Some protein kinases are known to acquire resistance to selective small molecule inhibitors upon mutation of a conserved threonine at the ATP binding site to a larger residue. Here, we performed a comprehensive mutational analysis of this structural element and determined the cellular sensitivities of several disease-relevant tyrosine kinases against various inhibitors. Mutant kinases possessing a larger side chain at the critical site showed resistance to most compounds tested, such as ZD1839, PP1, AG1296, STI571, and a pyrido[2,3-d]pyrimidine inhibitor. In contrast, indolinones affected both wild-type and mutant kinases with similar potencies. Resistant mutants were established for pharmacological analysis of betaPDGF receptor-mediated signaling and allowed the generation of a drug-inducible system of cellular Src kinase activity. Our data establish a conserved structural determinant of protein kinase sensitivity relevant for both signal transduction research and drug development.

Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC 50 values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKC␣ or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome. Molecular & Cellular Proteomics 3:490 -500, 2004.

Structural parameters of both inhibitors and kinases contribute to the complexity of designing kinase inhibitors. Kinase inhibitors that target the inactive state of a kinase have become a novel rule in the design of highly active and selective compounds. The combination of high-resolution structures of ligand-enzyme complexes with especially detailed kinetic studies will in the long-term help to develop new low-molecular weight type II inhibitors.

Structural parameters of both inhibitors and kinases contribute to the complexity of designing kinase inhibitors. The continued study of high-resolution structures of ligand-enzyme complexes in combination with a more dynamic understanding of accessible conformational states of the target proteins, supported by detailed kinetic studies, will in the long-term help in developing new low-molecular weight kinase inhibitors.

The hepatitis C virus (HCV) NS5A protein is highly phosphorylated by cellular protein kinases. To study how NS5A might be integrated in cellular kinase signalling, we isolated phosphoproteins from HuH-7 hepatoma cells that specifically interacted with recombinant NS5A protein. Subsequent mass spectrometry identified the adaptor protein amphiphysin II as a novel interaction partner of NS5A. Mutational analysis revealed that complex formation is primarily mediated by a proline-rich region in the C-terminal part of NS5A, which interacts with the amphiphysin II Src homology 3 domain. Importantly, we could further demonstrate specific co-precipitation and cellular colocalization of endogenous amphiphysin II with NS5A in HuH-7 cells carrying a persistently replicating subgenomic HCV replicon. Although the NS5A-amphiphysin II interaction appeared to be dispensable for replication of these HCV RNAs in cell culture, our results indicate that NS5A-amphiphysin II complex formation might be of physiological relevance for the HCV life cycle.Chronic infection with hepatitis C virus (HCV) bears a substantial risk of developing severe liver disease such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The interactions between cellular proteins and HCV gene products may provide clues for novel approaches to interfere with virus propagation and pathogenesis. The HCV non-structural protein 5A (NS5A) became the focus of studies concerning cellular binding partners when it was reported to be involved in HCV resistance to IFN-a (Enomoto et al., 1995. NS5A is presumed to be a component of the membrane-associated complex of HCV proteins that replicates the plus-strand RNA genome via a minus-strand RNA intermediate (Brass et al., 2002; Hijikata et al., 1993;Shirota et al., 2002). Cell culture-adaptive mutations in the NS5A sequence significantly enhance the replication efficiency of HCV replicons, supporting its role in RNA replication (Blight et al., 2000;Krieger et al., 2001;Lohmann et al., 2001).NS5A proteins of some HCV isolates associate with IFNinduced double-stranded RNA-activated protein kinase (PKR) and inhibit PKR activity (Gale et al., 1997(Gale et al., , 1998. In addition, mechanisms for PKR-independent repression of IFN action by NS5A, such as the induction of IL-8 expression by transcriptional stimulation, have been reported (Polyak et al., 2001). Transcriptional activation mediated by NS5A is most pronounced for N-terminally truncated NS5A, which is transported into the nucleus, in contrast to the perinuclear, cytoplasmic full-length protein (Enomoto et al., 1996; Kato et al., 1997;Tanimoto et al., 1997). A cellular transcription factor (Ghosh et al., 2000), as well as a putative nucleoplasmic transporter, karyopherin b3 (Chung et al., 2000), were found as NS5A interaction partners in yeast two-hybrid screens. Ectopically expressed NS5A protein has been described to interact with Grb2 (Tan et al., 1999), p53 (Majumder et al., 2001, Cdk1 (Arima et al., 2001) and TRAF-2 (Park et al., 2002) and to cau...