Phosphodiesterases (PDEs) are a superfamily of enzymes that degrade the intracellular second messengers cyclic AMP and cyclic GMP. As essential regulators of cyclic nucleotide signalling with diverse physiological functions, PDEs are drug targets for the treatment of various diseases, including heart failure, depression, asthma, inflammation and erectile dysfunction. Of the 12 PDE gene families, cGMP-specific PDE5 carries out the principal cGMP-hydrolysing activity in human corpus cavernosum tissue. It is well known as the target of sildenafil citrate (Viagra) and other similar drugs for the treatment of erectile dysfunction. Despite the pressing need to develop selective PDE inhibitors as therapeutic drugs, only the cAMP-specific PDE4 structures are currently available. Here we present the three-dimensional structures of the catalytic domain (residues 537-860) of human PDE5 complexed with the three drug molecules sildenafil, tadalafil (Cialis) and vardenafil (Levitra). These structures will provide opportunities to design potent and selective PDE inhibitors with improved pharmacological profiles.
The c-jun N-terminal kinase (JNK) signaling pathway is regulated by JNK-interacting protein-1 (JIP1), which is a scaffolding protein assembling the components of the JNK cascade. Overexpression of JIP1 deactivates the JNK pathway selectively by cytoplasmic retention of JNK and thereby inhibits gene expression mediated by JNK, which occurs in the nucleus. Here, we report the crystal structure of human JNK1 complexed with pepJIP1, the peptide fragment of JIP1, revealing its selectivity for JNK1 over other MAPKs and the allosteric inhibition mechanism. The van der Waals contacts by the three residues (Pro157, Leu160, and Leu162) of pepJIP1 and the hydrogen bonding between Glu329 of JNK1 and Arg156 of pepJIP1 are critical for the selective binding. Binding of the peptide also induces a hinge motion between the Nand C-terminal domains of JNK1 and distorts the ATPbinding cleft, reducing the affinity of the kinase for ATP. In addition, we also determined the ternary complex structure of pepJIP1-bound JNK1 complexed with SP600125, an ATP-competitive inhibitor of JNK, providing the basis for the JNK specificity of the compound.
A number of eukaryotic viruses have evolved mechanisms to downregulate activity of the interferon-induced, double-stranded RNA-activated protein kinase (referred to as P68 based on its Mr of 68,000 in human cells). This control is essential because once activated, the P68 kinase phosphorylates its natural substrate, the alpha subunit of the eukaryotic protein synthesis initiation factor 2 (eIF-2), limiting functional eukaryotic protein synthesis initiation factor 2 available for protein synthesis initiation. We have previously shown that influenza virus encoded a specific mechanism to repress the autophosphorylation and activity of P68. Using in vitro assays for P68 inhibition, we now have purified, to near homogeneity, the P68 repressor from influenza virus-infected cells. The purified product inhibited both the autophosphorylation of P68 as well as phosphorylation of the alpha subunit of eukaryotic protein synthesis initiation factor 2 by the kinase. We tested for both protease and phosphatase activity but found neither activity associated with the purified inhibitor. Surprisingly we found the purified repressor, which had an apparent Mr of approximately 58,000, was a cellular and not a viral-encoded protein. Possible mechanisms by which influenza virus activates this cellular regulator of the protein kinase, thereby minimizing potential antiviral effects of interferon, are discussed.
The interferon-induced RNA-dependent protein kinase (PKR) is considered to play an important role in the r defense against viral infection and, in addition, has been suggested to be a tumor suppressor gene because of its growthsuppressive properties. Activation of PKR by double-stranded RNAs leads to the phosphorylation of the a subunit of eukaryotic initiation factor 2 (eIF-2a) and a resultant block to protein synthesis Initiation. To avoid the consequences of kinase activation, many viruses have developed strategies to down-regulate PKR. Recently, we reported on the purification and characterization of a cellular inhibitor of PKR (referred to as p58), which is activated during influenza virus infection. Subsequent cloning and sequencing has revealed that p58 is a member of the tetripeptide repeat (TPR) family of proteins. To further examine the physiological role of this PKR inhibitor, we stably ransfected NIH 3T3 cells with a eukaryotic expression plasmid contining p58 cDNA under control ofthe cytomegalovirus early promoter. By taking advantage of a recently characterized p58 species-specific monoclonal antibody, we isolated cell lines that overexpressed p58. These cells exhibited a transformed phenotype, owing at faster rates and higher saturation densities and exhibiting anchorage-independent growth. Most importantly, inoculation of nude mice with p58-overexpressing cells gave rise to the production of tumors. Finally, murine PKR activity and endogenous levels of eIF-2a phosphorylation were reduced in the p58-expressing cell lines compared with control cells. These data, taken together, suggest that p58 functions as an oncogene and that one meanism by which the protein induces malnt transformation is through the down-regulation of PKR and subsequent deregulation of protein synthesis. PKR (protein kinase, RNA dependent) is an interferoninduced protein kinase that plays a key role in the cellular defense against viral infection, the regulation of cellular gene expression, and control ofcell growth and proliferation (1-6). Activation by double-stranded RNAs (dsRNAs) or polyanions induces PKR autophosphorylation and, in turn, catalyzes phosphorylation of its natural substrate, the a subunit of the eukaryotic protein synthesis initiation factor, eIF-2a. These events lead to an often dramatic inhibition of protein synthesis initiation (1-3, 7). The activation of PKR can result from interactions with viral-specific RNAs (reviewed in refs.
PRL-3, a novel class protein of prenylated tyrosine phosphatase, is important in cancer metastasis. Due to its high levels of expression in metastatic tumors, PRL-3 may constitute a useful marker for metastasis and might be a new therapeutic target. Here, we present the solution structure of the phosphatase domain of a human PRL-3 (residues 1-162) in phosphatefree state. The nuclear magnetic resonance (NMR) structure of PRL-3 is similar to that of other known phosphatases with minor differences in the secondary structure. But the conformation and flexibility of the loops comprising the active site differ significantly. When phosphate ions or sodium orthovanadate, which is a known inhibitor, are added to the apo PRL-3, the NMR signals from the residues in the active site appeared and could be assigned, indicating that the conformation of the residues has been stabilized.
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