The lack of kinase activity of mutant proteins in these residues has led to the proposal that DYRKs require phosphorylation of the activation loop to achieve full activity, as shown for yeast Yak1p and Pom1p (9, 33); C. elegans MBK-2 (22); Drosophila dDyrk2 and Mnb (28); and mammalian DYRK1A, DYRK1B, DYRK3, and DYRK4 (refs. 34 –36 and unpublished results). This requirement has been challenged by the observation that a bacterially expressed DYRK1A protein that has no phosphorylated tyrosine residues due to treatment with phosphatases does not lose kinase activity in vitro (37). DYRKs were initially classified as proline-directed 451

Figure 2. DYRK proteins in mammals. A) Schematic representation of the protein structure of the 5 mammalian DYRKs. The different protein motifs identified are indicated: NLS, nuclear localization signal; DH, DYRK-homology box; NAPA, N-terminal autophosphorylation accessory region; Kinase, kinase domain; PEST, motif rich in proline, glutamic acid, serine, and threonine residues; His, polyhistidine stretch; and S/T, region enriched in serine and threonine residues. Red lines indicate protein regions affected by alternative splicing events. B) Sequence alignment of the region upstream of the kinase domain of the 5 human DYRK proteins. DH box and NAPA regions are indicated (according to ref. 29; a more exhaustive comparison of these regions in DYRKs from different organisms can be found in this reference). NLSs identified in DYRK1A (64) and DYRK2 (42) are boxed. Conserved/similar amino acids in all kinases are highlighted in green/yellow; conserved amino acids in class II kinases are highlighted in brown. C) Sequence alignment of the kinase domain of the 5 human DYRK proteins. Conserved amino acids are highlighted in green; activation loop is in yellow. Secondary structure (corresponding to DYRK1A kinase domain, as annotated in http://www.rcsb.org/ pdb/home/home.do) is shown below the protein alignment. Arrows indicate ␤ sheets; blue boxes indicate helical structures, named according to PKA crystal structure. Lines above the alignment indicate the kinase subdomains (I-XI), according to ref. 2, including the protein segment between subdomains X and XI, which is known as the CMGC insert. The following functional features are also indicated: ATP-anchor, structure covering and anchoring the nontransferable phosphates and the adenine ring of ATP; phosphate-anchor, lysine residue that helps to anchor and orient ATP by interacting with ␣- and ␤phosphates; catalytic loop, structure responsible for transferring the ␥-phosphate of the ATP to a hydroxyl residue in the substrate; cation binding site, structure responsible for chelating Mg2⫹, the metal cation that bridges the ATP ␤- and ␥-phosphates and aids in the orientation of the transferable phosphate; activation loop, domain necessary to stabilize the kinase in an active conformation; P ⫹ 1 loop, structure playing a major role in the recognition of peptide substrates.

kinases due to the preference for a proline residue at the P ⫹ 1 position, and a Pepscan analysis led to the identification of consensus phosphorylation sequences for DYRK1A (RPXS/TP) (38) and for DYRK2 and DYRK3 (RXS/TP) (39). Therefore, differences in substrate choice can be expected among DYRK kinases. Such a case has been shown for the phosphorylation of histone H2A by DYRK2 and DYRK3 but not by DYRK1A in vitro (31, 39) and can be implied by the minimal overlap (found thus far) among the substrates of different DYRKs (Supplemental Table S2). While the presence of an arginine at P ⫺ 3 or P ⫺ 2 and a proline at P ⫹ 1 has been a useful guide for the discovery of phosphorylation sites on substrates, it should be noted that not all of the phosphorylation sites identified to 452

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date match the consensus sequences (Supplemental Table S2). This finding suggests that other mechanisms might direct the specificity of DYRKs toward their substrates. One such mechanism could be the presence of docking sites in the DYRK proteins outside the kinase domain, as shown for the case of DYRK1A and Sprouty2 (40). Activation mechanism Unlike MAPKs, for which phosphorylation of the activation loop requires an upstream kinase, phosphorylation of the DYRK activation loop is an autocatalytic process. Elegant work from Cleghon and colleagues

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Figure 3. Genomic structure and splicing events of human DYRKs. Schematic representation of human DYRK1A (A), DYRK1B (B), DYRK2 (C), DYRK3 (D), and DYRK4 (E) genes: boxes represent exons; lines represent introns. Gene size and chromosomal location are indicated. For each gene, distinct splicing events are depicted. Untranslated and translated exons are shown in gray and orange, respectively. A partial scheme of the transcripts produced by the differential usage of distinct promoters and/or splicing events is included, and the predicted protein produced by each transcript is indicated. A) Experimental evidence suggests that DYRK1A contains 3 putative promoter regions: promoter M (pM) before exon 1M, promoter A (pA) before exon 1A, and promoter B (pB) before exon 1B (53). Their use produces distinct transcripts with different 5⬘ ends. Transcription from pM and pA would result in transcripts with an identical coding potential, whereas transcripts generated from pB could potentially encode a protein lacking the first 29 aa. B) DYRK1B contains 2 putative promoter regions, pA and pB, whose alternative usage produces different transcripts encoding different protein isoforms: a short isoform of 629 aa and a long isoform of 689 aa, respectively (60). Internal splicing events affecting the catalytic domain are also shown. C) Skipping of exon 2 in DYRK2 gives rise to two different transcripts encoding different protein isoforms. D) The two DYRK3 transcripts do not share the first exon; therefore, the existence of 2 distinct promoters can be predicted (63). E) The existence of two DYRK4 transcripts with different exon content at their 5⬘ ends also suggests the existence of 2 promoters (unpublished results). DYRK FAMILY OF PROTEIN KINASES

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(28) proposed a particular mechanism for DYRK activation based on in vitro studies with Drosophila DYRKs: during the translation of DYRK proteins, an active, transient intermediate form is autophosphorylated in cis at the second tyrosine within the activation loop, resulting in full kinase activation. The specificity of this intermediate for tyrosines is lost on completion of translation, and kinase activity becomes restricted to threonine and serine. The researchers argue that the differential specificity would be fixed by structural changes induced on phosphorylation of the activation loop, a hypothesis that needs to be validated by the comparison of crystal structures of the inactive and mature forms. Further work by the same group has established a link between evolutionary diversification in the DYRK family and kinase activation mode by showing that the tyrosine autophosphorylation process is mechanistically different between class I (or DYRK1 group) and class II (or DYRK2 group) DYRKs based on the requirement of the NAPA domain for tyrosine autophosphorylation. The NAPA motif, which is present in the N-terminal region of class II DYRKs only, provides a chaperone-like function for the intermediate form with tyrosine autophosphorylation activity (29). It is unclear yet whether a chaperone-like activity is provided by an external partner in the case of class I DYRKs.

SPRED1/2 (43– 45). Finally, control of subcellular localization (see below) or regulation of protein stability, as observed for DYRK2 phosphorylation by ATM in response to genotoxic stress (42), very likely represent regulatory mechanisms that might help in sequestering DYRKs away from their substrates when required. Future reports will surely shed light on the various mechanisms that regulate DYRKs activity. DYRKs as priming kinases One of the remarkable features of DYRK kinases is their ability to act as priming kinases, that is, DYRK phosphorylation of a given residue allows for more efficient phosphorylation of a different residue by a subsequent kinase. Considering the type and the relative position of the phosphorylated residue, priming mediated by different DYRK proteins belongs to 3 different types (Fig. 4). Classical GSK3 priming has been shown for several DYRK kinases, and in this regard, it is important to draw attention to the risk of attributing phosphorylation events to GSK3 based on the use of the inhibitor SB216763, because it also inhibits DYRK1A activity in vitro and in vivo (44, 46). Nonclassical GSK3 (or discon-

Regulation of DYRK activity Given that DYRK proteins are synthesized as constitutively active kinases, other mechanisms, apart from phosphorylation of the activation loop, might exist to regulate their cellular activity. Regulation of the enzymatic activity has been already observed for nonmammalian DYRKs. Fission yeast Pom1p, although present throughout the cell cycle, shows significant changes in its kinase activity with a transient decrease at and shortly after septation by a mechanism that is, at present, not known (9). Another member of the DYRK2 branch, C. elegans MBK-2, has been shown to require phosphorylation outside the catalytic domain for full activity (very likely to oppose the effects of negative regulators present in oocytes; ref. 22); moreover, MBK-2 is inhibited directly by the binding to its activation loop of the pseudophosphatases EGG-4 and EGG-5 (22). In contrast, information on changes in kinase activity in vivo for mammalian DYRKs is scarce. Nevertheless, several regulatory mechanisms can be envisaged. Dephosphorylation of the activation loop might turn off DYRK kinase activity, but no specific phosphatases have been described yet. In addition, phosphorylation events outside the activation loop might contribute to DYRKs full activity or to substrate recognition, and they could be the basis for DYRK1B regulation by MKK3 (41) or for DYRK2 phosphorylation by ATM within ␣-helix G in the catalytic domain (42). Modulation of kinase activity could also rely on interactions with regulatory proteins, as shown for the interaction of DYRK1A with 14-3-3 proteins or 454

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Figure 4. Priming activity in DYRKs. A) Classic GSK3 priming: GSK3 requires a priming phosphate at the P ⫹ 4 position to phosphorylate many of its substrates. DYRK1A and DYRK2 phosphorylate the P ⫹ 4 residue in several GSK3 substrates. B) Nonclassic (discontinuous) GSK3 priming: DYRK1A and DYRK2 phosphorylate the conserved Ser/Pro repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and Ser-rich region 1 (SRR1) motifs by GSK3 and casein kinase 1 (CK1), respectively (30, 76). OMA-1 phosphorylation by MBK-2 at T239 is required for the phosphorylation of T339 by GSK3 (19). C) PLK priming: PLK1/2 enhances the function of MEX-5/6 in vivo by binding, via their Polo-binding domains, to MBK-2-primed MEX-5/6 (24).

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tinuous) priming, in which phosphorylated residues are distant from the target site for GSK3, is a conserved activity of mammalian DYRK1A and DYRK2 and C. elegans MBK-2 (19, 30). Finally, polo-like kinase (PLK) priming is an event only described for MBK-2 (24), but possibly applicable to other DYRKs because DYRK preference for a proline at the P ⫹ 1 position could potentially create a polo box-binding motif (SpT/pSP) at the DYRK phosphorylation site. DYRK inhibitors The use of pharmacological inhibitors of DYRKs has been very helpful to elucidate the molecular mechanism of phosphorylation catalysis. For instance, different responses to purvanalol A and 4,5,6,7-tetrabromobenzotriazole suggest the existence of 2 structurally different active molecules of dDyrk2: one a transitional intermediate and the other a mature protein (28). The use of (⫺)-epigallocatechin-3-gallate (EGCG), one of the major catechin components of green tea and a DYRK inhibitor, has revealed the importance of the kinase subdomain XI for catalytic activity (47). However, none of these chemicals are useful to elucidate the in vivo functionality of DYRKs as they also inhibit other kinases (46). Recently, the alkaloid harmine, a classical inhibitor of monoamine oxidase, was identified as a novel and potent ATP-competitive DYRK1A inhibitor (46). Although harmine also inhibits DYRK1B, DYRK2, and DYRK3 (5-fold less efficiently for DYRK1B; 50-fold less, in the case of DYRK2 and DYRK3) (46, 48), and its use must be complemented with additional techniques, this alkaloid represents a very helpful tool to discover new DYRK1A functions, as illustrated in the recent identification of DYRK1A as a negative regulator of the intrinsic apoptotic pathway or cardiomyocyte hypertrophy (49 –51). DYRKs are expressed as different protein isoforms with tissue-specific patterns DYRK1A is the most ubiquitously expressed member of the DYRK family. In contrast, the other DYRK members have a more restricted and diverse expression pattern, although all are highly expressed in the testis (31, 52). Mammalian DYRK genes are all expressed as several transcripts, differing in their 5⬘ ends due to alternative promoter usage and first exon choice, potentially having distinct coding capabilities (Fig. 3). In addition, alternative splicing events within DYRK coding regions result in the production of distinct protein isoforms (Fig. 3). Some of the transcript variants show tissuespecific regulation, suggesting that the different protein isoforms might fulfill distinct roles. DYRK1A has 3 putative promoter regions: pA, pB, and pM (Fig. 3A) (53). pM and pB contain binding sites for activator protein 4 (AP4) that are able to recruit both AP4 and its corepressor partner geminin, a mechanism proposed to regulate DYRK1A expression during embryonic brain development (54). In addiDYRK FAMILY OF PROTEIN KINASES

tion, pB responds to E2F1 overexpression, although the responsive element for this transcription factor has not been identified (53). pA contains a putative CREBbinding site (53), although not experimentally verified, that is consistent with the identification of DYRK1A in a high-throughput analysis of CREB-dependent gene expression (55). In addition, an increase in the accumulation of DYRK1A in response to NFAT activation has also been described, and suggested to be part of a negative feedback loop (56); moreover, the possibility of DYRK1A expression being regulated by miRNAs has been also suggested (57). The use of alternative acceptor splicing sites in exon 4 gives rise to 2 protein isoforms that differ in the inclusion/exclusion of 9 aa in the N terminus of the protein (Fig. 3A). Experimental evidence suggests the existence of ⱖ3 DYRK1A protein variants that might be translated from the different transcripts (58, 59). However, no functional differences among these protein isoforms have been reported to date. DYRK1B contains 2 putative promoter regions, pA and pB, whose usage produces different transcripts encoding distinct protein isoforms (Fig. 3B). Expression driven by pA produces a 629-aa isoform (i.e., the p69 isoform), and the usage of pB results in a 689-aa isoform (i.e., the p75 isoform) (60). Both isoforms are present in mature mouse skeletal muscle but seem to be differentially regulated (60, 61). The 2 promoters contain E-boxes, but only the one in pA appears to respond during the differentiation of C2C12 myoblasts (61). Interestingly, pA induction is repressed by MEK1 activation, independently of the presence of E-boxes, and is induced by activation of RhoA (61). Additional splicing events affecting exon 9 contribute to the production of different variants with internal deletions of 40 or 25 aa (Fig. 3B). These deletions affect the CMGC-insert and ␣-helix H in subdomain XI and could potentially alter the kinase structure. Consequently, these variants have been shown to be kinase-dead proteins (60). DYRK2 and DYRK3 encode 2 protein isoforms, which differ in their N termini (Fig. 3C, D). In the case of DYRK3, the corresponding transcripts are expressed at different ratios during erythroid progenitor cell development (62, 63). Similarly, the human DYRK4 gene encodes 2 main protein isoforms (Fig. 3E) that are differentially expressed (unpublished results). Subcellular localization as a way of regulating substrate accessibility for DYRK kinases DYRKs have been historically classified as nuclear (DYRK1A and DYRK1B) or cytosolic (DYRK2, DYRK3, and DYRK4) proteins based on subcellular localization studies of ectopically expressed proteins in cell lines (31). Although useful in the initial steps of DYRK characterization, this classification does not actually reflect the much more complex scenario observed under physiological conditions. In overexpressing conditions, DYRK1A protein is 455

mainly found in the nucleus with a punctuated staining that colocalizes with the nuclear speckle marker SC35. By contrast, DYRK1B nuclear staining is nucleoplasmic diffuse (31, 64). The distinct staining pattern is due to the presence of a histidine stretch in DYRK1A, absent in DYRK1B, which is responsible for the accumulation of the protein in nuclear speckles (64). Endogenous DYRK1A is detected in both the nucleus and the cytosol of murine, human, and chicken neuronal cells (58, 59, 65), whereas DYRK1A localization in mouse glial cells is mostly restricted to the cytosol (58). These studies correlate with the observed nuclear-cytosolic partitioning of DYRK1A in mouse and rat brain by biochemical fractionation, which also indicates that DYRK1A associates to the synaptic plasma membrane and vesicle fraction (40, 66). Moreover, regulation of the nucleocytoplasmic distribution of DYRK1A is suggested by the transient nuclear translocation that is observed during the differentiation of Purkinje cells in mice (65). DYRK1B shows a predominant nuclear staining in several cell lines, whereas major cytosolic staining is observed in adult human muscle fibers, rhabdomyosarcomas, and pancreatic ductal carcinomas (67). This differential protein distribution has been associated with the distinct roles of DYRK1B in the nucleus, as a negative regulator of cell cycle progression, and in the cytosol, as a prosurvival factor. However, it cannot be ruled out that these staining patterns are the result of the differential expression of DYRK1B isoforms with distinct nucleocytoplasmic distribution. In the case of class II DYRKs, DYRK3 is predominantly nuclear (62), and DYRK2 is present in the cytosol, though it accumulates in the nucleus in response to genotoxic stress (68). This nuclear accumulation depends on an NLS in the N terminus of the protein and on an Mdm2-dependent increase in protein stability (42). Finally, alternative splicing in DYRK4 contributes to the generation of DYRK4 protein isoforms with distinct nucleocytoplasmic properties (unpublished results). These observations suggest that, similar to Yak1p, Pom1p, and MBK-2, the activity of mammalian DYRKs is spatially restricted, and changes in subcellular distribution likely represent a way of controlling substrate accessibility. Subcellular localization, therefore, emerges as a key regulatory mechanism for this family of kinases. DYRK proteins are pleiotropic regulators of cellular functions The participation of DYRK proteins in different cellular processes and signal transduction pathways has been inferred, in most cases, from the activity of their interactors and substrates (Supplemental Table S2). In this context, a note of caution should be given to the fact that many of the phosphorylation events described to date have only been demonstrated to occur in vitro (those substrates for which experimental evidence exists for in vivo phosphorylation are highlighted in 456

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Supplemental Table S2). The list of substrates indicates that mammalian DYRKs are pleiotropic proteins with widespread cellular functions, some of which seem to be conserved from their orthologous genes in lower eukaryotes. For instance, the prosurvival role and the differentiation-promoting activity of DYRK1A, DYRK1B, and DYRK3 are reminiscent of the protective roles of Yak1p and YakA in lower eukaryotes. Perhaps, the most striking example of functional conservation is represented by the strong effect in brain development that occurs when the expression of DYRK1A orthologues in flies, mice, or humans is reduced by mutation (26, 69, 70). A second source of information comes from the phenotypic analysis of loss-of-function mouse models. Whereas Dyrk1a-null embryos present a severe developmental delay and die around embryonic day 10.5 (69), Dyrk3- and Dyrk4-knockout mice are viable and fertile (52, 71), indicating an essential role for Dyrk1a and nonessential functions and/or functional redundancy for Dyrk3 and Dyrk4. Most of the DYRK functional relationships depend on direct phosphorylation of their targets, although some kinase-independent activities have also been noted, suggesting a role for DYRK kinases as scaffolds in certain processes (72–74). Phosphorylation by DYRKs affects several aspects of their targets’ biology (see Supplemental Table S2 for a detailed description), including changes in enzymatic activity (as in the case of SIRT1; ref. 75), in subcellular localization (as in the case of NFAT proteins; refs. 30, 76), and in protein stability (as in the case of katanin; ref. 74). In this regard, the recent finding of DYRK2 as a component of the E3 ubiquitin ligase EDVP deserves a special mention because the kinase fulfills 2 roles: as an adaptor for the formation of the ligase complex and as a kinase to phosphorylate the ligase substrates (74). Despite the wide variety of DYRK substrates acting on different cellular processes, a few common categories have emerged, and they are discussed below. Cell survival DYRK1A, DYRK1B, and DYRK3 have emerged as protective kinases against apoptosis, although they might not act through the same molecular targets. Conversely, DYRK2 behaves as a proapoptotic kinase. Two independent groups have defined the cysteineprotease caspase 9 as a DYRK1A substrate (49, 50). Seifert et al. (49) demonstrated that DYRK1A interacts with and phosphorylates caspase 9 at T125, which inhibits its processing and further activation in cells. Moreover, the researchers showed that DYRK1A is the major kinase phosphorylating this site during interphase, and it participates in caspase 9 phosphorylation in response to hyperosmotic stress (77). Laguna et al. (50), using both genetic manipulations and pharmacological approaches, demonstrated that DYRK1A is the major kinase for caspase 9 during mouse retina development, and caspase 9-mediated developmental cell death in the retina is a process sensitive to DYRK1A gene-dosage imbalance. Additional antiapoptotic roles

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have been reported for DYRK1A. First, p53-mediated survival during DNA damage is induced by DYRK1A (and DYRK3)-mediated phosphorylation and activation of the protein deacetylase SIRT1, an inhibitor of p53 (75). Second, Hip1 (huntingtin-interacting protein 1)mediated cell death during the differentiation of embryonic hippocampal neuroprogenitor cells is blocked by DYRK1A phosphorylation of Hip1 (78). Moreover, the increase in DYRK1A expression levels in human foreskin keratinocytes immortalized by infection with human papillomavirus type 16 appears to favor these cells to escape from apoptosis (79). These findings support a protective role against apoptosis for DYRK1A, both in physiological conditions and in viral-induced oncogenesis, by acting at different levels of the apoptotic response. The prosurvival activity of DYRK1B appears to be required for both nontransformed and tumor cells (67, 80, 81). The protective role of DYRK1B is exerted in G0 during normal cell differentiation and in damaged tumor cells arrested in a quiescent state to allow cellular repair (80, 82). The molecular mechanism for the antiapoptotic activity of DYRK1B has been proposed to rely on the phosphorylation of 2 CDK inhibitors, p21 and p27. DYRK1B phosphorylates p21, promoting its export to the cytoplasm, where it inhibits proteins with proapoptotic activities, such as procaspase 3 or ASK1 (80). DYRK1B also seems to be responsible for the BCL2/BCLx-mediated increase in p27 S10 phosphorylation, which is necessary for the maintenance of the G0 state (83). DYRK2 phosphorylation of nuclear p53 at S46 promotes its apoptotic activity (68). This residue is also phosphorylated by HIPK2, leading to the enhancement of p53-dependent transcriptional activity. However, while DYRK2 phosphorylation is required for UV- and adriamycin-induced apoptosis, HIPK2 phosphorylation is only necessary for UV-induced apoptosis (68). Cell differentiation Aside from their role as prosurvival kinases, DYRK1A, DYRK1B, and DYRK3 also play a role during cell differentiation in distinct cell types: neurons, muscle cells, and blood cells, respectively. The information available suggests that DYRKs perform distinct functions during the differentiation programs for the cells to acquire the specific morphological and/or functional features, and also they take part in decisions connecting cell cycle progression with cell differentiation. In this regard, DYRK1A and DYRK1B have been suggested to associate with activities during the G0/G1 phase of the cell cycle (84 – 87), but DYRK2 knockdown induces a G2/M arrest, supporting its participation in M-phase progression (74). Both in vivo and in vitro findings support a role for DYRK1A in neuritogenesis, though the molecular mechanisms underlying this function are still not well understood. Initial observations showed that DYRK1A is DYRK FAMILY OF PROTEIN KINASES

expressed in the neurites of differentiating neurons in chicks (65), and neocortical pyramidal cells from Dyrk1a heterozygous mice exhibit reduced complexity of dendritic trees (88). More recently, DYRK1A overexpression has been shown to result in a severe reduction of both dendritic growth and complexity in primary mouse cortical neurons (89). A similar phenotype is observed when DYRK1A is knocked down in primary rat cortical neurons or inhibited by harmine treatment in cultured hippocampal neurons (48, 90). These results indicate that any alteration in DYRK1A dosage could affect the differentiation program of neuronal cells. In this context, DYRK1A overexpression potentiates nerve growth factor-mediated neuronal differentiation in PC12 cells through an enhancement of Ras/MAPK signaling (73) and exit from the cell cycle in neuronal progenitors (87). Activation of p53 by DYRK1A phosphorylation on S15 has been proposed to be the target for the cell cycle arrest mediated by DYRK1A overexpression (86). Furthermore, DYRK1A gene dosage imbalance has been shown to severely modify the levels of neuron-restrictive silencer factor (NRSF or REST), a key regulator of pluripotency and neuronal differentiation gene expression programs (91), and recently, DYRK1A participation in a molecular pathway involved in EGF-dependent cell-fate decisions of adult neural stem cells has been reported (92). Some of these DYRK1A-dependent activities, and/or novel ones yetto-be-discovered, might be at the basis of the central nervous system phenotypes shown by animal models with DYRK1A dosage altered, and which support a key role for DYRK1A in brain development (refs. 50, 69; overexpression models were recently reviewed in ref. 93). DYRK1B promotes myoblast cell differentiation by mediating cell cycle arrest of proliferating myoblasts and by promoting the survival of differentiating myocytes (94). Two targets, p27 and cyclin D1, might contribute to the proliferative arrest, because their phosphorylation by DYRK1B promotes their stabilization and destabilization, respectively (84, 85). DYRK1B also phosphorylates histone deacetylases HDAC5 and HDAC9, resulting in the derepression of MEF2 and promoting myocyte differentiation (94). In addition, DYRK1A and DYRK1B are induced on osteoclast differentiation. DYRK1A blocks osteoclast differentiation by inhibiting NFAT activity, and DYRK1B has a currently unknown role in this process (56). DYRK3 is proposed to be a regulator of late erythropoiesis and as a survival factor for hematopoietic progenitor cells (36). The phenotype of knockout and transgenic mice for Dyrk3 supports such roles due to the altered number of mature reticulocytes after the induction of erythropoiesis by hemolytic anemia (71). In addition, DYRK3 expression is high in precursor cells from the erythroid lineage and is inhibited on GATA1-induced erythroid differentiation (62, 63). Despite lacking a clear mechanistic picture, DYRK3-induced phosphorylation and activation of the transcription factor CREB (in a kinase-independent manner and 457

depending on the activity of PKA) or the inhibition of NFAT transcriptional activity could be the basis of the prodifferentiation role of DYRK3 (36, 71). Gene transcription A relevant group of DYRK targets are proteins involved in the regulation of transcription and gene expression programs, an activity that may, indeed, have a great effect on cellular responses (Supplemental Table S2). Among transcription factors, NFAT proteins are common targets of several DYRK family members. Phosphorylation induces NFAT cytosolic translocation, and thereby, DYRKs act as negative regulators of these transcription factors in distinct cellular environments (30, 51, 56, 71, 76). Gli proteins are substrates of several DYRKs with distinct outcomes; while DYRK1A activates Gli1, DYRK2 induces degradation of Gli2 (95, 96). Similarly to DYRK2, DYRK1B acts as a suppressor of hedgehog signaling, but the exact molecular mechanism is still not known (97). The tumor suppressor p53 is also a common substrate of DYRK1A, DYRK2, and DYRK3, with these kinases phosphorylating different p53-activating residues: S15 for DYRK1A, and S46 for DYRK2 and DYRK3 (68, 86). In the case of the functional interaction of DYRK1A with p53, apparently contradictory results have been reported since both down-regulation and overexpression of the kinase result in increased levels of p53-target genes as a result of DYRK1A acting as a p53 inhibitor or activator, respectively (75, 86). Given that both reports used two very different experimental systems, DNA damage response and neuroprogenitors differentiation, respectively, it is possible that DYRK1A may exert a dual role on the regulation of p53, depending on the cellular context. DYRK-mediated regulation of transcription is not only restricted to transcription factors but also works through chromatin modification. For instance, DYRK1A is found in complexes with the SWI/SNF chromatin remodeling complex and cooperates with the SNF2-like ATPase Arip4 (72, 90). DYRK1B induces the cytosolic localization of the histone modifier HDAC5 (94), and both DYRK1A and DYRK3 potentiate the deacetylase activity of Sirt1, a known epigenetic regulator (75). Finally, DYRK1A has been proposed as a regulator of RNA processing based on DYRK1A localization in the splicing factor compartment (64) and on having several splicing factors as substrates, including ASF (Supplemental Table S2). In fact, DYRK1A phosphorylation of this alternative splicing factor prevents ASF-mediated inclusion of the alternatively spliced exon 10 in tau mRNA (98).

Furthermore, strong experimental evidence positions DYRK1A within the endocytic proteomic network as it has been functionally and physically related to proteins, such as the GTPase dynamin1, the phosphatase synaptojanin1, and the scaffold protein amphyphisin1. Phosphorylation of these proteins by DYRK1A modulates their interaction with other partners (see ref. 66 and references therein). However, functional data supporting the implication of DYRK1A in the endocytic process are still lacking. Conversely, DYRK3 was identified in a functional screen aimed at the detection of kinases involved in caveolae/raft-mediated endocytosis (99). Knockdown experiments revealed that DYRK3 controls the dynamics of caveolar vesicles and, therefore, the long-range cytoplasmic transport from the cell membrane (100). However, no DYRK3 targets relevant for this function have been identified to date.

DYRKS AND DISEASE DYRK1A represents the most relevant example of DYRK association with disease in humans, because nonpathological activity shows an exquisite dependence on gene dosage. DYRK1A expression is increased at the transcript and protein levels in Down syndrome (DS) individuals, and the analysis of mouse models of overexpression has led to the hypothesis of this protein as a candidate to explain some of the neuropathological traits of DS (93). Moreover, DYRK1A dosage sensitivity is highlighted by the fact that truncation mutants of DYRK1A result in clinical phenotypes, including microcephaly, intrauterine growth retardation, and developmental delay (70). The potential participation of DYRK1A in other pathologies, such as neurodegeneration, cardiac hypertrophy and bone homeostasis has also been suggested (51, 56, 101, 102). Overexpression of DYRK1B has been reported in solid tumors, including colon and lung cancer, pancreatic ductal adenocarcinomas, and rhabdomyosarcomas, and experimental data suggest that increased amounts of DYRK1B favor tumor development (67). An opposite scenario has been outlined for DYRK2 in gastric, lung, and esophageal adenocarcinomas, where DYRK2 overexpression has been related to a better prognosis (103), a fact likely linked to its positive role on the p53 proapoptotic pathway (68). Considering the participation of DYRK proteins in signaling pathways critical for developmental processes and cell homeostasis, the involvement of DYRKs in human pathological processes when deregulated or mutated is foreseen for the near future.

Endocytosis

PROSPECTS AND PREDICTIONS

One cytosolic process in which the participation of DYRK proteins could be relevant is endocytosis. DYRK1A has been detected in free vesicle-containing fractions during brain biochemical fractionation (40).

DYRK kinases are highly conserved during evolution from yeast to humans. The evolutive diversification process observed in the family might reflect the necessity of more specialized functions in higher eukaryotes

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or alternatively, be envisaged as a mechanism of genetic redundancy to ensure cell viability. Experimental evidence supports both possibilities. For instance, Yak1p and YakA are stress transducers, and a similar function is accomplished by DYRK2 in mammals when transducing DNA-damage stress or by DYRK1A-mediated caspase 9 phosphorylation in response to hyperosmolarity (68, 77). Likewise, the role of Yak1p as a regulator of gene expression by acting on transcription factors is shared by almost all mammalian DYRK members. Fission yeast Pom1p and C. elegans MBK-2 are necessary for the correct positioning of the mitotic spindle and the correct asymmetric division of cells, the latter of which is mediated through the phosphorylation of the microtubule-severing protein MEI-1. Notably, the MEI-1 orthologue in mammals is katanin, which is a substrate of DYRK2 (74). Thus, it is predictable that one or more mammalian DYRKs might play a role in cellular decisions leading to asymmetric divisions. Indeed, such a role has been recently shown for DYRK1A (92). Some relevant issues should be addressed in the future to better understand the functionality of DYRK kinases. First, identification of upstream regulators will help to define specific signaling pathways in which DYRK proteins might exert their role. In this sense, the mechanisms that control DYRK subcellular localization and how this correlates with specific cellular functions appear to have a special significance. Second, specific kinase inhibitors should be developed to address the functional relevance of these proteins in vivo. Third, the physiological functions of DYRK proteins should be tackled with genetically modified mouse models, which would help to determine the relevance and functional redundancy, if any, of the different DYRK members in various tissues. Finally, the participation of DYRKs in pathological processes when deregulated should be investigated. All of these approaches will surely lead to a better knowledge on DYRK kinases involvement in cell physiology.

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The authors thank all of the members of the M. L. Arbone´s (Center for Genomic Regulation, Barcelona, Spain) and S.L. groups for helpful comments and discussion. S.A. and A.L. were FI predoctoral fellows [Age`ncia de Gestio´ d’Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalunya, Barcelona, Spain]. S.L.’s group is supported by the Spanish Ministry of Science and Innovation (BFU2007-61043/BMC), AGAUR (2009SGR1464), EC AnEUploidy grant, and Foundation Jerome Lejeune. The authors apologize to any DYRK community members in the event that their published work has not been included in the reference list or in references associated with the supplemental tables.

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Received for publication May 20, 2010. Accepted for publication October 14, 2010.

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