The evolutionary age of the DYRK subfamilies.

<p>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.</p

Activation loop phosphorylation by ΔN1-DmDYRK2 cannot be rescued in <i>trans</i>.

<p>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.</p

NAPA-1 is the critical region for <i>trans</i>-complementation.

<p>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.</p

Class 2 DYRK from <i>Trypanosoma brucei</i>.

<p>(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).</p

Classification and statistics of the 21 eukaryotic genomes scanned for DYRKs.

<p>Classification and statistics of the 21 eukaryotic genomes scanned for DYRKs.</p

The NAPA-1 region is functionally conserved across species.

<p>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 <i>Drosophila</i> DmDYRK2 (DmD2 Nt), human DYRK2 (HsD2 Nt), or <i>T. brucei</i> TbDYRK2 (TbDYRK2 Nt), as indicated. (A) Proteins were immunoprecipitated from Sf9 cell lysates with anti-FLAG antibody, fractionated by SDS/PAGE, and analyzed on immunoblots. Protein levels were monitored by probing with anti-FLAG antibody (α-FLAG). (B) WT and ΔN2-DmDYRK2 (proteins containing the kinase domain) were immunoprecipitated from cell lysates with an antibody against the C-terminus of DmDYRK2, fractionated by SDS/PAGE, and analyzed on immunoblots. The ability of the full-length DmDYRK2, and N-terminal DYRK2 proteins from <i>Drosophila</i>, human and <i>Trypanosoma</i> to complement tyrosine activation loop phosphorylation by ΔN2-DmDYRK2 in <i>trans</i> was assessed by probing blots with anti-pTyr antibody (α-pTyr). Results are representative of three different experiments.</p

NAPA-1 and NAPA-2 are essential for TbDYRK2 activation loop phosphorylation.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029702#s2" target="_blank">Materials and Methods</a>. The results are presented as the means ± SD (n = 3).</p

Chemical Proteomic Analysis Reveals the Drugability of the Kinome of <i>Trypanosoma brucei</i>

The protozoan parasite <i>Trypanosoma brucei</i> is the causative agent of African sleeping sickness, and there is an urgent unmet need for improved treatments. Parasite protein kinases are attractive drug targets, provided that the host and parasite kinomes are sufficiently divergent to allow specific inhibition to be achieved. Current drug discovery efforts are hampered by the fact that comprehensive assay panels for parasite targets have not yet been developed. Here, we employ a kinase-focused chemoproteomics strategy that enables the simultaneous profiling of kinase inhibitor potencies against more than 50 endogenously expressed <i>T. brucei</i> kinases in parasite cell extracts. The data reveal that <i>T. brucei</i> kinases are sensitive to typical kinase inhibitors with nanomolar potency and demonstrate the potential for the development of species-specific inhibitors