DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K m ؍ 35 M) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogenactivated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P ؊3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.DYRK1A is a nuclear protein kinase that is ubiquitously expressed in rat tissues (1, 2). We have recently characterized a closely related isoform, DYRK1B, that is predominantly expressed in testis and muscle (3). The homolog of DYRK1A and DYRK1B in Drosophila, the protein kinase MNB, is encoded by the minibrain gene whose mutation results in specific defects in the development of the central nervous system (4). The human gene for DYRK1A is located in the "Down syndrome critical region" of chromosome 21, and the similarity of DYRK1A and MNB suggests that the triplication of the DYRK1A gene may play a role in mental retardation of patients with Down syndrome (5-9).DYRK1A, DYRK1B, and MNB belong to a subfamily of protein kinases with structurally related catalytic domains and similar enzymatic properties (2, 10). At least 7 different DYRKrelated kinases exist in mammals, of which DYRK1A and DYRK1B are targeted to the nucleus, whereas DYRK2 and DYRK3 are located in the cytoplasm (2, 3).1 Members of the DYRK family have also been found in lower eukaryotes, such as Yak1p in Saccharomyces cerevisiae (11), Pom1p in Schizosaccharomyces pombe (12), and YAKA in Dictyostelium discoideum (13). Although mutations in YAK1, pom1, and yakA have diverse phenotypic consequences, it appears reasonable to generalize that DYRK-related kinases are involved in the regulation of growth and development.The enzymatic activity of DYRK1A has been shown to depend on the presence of tyrosine residues in the activation loop, a result that suggests an activation mechanism similar to that of the MAP 2 kinases (1). However, the participation of DYRK1A or other DYRK-related kinases in a particular signal transduction pathway has not been eluci...
The assembly of the polymerase complex of influenza A virus from the three viral polymerase subunits PB1, PB2, and PA is required for viral RNA synthesis. We show that peptides which specifically bind to the protein-protein interaction domains in the subunits responsible for complex formation interfere with polymerase complex assembly and inhibit viral replication. Specifically, we provide evidence that a 25-amino-acid peptide corresponding to the PA-binding domain of PB1 blocks the polymerase activity of influenza A virus and inhibits viral spread. Targeting polymerase subunit interactions therefore provides a novel strategy to develop antiviral compounds against influenza A virus or other viruses.Influenza A viruses are human pathogens that are responsible for both annual epidemics and recurring devastating pandemics. The recent emergence of highly pathogenic avian influenza virus strains of the H5N1 subtype and the lethality associated with these viruses reflect the continuing threat of influenza viruses. Despite the existence of vaccines against annually recurring influenza A and B virus strains, as well as antiviral drugs targeting either the viral neuraminidase (9) or M2 proteins (14), the protection against epidemic and pandemic influenza is still incomplete. The viral RNA-dependent RNA polymerase consisting of three subunits, PA, PB1, and PB2 (the P proteins), is a potential target for the development of new anti-influenza drugs. PB1 is the central protein, containing two different domains interacting with the PB2 and the PA subunits (1,7,13,16). We hypothesized that viral RNA synthesis could be blocked by the specific inhibition of viral polymerase complex formation by using small peptides which bind to the protein-protein interaction domains responsible for hetero-oligomerization between the individual subunits. Here, we describe the characterization of a PB1-derived short peptide that fulfils these criteria.The first 25 aa of PB1 fused to GFP bind to the viral polymerase subunit PA and inhibit polymerase activity. To design an inhibitory peptide, we selected the region encompassing amino acids (aa) 1 to 25 of the PB1 polymerase subunit of influenza A virus (A/WSN/33), which has been demonstrated previously to bind the PA subunit (16, 17) and is highly conserved among influenza A virus strains (Fig. 1A). To confirm that this region indeed binds PA, we fused this sequence to the N terminus of the green fluorescent protein (GFP), transiently expressed the fusion protein (PB1 1-25 -GFP) together with hemagglutinin (HA)-tagged PA (PA-HA) in HEK293T cells, and immunoprecipitated PA-HA from cell extracts by using anti-HA antibodies. As shown in Fig. 1B, PB1 1-25 -GFP, but not Flag-tagged GFP, was specifically immunoprecipitated with PA-HA (lane 3). In a similar experiment, we also tested whether the PB1 aa 715 to 740, which are embedded in a larger PB2-binding domain (13), were sufficient for binding PB2 when fused to GFP (PB1 715-740 -GFP). However, PB1 715-740 -GFP could not be immunoprecipitated with HA-t...
Arrays of octameric peptide libraries on cellulose paper were screened by using 32 protein kinase inhibitor ͉ combinatorial libraries ͉ SPOT method ͉ membrane translocation signal ͉ smooth muscle T he cGMP-dependent protein kinases type I␣ and I (cGPK) act directly downstream in the NO-mediated signaling pathway, controlling a variety of cellular responses, ranging from smooth muscle cell relaxation to neuronal synaptic plasticity (1, 2). The structural similarity of cGPK and its closest relative, the cAMP-dependent protein kinase (cAPK), has made it difficult to study cGPK pathways independent of those mediated by cAPK, primarily because of the lack of potent and selective cGPK inhibitors. Because recent studies have suggested that cAMP and cGMP are each able to cross-activate either cGPK or cAPK under physiological conditions, the specific role for cGPK within the NO͞cGMP-mediated signaling pathway remains obscure (for a review see ref. 1). However, recent advances have clearly identified specific intracellular targets for the cGPK isozymes I␣ and I (3, 4). Also, inactivation of the genes for cGPK I␣͞I and cGPK II showed that the cGPK isozymes regulate distinct cellular functions by pathways separate from those mediated by cAPK (5, 6).Attempts to identify cGPK-selective inhibitor peptides based on the autoinhibitory domain of the enzyme or in vivo substrates have been tedious at best, because of the lack of a well defined consensus sequence. Only a relative preference for basic residues surrounding the phosphate acceptor site has been established (7). Various synthetic peptides have been used to analyze the sequence requirements for cGPK substrates (8-11). Recently, we developed an iterative approach using phosphorylation of peptide libraries on cellulose paper to determine a priori the substrate specificity of cGPK versus cAPK. Consequently, we identified the cGPK substrate sequence TQAKRKKSLAM-FLR, in which the serine represents the phosphate-acceptor site (12, 13). Substitution of this serine by alanine yielded cGPK inhibitors with K i values of 7.5-22 M (13) and improved cGPK͞cAPK selectivity, as has been reported with other synthetic peptide derivatives (14,15). However, all cGPK peptide inhibitors known so far lack satisfactory potency and selectivity.Here we report a peptide library screen specifically designed to select for tight binding peptides rather than substrate peptides. First, we took advantage of the autophosphorylation properties of cGPK, which provides the means to study the transient enzyme-peptide interactions. Second, we used peptide libraries that lack the phosphate acceptor residues serine and threonine to select for peptide binding over phosphorylation. Linking the best sequence from this screen to membrane translocation signals (MTS) for intracellular delivery resulted in the highly effective cGPK I␣ inhibitors DT-2 and DT-3. Finally, we have demonstrated that both peptides are powerful tools for studying the specific functional roles of cGPK in smooth muscle. MethodsEnzyme Prepa...
There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds.
An iterative approach to the a priori determination of the substrate specificity of cAMP- and cGMP-dependent protein kinases (PKA and PKG) by the use of peptide libraries on cellulose paper is described. The starting point of the investigation was an octamer library with the general structure Ac-XXX12XXX, where X represents mixtures of all 20 natural amino acids and 1 and 2 represent individual amino acid residues. The library thus contained all possible 2.56 x 10(10) octamers, divided into 400 sublibraries with defined amino acids 1 and 2 each consisting of 6.4 x 10(7) sequences. After phosphorylation with the kinases in the presence of [gamma-32P]ATP, the sublibrarys Ac-XXXRRXXX and Ac-XXXRKXXX were identified as the best substrates for PKA and PKG, respectively. The second-generation libraries had the structures Ac-XXXRR12X and Ac-XXXRK12X for PKA and PKG and resulted in the most active sequence pools Ac-XXXRRASX and Ac-XXXRKKSX. After delineation of every position in the octameric sequence and extension of the investigation to decameric peptides, the best sequences, Ac-KRAERKASIY and Ac-TQKARKKSNA, were obtained for PKA and PKG, respectively. Promising octameric and decameric peptides were assembled 5 or 10 times each and assayed in order to determine the experimental scatter inherent in the approach. The kinetic data of several octameric and decameric sequences were determined in solution and compared to data for known substrates. The recognition motif of PKA was confirmed by this approach, and a novel substrate sequence for PKG was identified.(ABSTRACT TRUNCATED AT 250 WORDS)
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