An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation

Rominger, Cynthia M.; Schaber, Michael D.; Yang, Jing; Gontarek, Richard R.; Weaver, K.; Broderick, Timothy J.; Carter, Luke; Copeland, Robert A.; May, Earl W.

doi:10.1016/j.abb.2007.04.004

Cited by 22 publications

(19 citation statements)

References 16 publications

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“…As the incubation times increase, the EC 50 values of the activation curves shift to lower values and the slopes dramatically increase, which are indicative of a highly cooperative activation process consistent with the feedback activation proposed in Fig. 1E (26). …”

supporting

confidence: 78%

Small-Molecule Activators of a Proenzyme

Wolan

Zorn

Gray

et al. 2009

Science

145

167

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Virtually all of the 560 human proteases are stored as inactive proenyzmes and are strictly regulated. We report the identification and characterization of small molecules that directly activate the apoptotic proenzymes, procaspases-3 and -6. Surprisingly, these compounds induce autoproteolytic activation by stabilizing a conformation that is both more active and more susceptible to intermolecular proteolysis. These procaspase activators bypass the normal upstream proapoptotic signaling cascades and induce rapid apoptosis in a variety of cell lines. Systematic biochemical and biophysical analyses identified a cluster of mutations in procaspase-3 that resist small molecule activation both in vitro and in cells. Compounds that induce gain-of-function are rare and the activators reported here will enable direct control of the homodimeric executioner caspases in apoptosis and in cellular differentiation.Activation of proteases trigger a myriad of fate-determining biological events, such as apoptosis and blood clotting, both inside and outside of the cell (1). Proteases are generally stored as inactive proenzymes that are usually activated by upstream proteases or by themselves. These activation events may sometimes involve binding a protein partner or, in rare instances, interaction with a natural small molecule (2) or peptide (3). In the case of autoproteolysis, the proenzyme must achieve not only an active state, but also one in which the sites of proteolysis are exposed. In situ activation of specific proproteases with synthetic small molecules would uncover new molecular principles in zymogen activation and facilitate direct control of these important processes in biology.The executioner procaspases, consisting of procaspases-3, -6 and -7, represent excellent initial candidates for discovery of small molecule protease activators. Caspases are a family of homodimeric cysteine proteases responsible for many of the fate-determining processes in cell biology including apoptosis, innate immune signaling, early stages of stem cell differentiation, and cellular remodeling (4-6). As with most proteases, caspases are synthesized as inactive procaspases, or zymogens, and are activated by upstream proteolysis or autoproteolysis. Previous studies have shown that the mature active caspases are intrinsically dynamic (7,8) and sample both an "on-state" and an "off-state" that structurally resembles the zymogen-like conformation (9,10). Small molecules have been found to trap these two forms of the mature enzyme (11,12). We reasoned that, if the procaspases existed in a similar dynamic equilibrium of off-and on-states, it might be possible to find small molecules that promote autoproteolytic activation via stabilization of an on-state conformation. Executioner procaspases are particularly good targets as they are susceptible to rapid activation by both upstream proteases and self-proteolysis. Thus, any activation would be accentuated in trans by autocatalytic activation. Moreover, these particular caspases are essential...

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supporting

confidence: 78%

Small-Molecule Activators of a Proenzyme

Wolan

Zorn

Gray

et al. 2009

Science

145

167

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“…It should be noted that the Km for ATP differed by 10-fold between the ATPase and the transphosphorylation assay (30 vs. 3 μM), highlighting the importance of experimentally determining Km for ATP when developing an ATPase kinase assay. This magnitude of shift in ATP Km for kinase intrinsic ATPase activity has been previously observed 25 .…”

Section: Discussionsupporting

confidence: 81%

“…Intrinsic ATPase activity has been observed for a number of kinases using in vitro biochemical assays and purified kinase. This ATPase activity has been described for several MAPKs, including p38α, p38γ, ERK2, JNK3 and MEK1 24–25 . A format comparison study compared screening results using an antibody-based disassociation-enhanced lanthanide fluoroimmunoassay (DELFIA), an ATP-consumption intrinsic ATPase assay, and a fluorescence polarization binding assay for ITK 26 .…”

Section: Introductionmentioning

confidence: 56%

Development and Validation of a High-Throughput Intrinsic ATPase Activity Assay for the Discovery of MEKK2 Inhibitors

et al. 2013

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The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. In order to identify inhibitors of MEKK2 kinase activity, we have developed a novel activity assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase activity. This MEKK2 ATPase assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase assay IC50 data for multiple inhibitors to literature values and to IC50 data generated using MEKK2 binding and transphosphorylation assays. Taken together, these data indicated that genuine MEKK2 activity was being measured in this assay and no other ATPases contributed to the signal. A miniaturized version of the assay was validated for high throughput screening and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding and transphosphorylation assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 activity with IC50 values of <100 nM. Thus, this assay has utility for the discovery of small molecule inhibitors of MEKK2 activity.

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“…Indeed, positive charges including a Lys side chain and Mg ion are pointed toward this β-phosphate and are observed in many kinase crystal structures in an arrangement that could stabilize the buildup of negative charge in the leaving group. It is also true that many kinases show substantial ATP-hydrolytic activity, which is consistent with generation of a metaphosphate-like species that can be intercepted by water in the absence of precise positioning of a protein hydroxyl (Rominger et al, 2007). …”

Section: Chemical Mechanism Of Kinase Phosphoryl Transfermentioning

confidence: 80%

Catalytic Mechanisms and Regulation of Protein Kinases

Wang

Cole

2014

Methods in Enzymology

131

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Protein kinases transfer a phosphoryl group from ATP onto target proteins and play a critical role in signal transduction and other cellular processes. Here, we review the kinase kinetic and chemical mechanisms and their application in understanding kinase structure and function. Aberrant kinase activity has been implicated in many human diseases, in particular cancer. We highlight applications of technologies and concepts derived from kinase mechanistic studies that have helped illuminate how kinases are regulated and contribute to pathophysiology.

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An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation

Cited by 22 publications

References 16 publications

Small-Molecule Activators of a Proenzyme

Small-Molecule Activators of a Proenzyme

Development and Validation of a High-Throughput Intrinsic ATPase Activity Assay for the Discovery of MEKK2 Inhibitors

Catalytic Mechanisms and Regulation of Protein Kinases

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