Inhibitor Scaffolds as New Allele Specific Kinase Substrates

Kraybill, Brian; Elkin, Lisa; Blethrow, Justin D.; Morgan, David; Shokat, Kevan M.

doi:10.1021/ja0264798

Cited by 53 publications

(58 citation statements)

References 39 publications

(60 reference statements)

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“…1B). This is consistent with previous observations where a similar reduction in catalytic activity was reported for analog-sensitive mutants of v-Src (21). We therefore sought to improve the catalytic activity of RapR-Src-as2 by modifying our approach for kinase activation.…”

Section: Engineering Src Kinase With Independent Activation and Inactsupporting

confidence: 89%

Mimicking transient activation of protein kinases in living cells

Klomp

Huyot

Ray

et al. 2016

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

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Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two proteinengineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1-and Src-PI3K-signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.any physiological and pathological processes are regulated by protein kinases. Therefore, dissection of kinase functions helps to uncover molecular mechanisms and guides the development of new therapeutic strategies (1). However, progress in our understanding of kinase-mediated signaling is often hampered by the limitations of available tools (2). Pharmacological inhibitors of kinases have been valuable for the study of kinase functions, but they often do not provide the desired specificity and they are not available for the majority of kinases (1, 3). Also, the application of inhibitors is limited to the identification of processes affected by the inactivation of the kinase. Activation of a kinase is often achieved by the treatment of cells with growth factors or other extracellular stimuli. However, this approach triggers a multitude of parallel signaling pathways, which significantly complicates the analysis of individual kinase functions (4). Specific activation or inactivation of a kinase can be achieved by genetic modifications. However, this method is susceptible to compensatory artifacts and does not allow us to control the level and timing of kinase activation. Thus, development of novel tools that combine high specificity and tight temporal control of kinase activity remains a necessity. Under physiological conditions, kinases are activated for a finite period, and the duration of this activation is critical for eliciting specific biological outcomes (5, 6). Therefore, to mimic the biological activity of a kinase we need to use methods that allow for transient activation of a kinase with precise temporal control. An optogenetic approach has been successfully used for transient regulation of Raf kinase by dimerization (7). However, this method relies on the fact that Raf dimerization is required for its activation and thus has limited applicability to other kinases. Therefore, development of broadly applicable methods for tightly contro...

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Section: Engineering Src Kinase With Independent Activation and Inactsupporting

confidence: 89%

Mimicking transient activation of protein kinases in living cells

Klomp

Huyot

Ray

et al. 2016

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

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“…[17,28] In contrast, many serine/threonine kinases already have a bulky hydrophobic amino acid at this position (F80 in cdk2). Our enzymatic analysis, in agreement with previous studies, [16] does not indicate any substantial decline in enzymatic activity of cdk2 upon substitution of the gatekeeper residue with an amino acid lacking a side chain. The interaction with a cyclin assists in holding the cdk in an active conformation and this may be a factor in the retention of activity with F80G cdk2.…”

Section: Discussionsupporting

confidence: 93%

“…[15] With respect to serine/threonine kinases, there is limited kinetic data already published with N 6 -modified ATP analogues. [16] In addition, recent work has demonstrated that substitution at the gatekeeper residue can have profound effects on kinase catalytic efficiency. [17] In order to clarify these issues Chemical genetic studies with enlarged ATP binding sites and unnatural ATP analogues have been applied to protein kinases for characterisation and substrate identification.…”

Section: Introductionmentioning

confidence: 99%

“…[ [16,18,19] and have assessed a range of ATP analogues and a variety of known substrates. Although some kinetic evaluation has been performed with wild-type and analogue-sensitive vSrc and Cdk2/cyclin E previously [16] our work here substantially expands on this by examining multiple protein substrates and ATP analogues and quantitatively comparing the kinetics of two cdk2 holoenzymes with distinct cyclin subunits.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Quantitative Comparison of Wild‐Type and Gatekeeper Mutant Cdk2 for Chemical Genetic Studies with ATP Analogues

et al. 2009

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Chemical genetic studies with enlarged ATP binding sites and unnatural ATP analogues have been applied to protein kinases for characterisation and substrate identification. Although this system is becoming widely used, there are limited data available about the kinetic profile of the modified system. Here we describe a detailed comparison of the wild-type cdk2 and the mutant gatekeeper kinase to assess the relative efficiencies of these kinases with ATP and unnatural ATP analogues. Our data demonstrate that mutation of the kinase alters neither the substrate specificity nor the phosphorylation site specificity. We find comparable K(M)/V(max) values for mutant cdk2 and wild-type kinase. Furthermore, F80G cdk2 is efficiently able to compensate for a defective cdk in a biological setting.

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“…N 6 -benzyl-[␥-32 P]ATP was prepared enzymatically from N 6 -benzyl-ADP by using a modified version of the protocol described in ref. 19. Two hundred microliters of a 1:1 slurry of Co 2ϩ -charged iminodiacetic acid-Sepharose beads in Hepes-buffered saline (HBS) (50 mM Hepes, pH 7.4͞150 mM NaCl) were added to an empty 2-ml BioSpin column (Bio-Rad) and washed twice with 1 ml of HBS.…”

Section: Methodsmentioning

confidence: 99%

Combining chemical genetics and proteomics to identify protein kinase substrates

Dephoure

Howson²,

Blethrow³

et al. 2005

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

Self Cite

111

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Phosphorylation is a ubiquitous protein modification important for regulating nearly every aspect of cellular biology. Protein kinases are highly conserved and constitute one of the largest gene families. Identifying the substrates of a kinase is essential for understanding its cellular role, but doing so remains a difficult task. We have developed a high-throughput method to identify substrates of yeast protein kinases that employs a collection of yeast strains each expressing a single epitope-tagged protein and a chemical genetic strategy that permits kinase reactions to be performed in native, whole-cell extracts. Using this method, we screened 4,250 strains expressing epitope-tagged proteins and identified 24 candidate substrates of the Pho85-Pcl1 cyclin-dependent kinase, including the known substrate Rvs167. The power of this method to identify true kinase substrates is strongly supported by functional overlap and colocalization of candidate substrates and the kinase, as well as by the specificity of Pho85-Pcl1 for some of the substrates compared with another Pho85-cyclin kinase complex. This method is readily adaptable to other yeast kinases.cell signaling ͉ phosphorylation ͉ cyclin-dependent kinase T he general criteria for establishing that a protein is a substrate of a given kinase are the ability of the kinase to phosphorylate the substrate in vitro and the dependence on the activity of the kinase for phosphorylation of the substrate in vivo (1). In vivo validation of kinase substrates continues to be laborious and time-consuming. Thus, it is crucial to be able to efficiently identify candidates before committing to this step. Indirect data, such as genetic and physical interactions, can provide insights into potential substrates, but many interacting proteins and genes are not substrates (1, 2). More direct approaches using in vitro kinase reactions have been attempted in many permutations (1, 2). Purified component reactions directly measure the ability of a kinase to phosphorylate a particular substrate and can be scaled for high-throughput formats, but such conditions often compromise reaction specificity and produce false-positive results (1, 2).We sought to improve on these techniques by carrying out a biochemical screen in an environment that more closely resembles the in vivo state. To do so, we carried out kinase reactions with near physiological levels of exogenous kinase in native whole-cell extracts. These extracts maintain protein-protein interactions that may affect substrate presentation and preserve a nearly full complement of cellular proteins, including potential adaptor proteins and cofactors that could affect the reaction. These reaction conditions also preserve the native relative protein abundances and a natural competition among substrates for limited kinase.A challenge of carrying out protein kinase reactions in wholecell lysates is that one cannot attribute phosphorylation of a protein to a particular kinase in the reaction, because all kinases in the extract are capable of cat...

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Inhibitor Scaffolds as New Allele Specific Kinase Substrates

Cited by 53 publications

References 39 publications

Mimicking transient activation of protein kinases in living cells

Mimicking transient activation of protein kinases in living cells

A Quantitative Comparison of Wild‐Type and Gatekeeper Mutant Cdk2 for Chemical Genetic Studies with ATP Analogues

Combining chemical genetics and proteomics to identify protein kinase substrates

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