Monitoring Native p38α:MK2/3 Complexes via Trans Delivery of an ATP Acyl Phosphate Probe

Okerberg, Eric; Brown, Heidi E.; Minimo, Lauro; Alemayehu, Senait; Rosenblum, Jonathan S.; Patricelli, Matt; Nomanbhoy, Tyzoon; Kozarich, John W.

doi:10.1021/ja4129907

Cited by 8 publications

(8 citation statements)

References 20 publications

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“…Although we cannot rule out the possibility of alternative mechanisms, e.g. probe binding due to domain- (Nordin et al, 2015) or protein-interactions (Okerberg et al, 2014), we do provide evidence that the C1 domain serves as a ligand binding site for ritanserin distinct from the ATP binding region of DGKα (Figure 5A and 6A). The overlapping (DAGKa) and distinct (C1) binding sites of ritanserin compared with ATP helps explain previous kinetic findings of a mixed competitive mechanism of inhibition whereby ritanserin prefers to bind a DGKα-ATP complex (Boroda et al, 2017).…”

Section: Discussionmentioning

confidence: 69%

The Ligand Binding Landscape of Diacylglycerol Kinases

Franks

Campbell

Purow

et al. 2017

Cell Chemical Biology

109

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SUMMARY Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases. Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP- and small molecule-binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGK-alpha (DGKα) inhibitor ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region. Unexpectedly, deconstruction of ritanserin yielded a fragment compound that blocks DGKα activity through a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.

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Section: Discussionmentioning

confidence: 69%

The Ligand Binding Landscape of Diacylglycerol Kinases

Franks

Campbell

Purow

et al. 2017

Cell Chemical Biology

109

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“…For determination of native kinase engagement, compounds were profiled in cell lysates or live cells using the KiNativ chemoproteomics platform (ActivX Biosciences) (27,28,51). For flow cytometry, ERK5 auto-phosphorylation, immunoanalyses, and RNA-Seq, cells were treated for 1 h with compounds before agonist stimulation, followed by standard protocols.…”

Section: Methodsmentioning

confidence: 99%

ERK5 kinase activity is dispensable for cellular immune response and proliferation

Lin¹,

Amantea²,

Nomanbhoy³

et al. 2016

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

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104

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Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.) is a member of the mitogen-activated protein kinase (MAPK) family, which includes ERK1/2, JNK1/2/3, and p38α/β/δ/γ (1). However, unlike the other MAPK members, ERK5 contains a unique 400-amino-acid C-terminal domain in addition to the kinase domain. Through the MAPK signaling cascade, mitogenactivated protein kinase kinase 5 (MEK5) activates ERK5 by phosphorylating the TEY motif in the N-terminal activation loop (2). This event unlocks the N-and C-terminal halves, allowing ERK5 to auto-phosphorylate multiple sites in its C-terminal region, which can then regulate nuclear shuttling and gene transcription (3, 4). Noncanonical pathways (including cyclin-dependent kinases during mitosis and ERK1/2 during growth factor stimulation) also exist for phosphorylation of sites in the ERK5 tail (5-7). Although ERK5 has been demonstrated to directly phosphorylate transcription factors (8-10), the noncatalytic C-terminal tail of ERK5 can also interact with transcription factors and influence gene expression (4,11,12).ERK5 can be activated in response to a range of mitogenic stimuli [e.g., growth factors, G protein-coupled receptor (GPCR) agonists, cytokines] and cellular stresses (e.g., hypoxia, shear stress) (13). Like most kinases including MAPK members, ERK5 function is assumed to be driven by its kinase activity. ERK5 deletion is embryonic lethal in mice and a variety of tissue-or development-stage restricted KOs have shown clear phenotypes, suggesting that the catalytic function and/or an aspect of the nonkinase domain(s) have key roles in development and mature organ function (14-18). The availability of the first ERK5 inhibitor ...

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“…4 In another example, acylation of p38 α at K15, external to the ATP binding site, occurs only in the presence of MAPKAPK-2 and/or MAPKAPK-3, known binding partners of p38 α . 5 Protein–protein interactions orient K15 from p38 α into the ATP binding site of the respective MAPKAPK binding partner, resulting in transacylation of K15. With few exceptions, nucleotide acyl phosphates affect acylation of lysines in a proximity-driven manner that requires nucleotide binding of the probe for efficient delivery of the acyl group.…”

mentioning

confidence: 99%

Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6

Okerberg¹,

Dagbay

Green³

et al. 2019

Biochemistry

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Acyl phosphates of ATP (ATPAc) and related nucleotides have proven to be useful for the interrogation of known nucleotide binding sites via specific acylation of conserved lysines (K). In addition, occasional K acylations are identified in proteins without such known sites. Here we present a robust and specific acylation of procaspase-6 by ATPAc at K133 in Jurkat cell lysates. The K133 acylation is dependent on π–π stacking interactions between the adenine moiety of ATPAc and a conserved Y198–Y198 site formed at the homodimeric interface of procaspase-6. Significantly, the Y198A mutation in procaspase-6 abolishes K133 acylation but has no effect on the proteolytic activity of the mature, active caspase-6 Y198A variant. Additional in vitro studies show that ATP can inhibit the autoproteolytic activation of procaspase-6. These observations suggest that ATP, and possibly other nucleotides, may serve as the endogenous ligands for the allosteric site at the procaspase-6 dimer interface, a site that has persisted in its “orphan” status for more than a decade.

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Monitoring Native p38α:MK2/3 Complexes via Trans Delivery of an ATP Acyl Phosphate Probe

Cited by 8 publications

References 20 publications

The Ligand Binding Landscape of Diacylglycerol Kinases

The Ligand Binding Landscape of Diacylglycerol Kinases

ERK5 kinase activity is dispensable for cellular immune response and proliferation

Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6

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