An efficient proteomics method to identify the cellular targets of protein kinase inhibitors

Godl, Klaus; Wissing, Josef; Kurtenbach, Alexander; Habenberger, Peter; Blencke, Stephanie; Gutbrod, Heidrun; Salassidis, Kostadinos; Stein-Gerlach, Matthias; Missio, Andrea; Cotten, Matthew; Daub, Henrik

doi:10.1073/pnas.2535024100

Cited by 323 publications

(280 citation statements)

References 30 publications

(34 reference statements)

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“…Now, even data using low concentrations of the drug need to be re-evaluated, since it has been reported to inhibit some targets as effectively as and more effectively than it does p38 (11). Therefore, it is necessary to substantiate the results obtained with this inhibitor by alternative methods.…”

Section: Resultsmentioning

confidence: 99%

“…The use of SB203580 has implicated a p38 contribution to axotomy-induced apoptosis of retinal ganglion cells (8), glutamate-induced apoptosis of cerebellar granule neurons (9) and ceramide-induced death of cortical neurons (10). However, it has recently been revealed that SB203580 exhibits higher specificity for kinases other than p38 (11), and at the concentrations typically used to target p38 it can even inhibit JNK-dependent death (13). Therefore, conclusions relying on SB203580 as the only strategy to inhibit p38 must now be reconsidered.…”

Section: Discussionmentioning

confidence: 99%

“…There are proposals that p38 contributes to axotomy-induced apoptosis of retinal ganglion cells, excitotoxicity-induced apoptosis of cerebellar granule neurons, and ceramide-induced death of cortical neurons (8 -10). However, these reports rely on SB203580, the specificity of which has recently been called into question, since it inhibits other kinases more potently than it does p38 (11), and it discriminates poorly between p38 and certain JNK isoforms (12,13). Furthermore, the nature of the cell death under investigation, whether apoptotic, necrotic, or intermediate, is often unclear.…”

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confidence: 99%

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Distinct Requirements for p38α and c-Jun N-terminal Kinase Stress-activated Protein Kinases in Different Forms of Apoptotic Neuronal Death

Cao

Semenova

Solovyan

et al. 2004

Journal of Biological Chemistry

104

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The stress-activated protein kinases c-Jun-activated kinase (JNK) and p38 are implicated in neuronal apoptosis. Early studies in cell lines suggested a requirement for both in the apoptosis induced by withdrawal of nerve growth factor. However, studies in neuronal cells typically implicate JNK but not p38 in apoptosis. In some cases, p38 is implicated, but the role of JNK is undefined. It remains unclear whether p38 and JNK have differing roles dependent on cell type, apoptotic stimulus, or mechanism of cell death or whether they are redundant and each sufficient to induce identical forms of cell death. We investigate the relative roles of these protein kinases in different death mechanisms in a single system, cultured cerebellar granule neurons. Apoptosis induced by withdrawal of trophic support and glutamate are mechanistically different in terms of caspase activation, DNA fragmentation profile, chromatin morphology, and dependence on de novo gene expression. Caspase-independent apoptosis induced by glutamate is accompanied by strong activation of p38, and dominant negatives and inhibitors of the p38 pathway prevent this apoptosis. In contrast, withdrawal of trophic support induces caspase-dependent death accompanied by JNK-dependent phosphorylation of c-Jun, and inhibition of JNK is sufficient to prevent the death induced by withdrawal of trophic support. Inhibition of p38 does not block withdrawal of trophic support-induced death, nor does inhibition of JNK block glutamate-induced death. We propose that mechanistically different forms of apoptosis have differing requirements for p38 and JNK activities in neurons and demonstrate that only inhibition of the appropriate kinase will prevent neurons from undergoing apoptosis.Stress-activated protein kinases (SAPKs) 1 are believed to play an obligatory role in neuronal cell death; however, the relative roles of the JNK and p38 kinase groups have been unclear. Initial studies in the PC12 cell line suggested a role for both JNK and p38 in caspase-dependent cell death induced by withdrawal of nerve growth factor (1). Subsequent studies in primary cultured neurons failed to demonstrate a requirement for p38 in apoptosis induced by deprivation of trophic support, whereas a role for JNK in developmental, trophic withdrawalinduced, excitotoxic, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced death has been substantiated in a variety of neuronal systems (2-7). There are proposals that p38 contributes to axotomy-induced apoptosis of retinal ganglion cells, excitotoxicity-induced apoptosis of cerebellar granule neurons, and ceramide-induced death of cortical neurons (8 -10). However, these reports rely on SB203580, the specificity of which has recently been called into question, since it inhibits other kinases more potently than it does p38 (11), and it discriminates poorly between p38 and certain JNK isoforms (12, 13). Furthermore, the nature of the cell death under investigation, whether apoptotic, necrotic, or intermediate, is often unclear. Whether JNK and p38 ...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Distinct Requirements for p38α and c-Jun N-terminal Kinase Stress-activated Protein Kinases in Different Forms of Apoptotic Neuronal Death

Cao

Semenova

Solovyan

et al. 2004

Journal of Biological Chemistry

104

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“…Classically, identifying targets of SMs through biochemical purification relied on large amounts of starting protein, extensive protein fractionation, stringent wash conditions, gel visualization and excision of specific bands to yield only the most directly and tightly bound proteins (13,16,17). With proteomic MS approaches (18), even affinity pull-down experiments generate large protein catalogs, inflating the list of candidate ''hits'' and requiring, sometimes arbitrary, prioritization of these proteins for validation.…”

mentioning

confidence: 99%

Identifying the proteins to which small-molecule probes and drugs bind in cells

Ong

Schenone

Margolin³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

279

265

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Most small-molecule probes and drugs alter cell circuitry by interacting with 1 or more proteins. A complete understanding of the interacting proteins and their associated protein complexes, whether the compounds are discovered by cell-based phenotypic or targetbased screens, is extremely rare. Such a capability is expected to be highly illuminating-providing strong clues to the mechanisms used by small-molecules to achieve their recognized actions and suggesting potential unrecognized actions. We describe a powerful method combining quantitative proteomics (SILAC) with affinity enrichment to provide unbiased, robust and comprehensive identification of the proteins that bind to small-molecule probes and drugs. The method is scalable and general, requiring little optimization across different compound classes, and has already had a transformative effect on our studies of small-molecule probes. Here, we describe in full detail the application of the method to identify targets of kinase inhibitors and immunophilin binders.SILAC ͉ small molecules ͉ target identification

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“…However, several recent studies have shown that both of these molecules are more promiscuous than previously believed. [3][4][5] In addition to their roles as probes of basic biological processes, many drugs take advantage of differences in the expression patterns of target proteins to achieve the desired therapeutic effects. This is particularly true for antimicrobial, antiviral, and anticancer agents.…”

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confidence: 99%

Engineering small molecule specificity in nearly identical cellular environments

Sellmyer

Stankunas

Briesewitz

et al. 2007

Bioorganic & Medicinal Chemistry Letters

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Methotrexate (MTX), an inhibitor of dihydrofolate reductase, was tethered to an FKBP12 ligand (SLF), and the resulting bifunctional molecule (MTXSLF) potently inhibits either enzyme but not both simultaneously. MTXSLF is cytotoxic to fibroblasts derived from FKBP12-null mice but is detoxified 40-fold by FKBP12 in wild-type fibroblasts. These studies demonstrate that non-target proteins in an otherwise identical genetic background can be used to predictably regulate the biological activity of synthetic molecules. KeywordsSmall molecule specificity; Selective detoxification; Methotrexate; SLF; FKBP Cell-permeable small molecules that perturb the functions of proteins have proven utility as therapeutic agents as well as probes of basic biological processes. 1 In certain therapeutic cases a lack of specificity can be valuable, as inhibition of several related proteins may contribute to the desired outcome. 2 The issue of specificity is more pressing when these probes are used as tools for basic science. When interpreting the results of studies using cell-permeable probes, one would like to be confident that the observed biological responses are directly related to the putative target(s) of a given probe. Wortmannin has been widely used to probe PI3K function, and SB 203580 has similarly been used as an inhibitor of p38 MAP kinase. However, several recent studies have shown that both of these molecules are more promiscuous than previously believed. 3-5In addition to their roles as probes of basic biological processes, many drugs take advantage of differences in the expression patterns of target proteins to achieve the desired therapeutic effects. This is particularly true for antimicrobial, antiviral, and anticancer agents. For example,β-lactam antibiotics inhibit transpeptidase enzymes to achieve their antimicrobial activity, and the lack of similar enzymatic targets in mammals renders these antibiotics quite selective. Similarly, the thymidine kinase enzyme of herpes simplex virus activates the *Corresponding author. Tel +1 650 723 4005; fax +1 650 725 4665; email: wandless@stanford.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. prodrug, acyclovir, in infected cells to achieve antiviral selectivity. However, the therapeutic window is typically much smaller when the target protein is similarly expressed in both target and non-target cells. NIH Public AccessRecently there has been increased research emphasis on modulating the effects of small molecules through covalent linkage of two ligands to create bifunctional molecules. 6 This strategy has been used to block ...

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An efficient proteomics method to identify the cellular targets of protein kinase inhibitors

Cited by 323 publications

References 30 publications

Distinct Requirements for p38α and c-Jun N-terminal Kinase Stress-activated Protein Kinases in Different Forms of Apoptotic Neuronal Death

Distinct Requirements for p38α and c-Jun N-terminal Kinase Stress-activated Protein Kinases in Different Forms of Apoptotic Neuronal Death

Identifying the proteins to which small-molecule probes and drugs bind in cells

Engineering small molecule specificity in nearly identical cellular environments

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