Search

The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 microM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates.

Small molecule inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were developed as antagonists of protein tyrosine kinases implicated in cancer progression. Derivatives from this compound class are effective against most of the imatinib mesylate-resistant BCR-ABL mutants isolated from advanced chronic myeloid leukemia patients. Here, we established an efficient proteomics method employing an immobilized pyrido[2,3-d]pyrimidine ligand as an affinity probe and identified more than 30 human protein kinases affected by this class of compounds. Remarkably, in vitro kinase assays revealed that the serine/threonine kinases Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) and p38alpha were among the most potently inhibited kinase targets. Thus, pyrido[2,3-d]pyrimidines did not discriminate between tyrosine and serine/threonine kinases. Instead, we found that these inhibitors are quite selective for protein kinases possessing a conserved small amino acid residue such as threonine at a critical site of the ATP binding pocket. We further demonstrated inhibition of both p38 and RICK kinase activities in intact cells upon pyrido[2,3-d]pyrimidine inhibitor treatment. Moreover, the established functions of these two kinases as signal transducers of inflammatory responses could be correlated with a potent in vivo inhibition of cytokine production by a pyrido[2,3-d]pyrimidine compound. Thus, our data demonstrate the utility of proteomic methods employing immobilized kinase inhibitors for identifying new targets linked to previously unrecognized therapeutic applications.

The conversion of the cellular prion protein (PrP(c)) into pathologic PrP(Sc) and the accumulation of aggregated PrP(Sc) are hallmarks of prion diseases. A variety of experimental approaches to interfere with prion conversion have been reported. Our interest was whether interference with intracellular signaling events has an impact on this conversion process. We screened approximately 50 prototype inhibitors of specific signaling pathways in prion-infected cells for their capacity to affect prion conversion. The tyrosine kinase inhibitor STI571 was highly effective against PrP(Sc) propagation, with an IC(50) of < or =1 microM. STI571 cleared prion-infected cells in a time- and dose-dependent manner from PrP(Sc) without influencing biogenesis, localization, or biochemical features of PrP(c). Interestingly, this compound did not interfere with the de novo formation of PrP(Sc) but activated the lysosomal degradation of pre-existing PrP(Sc), lowering the half-life of PrP(Sc) from > or =24 h to <9 h. Our data indicate that among the kinases known to be inhibited by STI571, c-Abl is likely responsible for the observed anti-prion effect. Taken together, we demonstrate that treatment with STI571 strongly activates the lysosomal degradation of PrP(Sc) and that substances specifically interfering with cellular signaling pathways might represent a novel class of anti-prion compounds.

The adapter kinase receptor interacting protein-like interacting caspase-like apoptosis regulatory protein kinase (RICK, also called RIP2 and CARDIAK) was found to be elevated at both the protein and RNA levels during human cytomegalovirus (HCMV) replication, suggesting either that the virus may require RICK for replication or that RICK is part of an unsuccessful host attempt to inhibit HCMV replication. It is demonstrated here that forced expression of RICK in either a kinase active or inactive form activates nuclear factor (NF)-kappaB by means of its intermediate domain and potently blocks HCMV replication in human fibroblasts. Importantly, NF-kappaB activation, which exerted a modestly positive effect on the early phase of infection, clearly had a strongly negative impact during later viral steps. A stable inhibitor of NF-kappaB (IkappaB) reverses the RICK inhibitory effect, and activation of NF-kappaB by IkappaB kinase beta expression is inhibitory to HCMV, demonstrating that NF-kappaB activation is part of a potent anti-HCMV response. Supernatant transfer experiments identified interferon-beta as a downstream component of the RICK inhibitory pathway. RICK expression was found to synergize with HCMV infection in the induction of interferon-beta expression. This study identifies an endogenous RICK-activated, NF-kappaB- and interferon-beta-dependent antiviral pathway that is either inhibited or faulty under normal HCMV replication conditions; efforts to bolster this pathway may lead to novel anti-viral approaches.

Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38alpha whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action.

SHP-2, a cytosolic protein tyrosine phosphatase with two SH2 domains and multiple tyrosine phosphorylation sites, contributes to signal transduction as an enzyme and/or adaptor molecule. Here we demonstrate that prolactin (PRL) stimulation of the PRL-responsive Nb2 cells, a rat lymphoma cell line, and T47D cells, a human breast cancer cell line, lead to the complex formation of SHP-2 and growth factor receptor-bound protein-2 (grb2). Using transient co-overexpression studies of the prolactin receptor (PRLR) and several tyrosine to phenylalanine mutants of SHP-2, we show that grb2 associates with SHP-2 through the C-terminal tyrosine residues of SHP-2, Y(546) and Y(584). Furthermore, in this study, we found a highly phosphorylated, 29-kDa protein (p29), a substrate of SHP-2. The recruitment of p29 to SHP-2 requires the carboxy-terminal tyrosine residues of SHP-2 (Y(546) and Y(584)). Together, our results indicate that SHP-2 may function as an adaptor molecule downstream of the PRLR and highlight a new recruitment mechanism of SHP-2 substrates., (Copyright 2002 Elsevier Science Inc.)

The protein kinase pUL97, encoded by human cytomegalovirus (HCMV), is an important determinant of virus replication. Recently, indolocarbazoles were identified as a class of substances that inhibit the pUL97 kinase activity in vitro. In parallel, it was shown that indolocarbazoles interfere with HCMV replication; however, the causal relationship between inhibition of pUL97 kinase activity and virus replication has not been clarified. Here evidence is provided that indolocarbazole-mediated inhibition of virus replication is a direct result of diminished pUL97 protein kinase activity. In cell culture infections, a strong and selective antiviral activity was measured with respect to several strains of HCMV in contrast with other related or non-related viruses. For fine quantification, recombinant HCMVs expressing green fluorescent protein were used, demonstrating the high sensitivity towards compounds NGIC-I and Gö6976. Interestingly, a ganciclovir-resistant virus mutant (UL97-M460I) showed increased sensitivity to both compounds. Supporting this concept, transfection experiments with cloned pUL97 revealed that ganciclovir-resistant mutants were characterized by reduced levels of autophosphorylation compared with wild-type and possessed particularly high sensitivity to indolocarbazoles. Moreover, the Epstein-Barr virus-encoded homologous kinase, BGLF4, which showed a similar pattern of autophosphorylation and ganciclovir phosphorylation activities, was not inhibited. Importantly, a cytomegalovirus deletion mutant, lacking a functional UL97 gene and showing a severe impairment of replication, was completely insensitive to indolocarbazoles. Thus, our findings indicate that a specific block in the activity of pUL97 is the critical step in indolocarbazole-mediated inhibition of virus replication and that pUL97 might be targeted very efficiently by a novel antiviral therapy.

The UL97-encoded protein kinase (pUL97) of human cytomegalovirus (HCMV) plays a critical role in the control of virus replication. Deletion of the UL97 gene results in a drastic reduction in the replication efficiency. Although the exact function of pUL97 remains unclear and its sensitivity to specific inhibitors is speculative, protein kinase inhibitors of the indolocarbazole class are effective inhibitors of cytomegalovirus. Based on the phosphorylation of ganciclovir (GCV), a novel quantification system for pUL97 kinase activity was established: the phosphorylated form of GCV exerts an easily quantifiable cytotoxic effect in transfected cells. Importantly, the addition of indolocarbazole compounds, Gö6976 and NGIC-I, which were highly effective at nanomolar concentrations while other protein kinase inhibitors were not, led to a significant reduction of pUL97 kinase activity. It was also demonstrated that a catalytically inactive mutant of pUL97, K355M, and a GCV-resistant mutant, M460I, were both negative for GCV phosphorylation, although protein phosphorylation remained detectable for the latter mutant. In vitro kinase assays were used to confirm the levels of pUL97-mediated phosphorylation recorded. To generate a tool for screening large numbers of putative inhibitors that preferentially interfere with GCV as well as protein phosphorylation, pUL97-expressing cell clones with stable pUL97 kinase activity were selected. This study demonstrates that certain indolocarbazole compounds are potent pUL97 inhibitors and, therefore, represent novel candidates for antiviral drugs that target viral protein kinase functions.

In Rat-1 fibroblasts epidermal growth factor (EGF), but not platelet-derived growth factor (PDGF) stimulates the activity of the c-Jun N-terminal kinase (JNK). Moreover, PDGF induced suppression of EGF-mediated JNK activation, apparently through protein kinase C (PKC) activation. Further analysis revealed that PKD was specifically activated by PDGF but not EGF in Rat-1 cells. In SF126 glioblastoma cells, however, EGF and PDGF synergistically activated JNK, while neither PDGF nor EGF stimulated PKD activity. In this cell line, overexpression of PKD blocked EGF- and PDGF-induced JNK activation. Mutational analysis further revealed that the EGFR mutant (T654/669E) was incapable of activating JNK and provided evidence that PKD-mediated dual phosphorylation of these critical threonine residues leads to suppression of EGF-induced JNK activation. Our results establish a novel crosstalk mechanism which allows signal integration and definition in cells with many different RTKs.

Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor beta-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(L-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D.