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Sc from >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 antiprion 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 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, Go$ 6976 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.

(1995) J. Biol. Chem. 270, 21277-21284) was investigated with respect to the involved structural elements of SHP-1. Various mutants of SHP-1 were transiently expressed in 293 or COS-7 cells and analyzed for their capacity to associate with immobilized autophosphorylated EGF receptor in vitro and to dephosphorylate coexpressed EGF receptor in intact cells. Inactivating point mutation of the C-terminal SH2 domain reduced the association weakly, point mutation of the N-terminal SH2 domain reduced association strongly and the respective double mutation abolished association totally. The capacity of SHP-1 to dephosphorylate coexpressed EGF receptor was impaired by all point mutations. Truncation of the N-terminal or of both SH2 domains strongly reduced or abolished association, respectively, but the truncated SHP-1 derivatives still dephosphorylated coexpressed EGF receptor effectively.Various chimeric protein-tyrosine phosphatases constructed from SHP-1 and the closely homologous SHP-2 dephosphorylated the EGF receptor when they contained the catalytic domain of SHP-1. As native SHP-2, the chimera lacked activity toward the receptor when they contained the catalytic domain of SHP-2, despite their capacity to associate with the receptor and to dephosphorylate an artificial phosphopeptide. We conclude that the differential interaction of SHP-1 and SHP-2 with the EGF receptor is due to the specificity of the respective catalytic domains rather than to the specificity of the SH2 domains. Functional interaction of native SHP-1 with the EGF receptor requires association mediated by both SH2 domains.The phosphorylation level of activated growth factor receptors with endowed tyrosine kinase activity is the net result of tyrosine specific autophosphorylation (1) and a rapid dephosphorylation by phosphotyrosine-specific phosphatases (PTPs). 1Since important aspects of receptor signaling depend on receptor autophosphorylation, the dephosphorylation reaction is believed to attenuate the receptor signal. Therefore, the identification and characterization of the PTPs involved in growth factor receptor dephosphorylation is of high interest. The SH2 domains containing PTPs SHP-1 and SHP-2 (2, 3) have been shown to interact with multiple growth factor receptors and to modulate their signaling activity. Transient coexpression of SHP-1 with different tyrosine kinase receptors results in complete or partial dephosphorylation of PDGF ␣-and ␤-receptor, insulin-like growth receptor-1 receptor, Kit/SCF receptor, insulin receptor, EGF receptor, and HER2 (4). SHP-2 has little activity with respect to dephosphorylation of associated receptors (4, 5) and rather seems to mediate a receptor signal via as yet not fully understood mechanisms (6 -9). One important pathway seems to involve tyrosine phosphorylation of receptorbound SHP-2 at the C terminus, which leads to subsequent association of Grb2 and activation of the Sos/Ras/Raf/MAPKsignaling cascade (10, 11). Depending on the cellular context, however, SHP-2 may be involved in silencing of cer...

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