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• W2152705959 abstract "•Crystal structures of DYRK1A with ATP-mimetic inhibitor and peptide substrate•Crystal structure of DYRK2 including NAPA and DH box region•Determination of consensus substrate motifs for DYRK1A and DYRK2•Observation of DYRK1A autophosphorylation on tyrosine in vitro Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK subfamily: DYRK1A with an ATP-mimetic inhibitor and consensus peptide, and DYRK2 including NAPA and DH (DYRK homology) box regions. The current activation model suggests that DYRKs are Ser/Thr kinases that only autophosphorylate the second tyrosine of the activation loop YxY motif during protein translation. The structures explain the roles of this tyrosine and of the DH box in DYRK activation and provide a structural model for DYRK substrate recognition. Phosphorylation of a library of naturally occurring peptides identified substrate motifs that lack proline in the P+1 position, suggesting that DYRK1A is not a strictly proline-directed kinase. Our data also show that DYRK1A wild-type and Y321F mutant retain tyrosine autophosphorylation activity. Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK subfamily: DYRK1A with an ATP-mimetic inhibitor and consensus peptide, and DYRK2 including NAPA and DH (DYRK homology) box regions. The current activation model suggests that DYRKs are Ser/Thr kinases that only autophosphorylate the second tyrosine of the activation loop YxY motif during protein translation. The structures explain the roles of this tyrosine and of the DH box in DYRK activation and provide a structural model for DYRK substrate recognition. Phosphorylation of a library of naturally occurring peptides identified substrate motifs that lack proline in the P+1 position, suggesting that DYRK1A is not a strictly proline-directed kinase. Our data also show that DYRK1A wild-type and Y321F mutant retain tyrosine autophosphorylation activity. The dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an evolutionarily conserved family of kinases with five human members (DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4). They belong to the CMGC family of serine/threonine (S/T) kinases and are categorized as class I (DYRK1A and DYRK1B) and class II (DYRK2, DYRK3, and DYRK4) DYRKs. The best-studied member of the DYRK family is DYRK1A, owing to its role in the pathology of Down syndrome and the early onset of neurodegeneration. DYRK members have been clearly shown to participate in important signaling pathways that control postembryonic neurogenesis, developmental processes, cell survival, differentiation, and death (Arron et al., 2006Arron J.R. Winslow M.M. Polleri A. Chang C.-P. Wu H. Gao X. Neilson J.R. Chen L. Heit J.J. Kim S.K. et al.NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21.Nature. 2006; 441: 595-600Crossref PubMed Scopus (546) Google Scholar; Mercer et al., 2005Mercer S.E. Ewton D.Z. Deng X. Lim S. Mazur T.R. Friedman E. Mirk/Dyrk1B mediates survival during the differentiation of C2C12 myoblasts.J. Biol. Chem. 2005; 280: 25788-25801Crossref PubMed Scopus (72) Google Scholar; Tejedor et al., 1995Tejedor F. Zhu X.R. Kaltenbach E. Ackermann A. Baumann A. Canal I. Heisenberg M. Fischbach K.F. Pongs O. minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila.Neuron. 1995; 14: 287-301Abstract Full Text PDF PubMed Scopus (308) Google Scholar). In addition, recent studies show DYRK1A and DYRK2 phosphorylate NFATc, countering the effect of calcium signaling and maintaining inactive NFATc (Arron et al., 2006Arron J.R. Winslow M.M. Polleri A. Chang C.-P. Wu H. Gao X. Neilson J.R. Chen L. Heit J.J. Kim S.K. et al.NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21.Nature. 2006; 441: 595-600Crossref PubMed Scopus (546) Google Scholar; Gwack et al., 2006Gwack Y. Sharma S. Nardone J. Tanasa B. Iuga A. Srikanth S. Okamura H. Bolton D. Feske S. Hogan P.G. Rao A. A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT.Nature. 2006; 441: 646-650Crossref PubMed Scopus (316) Google Scholar; Lee et al., 2009Lee Y. Ha J. Kim H.J. Kim Y.-S. Chang E.-J. Song W.-J. Kim H.-H. Negative feedback inhibition of NFATc1 by DYRK1A regulates bone homeostasis.J. Biol. Chem. 2009; 284: 33343-33351Crossref PubMed Scopus (44) Google Scholar). The first evidence for the key role of DYRK1A in neural proliferation and neurogenesis of the developing brain was provided by mutational analysis of the DYRK Drosophila ortholog minibrain (mnb), where loss-of-function mutations resulted in reduced brain size (Tejedor et al., 1995Tejedor F. Zhu X.R. Kaltenbach E. Ackermann A. Baumann A. Canal I. Heisenberg M. Fischbach K.F. Pongs O. minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila.Neuron. 1995; 14: 287-301Abstract Full Text PDF PubMed Scopus (308) Google Scholar). DYRK1A is localized in the Down syndrome (DS) critical region of chromosome 21 that has been linked to the development of DS phenotypes when triplicated (Delabar et al., 1993Delabar J.M. Theophile D. Rahmani Z. Chettouh Z. Blouin J.L. Prieur M. Noel B. Sinet P.M. Molecular mapping of twenty-four features of Down syndrome on chromosome 21.Eur. J. Hum. Genet. 1993; 1: 114-124Crossref PubMed Scopus (394) Google Scholar; Sinet et al., 1994Sinet P.M. Théophile D. Rahmani Z. Chettouh Z. Blouin J.L. Prieur M. Noel B. Delabar J.M. Mapping of the Down syndrome phenotype on chromosome 21 at the molecular level.Biomed. Pharmacother. 1994; 48: 247-252Crossref PubMed Scopus (54) Google Scholar). Indeed, triplication of the DYRK1A locus in DS results in overexpression of DYRK1A in the fetal as well as adult brain and strongly implicates DYRK1A in neurodevelopmental alterations linked to some DS pathologies and disease predispositions (Dowjat et al., 2007Dowjat W.K. Adayev T. Kuchna I. Nowicki K. Palminiello S. Hwang Y.W. Wegiel J. Trisomy-driven overexpression of DYRK1A kinase in the brain of subjects with Down syndrome.Neurosci. Lett. 2007; 413: 77-81Crossref PubMed Scopus (138) Google Scholar). These links prompted studies on the role of DYRK1A in age-associated neurodegeneration and suggested DYRK1A as a target for the development of inhibitors (Mazur-Kolecka et al., 2012Mazur-Kolecka B. Golabek A. Kida E. Rabe A. Hwang Y.-W. Adayev T. Wegiel J. Flory M. Kaczmarski W. Marchi E. Frackowiak J. Effect of DYRK1A activity inhibition on development of neuronal progenitors isolated from Ts65Dn mice.J. Neurosci. Res. 2012; 90: 999-1010Crossref PubMed Scopus (28) Google Scholar; Park et al., 2009Park J. Song W.-J. Chung K.C. Function and regulation of Dyrk1A: towards understanding Down syndrome.Cell. Mol. Life Sci. 2009; 66: 3235-3240Crossref PubMed Scopus (140) Google Scholar). The binding modes of the inhibitors INDY and Harmine in DYRK1A have recently been published (Ogawa et al., 2010Ogawa Y. Nonaka Y. Goto T. Ohnishi E. Hiramatsu T. Kii I. Yoshida M. Ikura T. Onogi H. Shibuya H. et al.Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A.Nat. Commun. 2010; 1: 86Crossref PubMed Scopus (180) Google Scholar). Apart from the well-studied DYRK1A isozyme, studies have provided evidence for the roles of DYRK1B in the development of various sarcomas (Deng et al., 2006Deng X. Ewton D.Z. Li S. Naqvi A. Mercer S.E. Landas S. Friedman E. The kinase Mirk/Dyrk1B mediates cell survival in pancreatic ductal adenocarcinoma.Cancer Res. 2006; 66: 4149-4158Crossref PubMed Scopus (47) Google Scholar) and in skeletal muscle differentiation (Deng et al., 2003Deng X. Ewton D.Z. Pawlikowski B. Maimone M. Friedman E. Mirk/dyrk1B is a Rho-induced kinase active in skeletal muscle differentiation.J. Biol. Chem. 2003; 278: 41347-41354Crossref PubMed Scopus (70) Google Scholar, Deng et al., 2005Deng X. Ewton D.Z. Mercer S.E. Friedman E. Mirk/dyrk1B decreases the nuclear accumulation of class II histone deacetylases during skeletal muscle differentiation.J. Biol. Chem. 2005; 280: 4894-4905Crossref PubMed Scopus (57) Google Scholar). DYRK2 is reported to regulate key developmental and cellular processes such as neurogenesis, cell proliferation, cytokinesis, and cellular differentiation (Taira et al., 2007Taira N. Nihira K. Yamaguchi T. Miki Y. Yoshida K. DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage.Mol. Cell. 2007; 25: 725-738Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar; Woods et al., 2001Woods Y.L. Cohen P. Becker W. Jakes R. Goedert M. Wang X. Proud C.G. The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bε at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase.Biochem. J. 2001; 355: 609-615Crossref PubMed Scopus (254) Google Scholar; Yoshida, 2008Yoshida K. Role for DYRK family kinases on regulation of apoptosis.Biochem. Pharmacol. 2008; 76: 1389-1394Crossref PubMed Scopus (56) Google Scholar). Notably, DYRK2 may function in DNA damage signaling pathways, because it phosphorylates p53 at Ser46 in response to DNA damage, which induces cellular apoptosis after genotoxic stress (Taira et al., 2007Taira N. Nihira K. Yamaguchi T. Miki Y. Yoshida K. DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage.Mol. Cell. 2007; 25: 725-738Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). In addition, ataxia telangiectasia mutated was shown to phosphorylate nuclear DYRK2 upon DNA damage, which appeared to enable DYRK2 to protect itself from degradation that occurs due to its association with MDM2 under normal conditions (Taira et al., 2010Taira N. Yamamoto H. Yamaguchi T. Miki Y. Yoshida K. ATM augments nuclear stabilization of DYRK2 by inhibiting MDM2 in the apoptotic response to DNA damage.J. Biol. Chem. 2010; 285: 4909-4919Crossref PubMed Scopus (52) Google Scholar). Emerging studies show DYRK2 has important roles in protein proteolysis, proteosomal degradation, and tumor progression (Varjosalo et al., 2008Varjosalo M. Björklund M. Cheng F. Syvänen H. Kivioja T. Kilpinen S. Sun Z. Kallioniemi O. Stunnenberg H.G. He W.W. et al.Application of active and kinase-deficient kinome collection for identification of kinases regulating hedgehog signaling.Cell. 2008; 133: 537-548Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar; Maddika and Chen, 2009Maddika S. Chen J. Protein kinase DYRK2 is a scaffold that facilitates assembly of an E3 ligase.Nat. Cell Biol. 2009; 11: 409-419Crossref PubMed Scopus (134) Google Scholar; Taira et al., 2012Taira N. Mimoto R. Kurata M. Yamaguchi T. Kitagawa M. Miki Y. Yoshida K. DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.J. Clin. Invest. 2012; 122: 859-872Crossref PubMed Scopus (102) Google Scholar). As for DYRK3 and DYRK4, their physiological functions remain poorly understood. All DYRKs contain a conserved catalytic kinase domain preceded by the DYRK-characteristic DYRK homology (DH) box (Figure 1A; for a sequence alignment, see Figure S1 available online). DYRKs rapidly autoactivate during folding by phosphorylation on the second tyrosine residue of the conserved activation loop YxY motif (Tyr321 of DYRK1A). This tyrosine corresponds to the secondary activation loop phosphorylation site in the TxY motif in MAPKs. It was reported based on studies with Drosophila melanogaster DYRKs that this phosphorylation event occurs in cis while DYRK is still bound to the ribosome, and subsequently DYRKs lose tyrosine phosphorylation ability and retain only S/T phosphorylation ability (Lochhead et al., 2005Lochhead P.A. Sibbet G. Morrice N. Cleghon V. Activation-loop autophosphorylation is mediated by a novel transitional intermediate form of DYRKs.Cell. 2005; 121: 925-936Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). For the human DYRK1A, mutation of Tyr321 or dephosphorylation did not abolish kinase activity (Adayev et al., 2007Adayev T. Chen-Hwang M.-C. Murakami N. Lee E. Bolton D.C. Hwang Y.-W. Dual-specificity tyrosine phosphorylation-regulated kinase 1A does not require tyrosine phosphorylation for activity in vitro.Biochemistry. 2007; 46: 7614-7624Crossref PubMed Scopus (27) Google Scholar). DYRKs were initially assumed to be proline-directed S/T kinases with specificity for proline and arginine at P+1 and P−3 positions, respectively (Himpel et al., 2000Himpel S. Tegge W. Frank R. Leder S. Joost H.G. Becker W. Specificity determinants of substrate recognition by the protein kinase DYRK1A.J. Biol. Chem. 2000; 275: 2431-2438Crossref PubMed Scopus (185) Google Scholar). However, further investigations revealed DYRK cellular substrates (e.g., synuclein; Kim et al., 2006Kim E.J. Sung J.Y. Lee H.J. Rhim H. Hasegawa M. Iwatsubo T. Min D.S. Kim J. Paik S.R. Chung K.C. Dyrk1A phosphorylates α-synuclein and enhances intracellular inclusion formation.J. Biol. Chem. 2006; 281: 33250-33257Crossref PubMed Scopus (82) Google Scholar) with a wide variation in phosphorylation motifs (Aranda et al., 2011Aranda S. Laguna A. de la Luna S. DYRK family of protein kinases: evolutionary relationships, biochemical properties, and functional roles.FASEB J. 2011; 25: 449-462Crossref PubMed Scopus (207) Google Scholar). To understand the molecular mechanism of DYRK1A activation, the roles of Tyr321 phosphorylation and regulatory elements located N-terminal to the catalytic domain, as well as substrate recognition, we determined the structure of the phosphorylated DYRK1A and DYRK2 catalytic domain and N-terminal regulatory DH box sections. The autophosphorylation behavior of DYRK1A was analyzed, and the substrate specificity of DYRK1A, DYRK1B, and DYRK2 was investigated using a novel mass spectrometry methodology (Kettenbach et al., 2012Kettenbach A.N. Wang T. Faherty B.K. Madden D.R. Knapp S. Bailey-Kellogg C. Gerber S.A. Rapid determination of multiple linear kinase substrate motifs by mass spectrometry.Chem. Biol. 2012; 19: 608-618Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The structure of a ternary substrate complex of DYRK1A, the ATP-mimetic inhibitor DJM2005, and a consensus substrate peptide (RARPGT*PALRE) reveals how DYRK1A recognizes substrates and provides a model for the structure-based design of selective DYRK inhibitors. The crystal structures of DYRK1A and DYRK2 comprising the catalytic kinase domain and DH box were determined. The DYRK1A structure was determined from a construct expressing residues 127–485 of human DYRK1A (National Center for Biotechnology Information [NCBI] genInfo identifier [gi] number 18765758). Residues Val135–Lys480 comprising the DH box and kinase domain were resolved in the electron density. The structure was determined in complex with the ATP-competitive inhibitor (S)-N-(5-(4-amino-2-(3-chlorophenyl)butanamido)-1H-indazol-3-yl)benzamide (DJM2005) at 2.40 Å resolution (Figure 2A; Table 1). The inhibitor DJM2005 was kindly provided by the laboratory of Kevan Shokat; the chemical structure is shown in Figure S2.Table 1Data Collection and Refinement StatisticsDYRK1A-InhibitoraThe inhibitor structure is shown in Figure S2.DYRK1A-InhibitoraThe inhibitor structure is shown in Figure S2.-PeptideDYRK2PDB ID code2VX32WO63K2LCrystallization conditions4% (v/v) PEG 300, 0.1 M Li2SO4, 0.1 M Tris, pH 8.50.2 M sodium formate, 20% (w/v) PEG 3350, 10% ethylene glycol1.26 M (NH4)2SO4, 0.2 M Li2SO4, 0.1 M Tris, pH 8.5Space groupC2P65P42212No. of molecules in the asymmetric unit421Unit cell dimensions a, b, c (Å)264.2, 65.1, 140.3168.4, 168.4, 62.484.3, 84.3, 148.5 α, β, γ (°)90.0, 115.44, 90.090.0, 90.0, 120.090.0, 90.0, 90.0Data CollectionBeamlineSLS X10SADiamond I02Diamond I03Resolution range (Å)bValues within parentheses refer to the highest resolution shell.27.24–2.40 (2.53–2.40)55.13–2.50 (2.64–2.50)42.19–2.36 (2.49–2.36)Unique observationsbValues within parentheses refer to the highest resolution shell.85,770 (12,458)35,283 (5,093)22,801 (3,275)Average multiplicitybValues within parentheses refer to the highest resolution shell.3.4 (3.2)7.5 (6.9)6.2 (6.4)Completeness (%)bValues within parentheses refer to the highest resolution shell.99.9 (99.9)100.0 (100.0)99.8 (100.0)RmergebValues within parentheses refer to the highest resolution shell.0.10 (0.82)0.18 (0.57)0.08 (0.90)Mean (I)/σ(I)bValues within parentheses refer to the highest resolution shell.9.5 (1.9)10.8 (3.7)12.2 (2.1)RefinementResolution range (Å)26.00–2.4040.00–2.5042.19–2.36R value, Rfree0.19, 0.230.19, 0.230.23, 0.29Mean protein B values (Å2)5323.1cResidual after TLS parameterization.33.8cResidual after TLS parameterization.Mean ligand B values (Å2)46 (inhibitor)34 (inhibitor)71 (peptide)Rmsd from ideal bond length (Å)0.0140.0140.014Rmsd from ideal bond angle (°)1.521.531.60Ramachandran outliers (%)Most favored (%)0.150.00.096.196.395.0a The inhibitor structure is shown in Figure S2.b Values within parentheses refer to the highest resolution shell.c Residual after TLS parameterization. Open table in a new tab The DYRK2 structure was determined from a construct expressing residues 74–479 of human DYRK2 (NCBI gi number 4503427). Residues Gly74–Pro470 comprising NAPA1 (N-terminal autophosphorylation accessory 1), NAPA2, DH box, and kinase domain were resolved in the electron density, as well as part of the N-terminal purification tag. The structure was determined in the absence of inhibitor (apo form) at 2.36 Å resolution (Figure 2B; Table 1). For both DYRK1A and DYRK2 the entire catalytic domain was well ordered, including a long hairpin-like structure for the N-terminal DH box and an active kinase conformation with a fully ordered activation segment (Figure 2). Mass spectrometry showed that the purified DYRKs were heterogeneously phosphorylated in solution (data not shown). However, the electron density maps only showed clear evidence of phosphorylation of DYRK1A at the second tyrosine of the dual-phosphorylation motif YxY (Tyr321) and double phosphorylation of DYRK2 at Ser159 of the glycine-rich loop and Tyr309 of the activation loop. The other phosphorylation sites might either have had low occupancy or were located in unstructured regions of the protein. The DYRK1A and DYRK2 structures superimpose with a root-mean-square deviation (rmsd) of 1.03 Å over 297 Cα atoms (using chain A of the DYRK1A structure). In DYRK1A, the ATP-mimetic inhibitor DJM2005 binds to the ATP binding site, forming three hydrogen bonds with the hinge backbone and an additional two hydrogen bonds from the inhibitor’s primary amine with the side chains of Asn292 and the DFG motif aspartate Asp307 (Figure 2C). There is also an electrostatic interaction via an ion (modeled as chloride) linking an inhibitor amide nitrogen to the backbone nitrogen of Asp307 from the DFG motif, and hydrogen bonding via a water molecule to the backbone carbonyl of Glu291. There are various favorable hydrophobic interactions with DYRK1A active site residues, including at the entrance to the ATP site, where the side chain of Tyr243 packs against the inhibitor’s phenyl ring. All of the DYRK1A residues involved in hydrogen bonding to the inhibitor are conserved in DYRK2 (Figure S3); there are, however, some potential differences in the hydrophobic interactions, such as the replacement of Tyr243 with Met233 in DYRK2 as well as differences at the back of the pocket and the hydrophobic residue preceding the DFG motif. Analysis of changes in DYRK1A and DYRK2 temperature shift values (ΔTm) in the presence of a set of potential kinase inhibitors showed only weak correlation, and therefore that it is possible to have DYRK1A- or DYRK2-specific inhibitors, as shown by some of the inhibitors screened that give changes in Tm with only DYRK1A or only DYRK2 (Figure 2D). Interestingly, the inhibitor’s primary amine also interacts with a sulfate molecule from the DYRK1A crystallization buffer that is found in a similar location as an autophosphorylated serine residue in DYRK2 (pS159; Figure S3). This sulfate is also bound by the side chains of Asp307 of the DFG motif, Ser169 of the glycine-rich loop, and Lys289 of the catalytic loop, and is in a similar position as that of a hydrolyzed γ-phosphate from ATP bound to PKA (Protein Data Bank [PDB] ID code 1RDQ; Yang et al., 2004Yang J. Ten Eyck L.F. Xuong N.-H. Taylor S.S. Crystal structure of a cAMP-dependent protein kinase mutant at 1.26 Å: new insights into the catalytic mechanism.J. Mol. Biol. 2004; 336: 473-487Crossref PubMed Scopus (65) Google Scholar) or a bound phosphate in the structure of Haspin with a 5-iodotubercidin ligand (PDB ID code 3IQ7; Eswaran et al., 2009Eswaran J. Patnaik D. Filippakopoulos P. Wang F. Stein R.L. Murray J.W. Higgins J.M.G. Knapp S. Structure and functional characterization of the atypical human kinase haspin.Proc. Natl. Acad. Sci. USA. 2009; 106: 20198-20203Crossref PubMed Scopus (114) Google Scholar). Addition of negatively charged groups to inhibitors to exploit this conserved binding pocket may help inhibitor design for some of these kinases. The C-terminal lobe reveals several unique features that define the DYRK family. The MAP kinase characteristic insertion observed in the C lobe of DYRKs (Figure 2) is extended in comparison with other CMGC family members such as CLK1, CLK3 (Bullock et al., 2009Bullock A.N. Das S. Debreczeni J.É. Rellos P. Fedorov O. Niesen F.H. Guo K. Papagrigoriou E. Amos A.L. Cho S. et al.Kinase domain insertions define distinct roles of CLK kinases in SR protein phosphorylation.Structure. 2009; 17: 352-362Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), GSK3β (Dajani et al., 2001Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Crystal structure of glycogen synthase kinase 3β: structural basis for phosphate-primed substrate specificity and autoinhibition.Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar), or MAPKs (Canagarajah et al., 1997Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Activation mechanism of the MAP kinase ERK2 by dual phosphorylation.Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar). In DYRK1A, this insert forms an elaborate subdomain of 40 residues comprising two short helices followed by an antiparallel β sheet that is conserved in vertebrate DYRK1 family members but not in Drosophila mnb. In DYRK2, this insert also forms a distinctive subdomain with two short helices and three short antiparallel β sheets. Along with the structural divergence between DYRK1A and DYRK2 in this insertion, this region is also the place of greatest divergence among the other DYRK family members. Deletion of the entire region of DYRK1A N-terminal to the kinase domain (1–148) has been shown to decrease catalytic activity (Himpel et al., 2001Himpel S. Panzer P. Eirmbter K. Czajkowska H. Sayed M. Packman L.C. Blundell T. Kentrup H. Grötzinger J. Joost H.G. Becker W. Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A.Biochem. J. 2001; 359: 497-505Crossref PubMed Scopus (138) Google Scholar). In Drosophila, the DH box was required for phosphorylation of SNR1 by DYRK2 but not by DYRK1 (Kinstrie et al., 2006Kinstrie R. Lochhead P.A. Sibbet G. Morrice N. Cleghon V. dDYRK2 and Minibrain interact with the chromatin remodelling factors SNR1 and TRX.Biochem. J. 2006; 398: 45-54Crossref PubMed Scopus (21) Google Scholar). With Drosophila DYRKs, the NAPA regions are required for the transient intramolecular tyrosine kinase activity of DYRKs (Kinstrie et al., 2010Kinstrie R. Luebbering N. Miranda-Saavedra D. Sibbet G. Han J. Lochhead P.A. Cleghon V. Characterization of a domain that transiently converts class 2 DYRKs into intramolecular tyrosine kinases.Sci. Signal. 2010; 3: ra16Crossref PubMed Scopus (32) Google Scholar) and are conserved across a wide range of eukaryotes, including for Trypanosoma brucei DYRK2, where the NAPA1 and NAPA2 regions are required for tyrosine autophosphorylation (Han et al., 2012Han J. Miranda-Saavedra D. Luebbering N. Singh A. Sibbet G. Ferguson M.A.J. Cleghon V. Deep evolutionary conservation of an intramolecular protein kinase activation mechanism.PLoS One. 2012; 7: e29702Crossref PubMed Scopus (18) Google Scholar). In the following analysis, the DH boxes and NAPA regions are those defined by Kinstrie et al., 2010Kinstrie R. Luebbering N. Miranda-Saavedra D. Sibbet G. Han J. Lochhead P.A. Cleghon V. Characterization of a domain that transiently converts class 2 DYRKs into intramolecular tyrosine kinases.Sci. Signal. 2010; 3: ra16Crossref PubMed Scopus (32) Google Scholar. In both DYRK1A and DYRK2, the N-terminal region containing the DH box is positioned on top of the N-terminal lobe of the kinase domain and forms a large network of interactions with all five strands of the N lobe β sheet, providing considerable stabilization (Figures 3A and 3B ). The most highly conserved residues in the DH box are those essential for stabilization of its folded state, in particular the two central tyrosines, Tyr140 and Tyr147, in DYRK1A (Figure 3A). This compact folded DH box appears essential for the formation of tertiary structure in the remainder of the N terminus, especially for class II DYRKs, which have NAPA1 and NAPA2 regions (Figure 3C). Although many of the DH box interactions are conserved between DYRK1A and DYRK2, we observed more hydrogen-bonding interactions in the DYRK1A structure. In particular, in DYRK1A, the central tyrosine, Tyr147, interacts with the DYRK1A equivalent of the NAPA2 region, Glu153 and Trp155. These residues are not present in the standard NAPA2 region of DYRK2, and DYRK2 does not have an equivalent interaction between the DH box and NAPA2 regions (Figure 3B). Recent evidence suggests phosphorylation of DYRK1A at Tyr145 and Tyr147 may have important regulatory roles (Kida et al., 2011Kida E. Walus M. Jarząbek K. Palminiello S. Albertini G. Rabe A. Hwang Y.W. Golabek A.A. Form of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A nonphosphorylated at tyrosine 145 and 147 is enriched in the nuclei of astroglial cells, adult hippocampal progenitors, and some cholinergic axon terminals.Neuroscience. 2011; 195: 112-127Crossref PubMed Scopus (13) Google Scholar). Tyr145 is solvent exposed, but phosphorylation of Tyr147 would change its interactions significantly, although it is not possible to predict whether this would be favorable or unfavorable, because pTyr147 could maintain interactions with Arg231 and replace the interactions of Glu153. As well as the stabilization provided by the N-terminal region, the 11 residues of the DH box itself interact with the loop linking αC with β3, providing stabilization to an “αC-in” active kinase conformation by fixing the N-terminal end of αC in position and so preventing αC from moving outward, as sometimes seen in inactive kinase structures (Figure 3A). Interestingly, the interaction appears not to be charge dependent, unlike for the interaction of the N-terminal hairpin of CLK3 (Bullock et al., 2009Bullock A.N. Das S. Debreczeni J.É. Rellos P. Fedorov O. Niesen F.H. Guo K. Papagrigoriou E. Amos A.L. Cho S. et al.Kinase domain insertions define distinct roles of CLK kinases in SR protein phosphorylation.Structure. 2009; 17: 352-362Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) with the kinase N-terminal lobe (Figure S4). The DYRK2 NAPA1 and NAPA2 regions, which are separated in sequence, fold together into a small domain that stabilizes the N-terminal lobe of the kinase domain (Figures 3C and 3D). The larger NAPA1 region folds around the five residues of the NAPA2 region. As with the DH box, the most highly conserved residues (marked in Figure 3D) are those forming the core of this folded subdomain, in particular His144 and Tyr147 from NAPA2. It is notable that the residues on the end of strand β4 (DYRK2: Phe218, Phe220, Arg221), which interact with the NAPA2 region (Figure 3D), are conserved across all human DYRKs (Figure S1). For DYRK1A, an early folded intermediate is implicated in enabling transient Tyr autophosphorylation in cis (Lochhead et al., 2005Lochhead P.A. Sibbet G. Morrice N. Cleghon V. Activation-loop autophosphorylation is mediated by a novel transitional intermediate form of DYRKs.Cell. 2005; 121: 925-936Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). The presence of small N-terminal domains (DH/NAPA1/NAPA2) capable of folding independently and that stabilize the kinase domain may explain how during translation a stabilized and catalytically active conformation can be achieved before translation is complete. The structures of DYRK1A and DYRK2 both show a completely ordered activation segment in a similar conformation (Figure 2). The second tyrosine of the YxY dual-phosphorylation motif (DYRK1A: Tyr321; DYRK2A: Tyr309) is the main mediator of a network of interactions that stabilize the active conformation, including with Arg325 and Arg328 (numbering for DYRK1A) that precede the APE motif, and with the backbone carbonyl of the catalytically important Gln323 (Figure 4A). Related CMGC kinases such as ERK2 have a TxY motif, and phosphorylation of the first resid" @default.
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• W2152705959 date "2013-06-01" @default.
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• W2152705959 title "Structures of Down Syndrome Kinases, DYRKs, Reveal Mechanisms of Kinase Activation and Substrate Recognition" @default.
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• W2152705959 doi "https://doi.org/10.1016/j.str.2013.03.012" @default.
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