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Matthew P Patricelli,
Tyzoon K Nomanbhoy,
Jiangyue Wu,
Heidi Brown,
David Zhou,
Jianming Zhang,
Subadhra Jagannathan,
Arwin Aban,
Eric Okerberg,
Chris Herring,
Brian Nordin, Helge Weissig,
Qingkai Yang,
Jiing-Dwan Lee,
Nathanael S Gray,
John W Kozarich
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ABSTRACT: Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.
Chemistry & biology 06/2011; 18(6):699-710. · 6.52 Impact Factor
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Yongsheng Liu,
Jiangyue Wu, Helge Weissig,
Juan M Betancort,
Wen Zhi Gai,
Phillip S Leventhal,
Matthew P Patricelli,
Babak Samii,
Anna K Szardenings,
Kevin R Shreder,
John W Kozarich
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ABSTRACT: The synthesis and biochemical characterization of AX4697, a fluorescent, bisindolylmaleimide-derived probe for PKCalpha and beta, is described. AX4697 was able to quantify changes in PKC expression in drug-treated Jurkat cells and was shown to covalently label PKCalpha on C619, a residue that sits just outside the active site.
Bioorganic & medicinal chemistry letters 09/2008; 18(22):5955-8. · 2.65 Impact Factor
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ABSTRACT: The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.
Biochemistry 02/2007; 46(2):350-8. · 3.42 Impact Factor
