Conceived and designed the experiments: JH DM-S NL MAJF VC. Performed the experiments: JH DM-S NL AS GS VC. Analyzed the data: JH DM-S NL VC. Contributed reagents/materials/analysis tools: MAJF. Wrote the paper: JH DM-S VC.
The authors have declared that no competing interests exist.
DYRK-family kinases employ an intramolecular mechanism to autophosphorylate a critical tyrosine residue in the activation loop. Once phosphorylated, DYRKs lose tyrosine kinase activity and function as serine/threonine kinases. DYRKs have been characterized in organisms from yeast to human; however, all entities belong to the Unikont supergroup, only one of five eukaryotic supergroups. To assess the evolutionary age and conservation of the DYRK intramolecular kinase-activation mechanism, we surveyed 21 genomes representing four of the five eukaryotic supergroups for the presence of DYRKs. We also analyzed the activation mechanism of the sole DYRK (class 2 DYRK) present in
Phosphorylation of activation loop residues is a wide-spread and important mechanism for regulating the catalytic activity of a protein kinase. Activation loop phosphorylation may be carried out by a separate protein kinase as part of a signaling cascade, or a kinase can autophosphorylate using an intermolecular or intramolecular mechanism
The DYRK family can be further subdivided into five subfamilies: class 1 (or DYRK1), class 2 (or DYRK2), Yaks, HIPKs (homeodomain-interacting protein kinase), and PRP4s (pre-messenger RNA [mRNA]-processing protein 4)
Class 1 and 2 DYRKs are the best-characterized subfamilies, particularly with regard to understanding activation mechanisms or regulatory events controlling the kinase activity of these enzymes. Class 1 and 2 subfamily members share a conserved central kinase domain and an adjacent N-terminal DH-box (DYRK homology box) but have divergent extended N- and C-terminal regions
All class 1 and 2 DYRKs as well as YAKs characterized to date contain a YxY motif in the activation loop; but only the second tyrosine residue in this motif is phosphorylated, an autocatalytic event that is essential for full serine/threonine kinase activity of the protein
Eukaryotes derive from a common ancestor. The advent of modern molecular biology including DNA sequencing has provided new insights into the relatedness and evolution of eukaryotic organisms leading to a re-examination of the eukaryotic phylogenetic tree. Based on accumulating evidence from morphological comparisons, biochemical pathways, and molecular phylogenetic approaches current evolutionary hypotheses have assembled eukaryotes into five or six supergroups
In this report, we have taken advantage of the availability of completely sequenced genomes from highly diverse organisms to interrogate the evolutionary conservation and phylogenetic distribution of DYRK family members. We have also compared NAPA region function across evolutionary time, by cloning and analyzing the sole class 2 DYRK encoded by T.
Our analysis demonstrates the evolutionary ancient nature of the DYRK family, and provides evidence that class 2 DYRKs were present in the primordial or root eukaryote. Our functional analysis of TbDYRK2 provides further evidence for the conservation of the mechanism of intramolecular phosphorylation of the DYRK activation loop and the potential role of the NAPA domain in this process.
Anti-Flag M2-agarose and anti-Flag antibodies were purchased from Sigma (St. Louis, MO). Anti-phosphotyrosine 4G10, was purchased from Millipore (Temecula, CA). Horseradish peroxidase conjugated with anti-mouse IgG, protein G sepharase, were obtained from GE Healthcare (Amersham, UK). The QuickChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). Clean-blot IP detection reagent (HRP) was purchased from Thermo Scientific (Logan, Utah). The SuperSignal west pico chemiluminescent reagent for immunoblot detection was from Amersham Biosciences Inc. (Piscataway, NJ). All PCR and mutagenesis primers were synthesized by Integrated DNA Technologies (Skokie, IL).
The open reading frame from the DYRK2 gene of
The generation of baculoviruses expressing FLAG epitope-tagged WT-, K227M-, ΔN1-, and ΔN2-DmDYRK2 has been described previously
Human Nt-DYRK2 was constructed using the first 447 nucleotides of the Dyrk2 isoform 1 (NM_003583) open reading frame (encoding the first 149 amino acids of the protein). To the 5′-end of the coding sequences, we added an EcoR1 restriction site and consensus Kozak sequences; and in frame FLAG-epitope encoding sequence immediately following the initiating-Met ATG codon. At the 3′-end of the coding sequences, a stop codon followed by a second EcoR1 site was added. The EcoR1-EcoR1 fragment was then cloned into EcoR1 linearized pcDNA3 to generate HsNt-DYRK2.
Constructs encoding the various proteins were then subcloned into the baculoviral shuttle vector pVL1393 (BD Biosciences). The various pVL1393 constructs were then used in conjunction with BaculoGold DNA (BD Biosciences) to generate recombinant baculovirus for expression in Sf9 (Spodoptera frugiperda 9) cells as described previously
For most immunoprecipitations, Sf9 cell extracts (0.15 mg) were incubated with Protein G-Sepharose conjugated to anti-FLAG (Sigma) or anti-DmDYRK2 antibody for 1 h on a rotating platform at 4°C. Efficient immunoprecipitation of ΔN2-TbDYRK2 required longer incubation times with the anti-FLAG antibody (up to 10 h). Immunoprecipitates were pelleted, washed twice with 1 ml buffer A [20 mM tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, and 1% Igepal CA-630] and once with 1 ml of ice-cold TBS buffer (20 mM tris-HCl [pH 7.6], 137 mM NaCl). Immunoprecipitated proteins were separated by SDS–polyacrylamide gradient gel (4–20% NuPAGE gels) electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes, blocked with 3% bovine serum albumin in 20 mM tris-HCl (pH 7.6), 137 mM NaCl, containing 0.2% Tween-20 (TBS-T), and probed with a monoclonal anti-FLAG antibody (1∶5000 dilution) or anti-phosphotyrosine antibody (1∶2000 dilution). Immunoblots were then incubated with HRP horseradish peroxidase conjugate (1∶1000) dilution, and visualized by SuperSignal west pico chemiluminescent reagent detection according to the protocol of the manufacturer (Amersham Biosciences).
For peptide kinase assays, WT and mutant forms of TbDYRK2 were immunoprecipitated in triplicate, washed three times in lysis (1% NP40 buffer) buffer, washed once in kinase buffer (30 mM HEPES [pH 7.4], 10 mM MgCl2, 1 mM DTT), and incubated with kinase buffer containing ATP and 50 microM Woodtide as described previously
The identification of DYRKs was done by scanning the predicted peptide datasets from 21 distinct eukaryotic genomes (
Taxonomy and species | Common name/description | Source database (version) | Predicted number of protein genes | Number of DYRKs |
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JGI (2.0) | 14002 | 3 |
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Kinomer | 9766 | 2 |
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Kinomer | 22194 | 7 |
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JGI (1.0) | 9196 | 4 |
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JGI (1.0) | 27273 | 5 |
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JGI (4.1) | 27710 | 6 |
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Recently, genome sequencing projects have made available information that allows us to ask questions concerning the conservation and evolution of proteins and protein motifs. We were interested in comparing the evolutionary depth of the various DYRK subfamilies. Keeling et al
DYRK family members identified in our search of 21 sequenced genomes clustered into five separate subfamilies (class 1, class 2, HIPK, PRP4, and Yak). Individual DYRK subfamilies were then placed into their respective eukaryotic supergroup.
As this broad representation of class 2 DYRKs has not been previously compared, we performed a multiple protein alignment using our class 2 dataset. The NAPA regions as well as the DH-box were found to be conserved in the class 2 DYRKs identified in all four eukaryotic supergroups (
To functionally compare class 2 DYRKs across evolutionary time, we cloned, and analyzed the sole DYRK family member, a class 2 DYRK, identified in the excavate
(A) Amino acid sequence of TbDYRK2. The conserved NAPA-1, DH-box, and NAPA-2 regions are underlined; the central kinase domain is boxed in gray; and the atypical activation loop sequences D-L-G and F-T-Y are boxed in white. (B) Multiple protein alignment of the NAPA and DH-box regions of TbDYRK2, DmDYRK2 and HsDYRK2. Symbols * invariant, : conservative substitutions, and . semi-conserved substitutions. TbDYRK2 mutant proteins affecting the NAPA regions as described later include ΔNAPA-1 (removal of only the indicated NAPA-1 region), ΔN2 (all N-terminal residues up to lower triangle removed), and D103V (conserved Asp in NAPA-2 altered to Val indicated by lower arrow).
For an initial characterization of TbDYRK2, we compared levels of tyrosine phosphorylation and the kinase activity of the WT molecule to a kinase-inactive form of the protein (K138M). We also compared the WT protein to an activation loop mutant (Y269F) where we modified the analogous tyrosine residue shown to be phosphorylated in other DYRKs. To monitor expression of WT and mutant proteins, a Flag-epitope was fused to the N-terminus and the modified proteins were expressed using recombinant baculovirus-infected Sf9 cells. When approximately equal levels of the proteins were compared, WT-TbDYRK2 was found to be tyrosine phosphorylated whereas the kinase-inactive and activation loop mutants displayed little if any tyrosine phosphorylation (
WT and mutant forms of TbDYRK2 were expressed in Sf9 cells and immunoprecipitated from cell extracts with anti-FLAG antibody. TbDYRK2 proteins analyzed include full length (WT), kinase inactive (K138M), activation loop tyrosine to phenylalanine (Y269F), NAPA-1-deletion lacking residues 53–76 (ΔNAPA-1), and point mutation of an invariant NAPA-2 residue (D103V). (A) Immunoprecipitates were subjected to SDS/PAGE and immunoblot analysis. Levels of TbDYRK2 proteins were detected with anti-FLAG antibody (α-FLAG), and activation loop phosphorylation was monitored with anti-phosphotyrosine antibody (α-pTyr). Experiments were performed at least three times. (B) Immunocomplexes of these same TbDYRK2 proteins were prepared in triplicate as above, and the kinase activity was measured using Woodtide as substrate as described in
Next we compared the ability of the WT and mutant proteins to phosphorylate the DYRK substrate, Woodtide. For this assay, TbDYRK2 proteins were immunoprecipitated and the kinase activity of the immunocomplexes determined by incubation with the Woodtide peptide. The results paralleled the levels of tyrosine phosphorylation, the WT molecule was significantly more active than either the kinase-inactive or activation loop mutant (
We recently demonstrated that the NAPA-1 and 2 regions were essential for tyrosine autophosphorylation and the subsequent kinase activity of
Normally, the NAPA-1 region functions in
The N-terminus of DmDYRK2 used in
The activation loop phosphorylation-defective mutant ΔN2-DmDYRK2 (ΔN2) was expressed alone in Sf9 cells; or it was coexpressed with full-length WT DmDYRK2 (WT), or with the entire non-catalytic N-terminus of either
We also created an analogous mutation in TbDYRK2 (ΔN2-TbDYRK2; see
We also took this opportunity to explore the
The N-terminus (Nt) of DmDYRK2 was assessed for its ability to complement activation loop phosphorylation by ΔN1-DmDYRK2 (ΔN1) and ΔN2-DmDYRK2 (ΔN2). (A) ΔN1-DmDYRK2 and ΔN2-DmDYRK2 were expressed alone in Sf9 cells or they were co-expressed with Nt-DmDYRK2 (Nt), as indicated. WT- and kinase-inactive K227M-DmDYRK2 proteins were included as controls. (A and B) DmDYRK2 proteins containing the kinase domain were immunoprecipitated from cell lysates with anti-DmDYRK2 antibody, fractionated by SDS/PAGE and analyzed on immunoblots. (A) Levels of proteins were compared by probing blots with anti-FLAG antibody (α-FLAG) and (B) levels of activation loop autophosphorylation were assessed with anti-pTyr antibody (α-pTyr). (C) To confirm that all proteins were expressed, Sf9 cell lysates were fractionated by SDS/PAGE and the levels of the DmDYRK2 proteins determined by probing with anti-FLAG antibody (α-FLAG Lysates). The experiments shown were performed at least three separate times.
For
The N-terminal DmDYRK2 molecules shown schematically in (A) were assessed for their ability to complement activation loop phosphorylation by ΔN2-DmDYRK2. All proteins contain a FLAG epitope. The presence and location of the conserved NAPA-1, NAPA-2, and DH box regions within the truncated proteins are indicated. (B) ΔN2-DmDYRK2 was expressed alone in Sf9 cells or it was co-expressed with N-terminal truncated proteins as indicated. WT- and kinase-inactive K227M-DmDYRK2 proteins were included as controls. In the top two panels of B, DmDYRK2 proteins containing the kinase domain were immunoprecipitated from cell lysates with anti-DmDYRK2 antibody, fractionated by SDS/PAGE and analyzed on immunoblots. Levels of proteins were compared by probing blots with anti-FLAG antibody (α-FLAG) and levels of activation loop phosphorylation were assessed with anti-pTyr antibody (α-pTyr). To confirm that all proteins were expressed, Sf9 cell lysates were fractionated by SDS/PAGE and the levels of the DmDYRK2 proteins determined by probing with anti-FLAG antibody (α-FLAG Lysates). The experiments shown were performed at least three separate times.
Taken together, these results indicate that the NAPA-1 region appears to be both necessary and sufficient for trans-complementation of ΔN2-DmDYRK2. In contrast, these results indicate that the DH-box and NAPA-2 regions may be required in cis for efficient activation loop tyrosine autophosphorylation.
In this study we show that the DYRK-family of protein kinases has deep evolutionary roots with family members present in all four eukaryotic supergroups for which genome sequencing data is available (
The presence of DYRK-like proteins has been noted in trypanosomes
Previously, we identified regions (termed NAPA-1 and 2) in the non-catalytic N-terminus of
As noted, TbDYRK2 contains an atypical DLG triplet at the beginning of the activation loop, a sequence shared with
In this study we have provided evidence that WT-TbDYRK2 is tyrosine phosphorylated (
Nucleotide sequence and conceptual translation of TbDYRK2.
(DOC)
Phylogenetic tree of the DYRKs. DYRK family members identified in our search of 21 sequenced genomes clustered into five separate subfamilies: class 1, class 2, HIPK, PRP4, and Yak.
(TIF)
Multiple protein alignment of class 2 DYRKs.
(TIF)
WT and mutant forms of TbDYRK2 were expressed in Sf9 cells and immunoprecipitated with anti-FLAG antibody. TbDYRK2 proteins analyzed include full length (WT), kinase inactive (K138M), activation loop tyrosine to phenylalanine (Y269F), N-terminal -deletion lacking residues 1–78 (ΔN2), and the entire non-catalytic N-terminus (Nt) of the molecule. ΔN2 was expressed alone in Sf9 cells; or it was coexpressed with Nt as indicated. Immunoprecipitates were subjected to SDS/PAGE and immunoblot analysis. Levels of TbDYRK2 proteins were detected with anti-FLAG antibody (α-FLAG), and activation loop phosphorylation was monitored with anti-phosphotyrosine antibody (α-pTyr). Large (*) and small (**) IgG background bands are indicated. Experiments were performed at least three times.
(TIF)
CLUSTAL W (1.83) multiple sequence alignment of class 2 DYRKs from the trypanosomatids
(DOC)
Identifiers of individual DYRK subfamily members categorized in each eukaryotic supergroup.
(DOCX)