Kinase‐Catalyzed Crosslinking and Immunoprecipitation (K‐CLIP) to Explore Kinase‐Substrate Pairs

Beltman, Rachel J.; Pflum, Mary Kay H.

doi:10.1002/cpz1.539

Cited by 6 publications

(5 citation statements)

References 40 publications

(54 reference statements)

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“…Only ATP-AFS 1d and ATP-HexBr 1e were successfully synthesized and purified, as described below. The ATP-aryl sulfonyl fluoride 1j was prone to hydrolysis and degraded during purification, ATP-propionyl bromide ( n = 2, 1g ) suffered from β-elimination of the bromide, and the other ATP-alkyl bromides either hydrolyzed ( n = 1, 1f ) or cyclized ( n = 3 and 4, 1h and 1i ) during purification, resulting in no desired product (data not shown) …”

Section: Resultsmentioning

confidence: 99%

“…The ATP-aryl sulfonyl fluoride 1j was prone to hydrolysis and degraded during purification, ATP-propionyl bromide (n = 2, 1g) suffered from β-elimination of the bromide, and the other ATP-alkyl bromides either hydrolyzed (n = 1, 1f) or cyclized (n = 3 and 4, 1h and 1i) during purification, resulting in no desired product (data not shown). 23 Synthesis of both ATP-AFS and ATP-HexBr began with the generation of the Boc-protected PEG linker (3), as previously published (Scheme S1). 24−26 27 After Boc deprotection under acidic conditions, the resulting amine was coupled to ATP at pH = 6.2−6.8 with Nmethylimidazole (NMI) 28 and EDCI to yield ATP-AFS (1d), which was characterized by NMR and HRMS (Figures S5− S10).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Affinity-Based Kinase-Catalyzed Crosslinking to Study Kinase–Substrate Pairs

Beltman,

Herppich,

Bremer

et al. 2023

Bioconjugate Chem.

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Phosphorylation of proteins by kinase enzymes is a post-translational modification involved in a myriad of biological events, including cell signaling and disease development. Identifying the interactions between a kinase and its phosphorylated substrate(s) is necessary to characterize phosphorylation-mediated cellular events and encourage development of kinase-targeting drugs. One method for substrate−kinase identification utilizes photocrosslinking γphosphate-modified ATP analogues to covalently link kinases to their substrates for subsequent monitoring. Because photocrosslinking ATP analogues require UV light, which could influence cell biology, we report here two ATP analogues, ATP-aryl fluorosulfate (ATP-AFS) and ATP-hexanoyl bromide (ATP-HexBr), that crosslink kinase−substrate pairs via proximity-mediated reactions without the need for UV irradiation. Both ATP-AFS and ATP-HexBr acted as cosubstrates with a variety of kinases for affinity-based crosslinking, with ATP-AFS showing more robust complexes. Importantly, ATP-AFS promoted crosslinking in lysates, which demonstrates compatibility with complex cellular mixtures for future application to kinase−substrate identification.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Affinity-Based Kinase-Catalyzed Crosslinking to Study Kinase–Substrate Pairs

Beltman,

Herppich,

Bremer

et al. 2023

Bioconjugate Chem.

Self Cite

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“…The second screen involved covalently crosslinking kinases to their substrates in a kinase-dependent manner, followed by identification of the crosslinked pairs by LC–MS/MS analysis. The kinase-catalyzed crosslinking and ion purification (K-CLiP) method 31 uses a γ-phosphoryl-modified adenosine 5’-triphosphate (ATP) analogue containing a photocrosslinking group to covalently link MIPS to both kinases and associated proteins. In K-CLiP, the ATP-photocrosslinker (ATP-PCL), ATP-arylazide, acts as a co-substrate of cellular kinases to transfer the arylazide photocrosslinker to 6xHis-Xpress-MIPS via kinase activity 32 .…”

Section: Resultsmentioning

confidence: 99%

Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase

Case,

Beltman,

Pflum

et al. 2023

Sci Rep

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Inositol depletion is a hypothesized mechanism of action of mood stabilization drugs used in the treatment of bipolar disorder. It was previously reported that the mood stabilizer valproate (VPA) increased phosphorylation of myo-inositol-3-phosphate synthases (MIPS), the rate limiting enzyme of inositol synthesis. Phosphosites were identified and examination of site-directed mutants suggested that phosphorylation leads to decreased enzymatic activity. In this study, we examined the extent of MIPS phosphorylation in response to VPA and used two interaction screens to identify protein kinases that interact with MIPS. Using an epitope tagged MIPS construct, we determined the fraction of phosphorylated MIPS to be very low (less than 2% of total), and we could not detect phosphorylation of untagged MIPS in response to VPA. In vitro analyses of phosphorylation revealed that putative protein kinases, PKC and CKII, have low specificity toward MIPS. These findings suggest that VPA likely depletes inositol via a mechanism other than MIPS phosphorylation. Consistent with this, mRNA levels of the MIPS-encoding gene INO1 and MIPS protein levels were significantly reduced during the mid-log growth phase in response to VPA treatment. These findings suggest that the mechanism whereby VPA causes inositol depletion is by reducing expression of the rate-limiting enzyme MIPS.

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“…Complexes containing crosslinked substrate and kinase can be purified by streptavidin pulldown or immunoprecipitation. Predicted KSRs can then be tested using western blots or, alternatively, unknown KSRs can be identified by proteomic analysis ( Dedigama-Arachchige and Pflum, 2016 ; Beltman and Pflum, 2022 ). Not all kinases can utilize unnatural ATP analogs to phosphorylate substrates and another disadvantage is that, because the ATP analogs do not cross cell membranes, these techniques cannot be performed in cells.…”

Section: Pharmacological Approaches To Define Kinase-substrate Relati...mentioning

confidence: 99%

Pharmacological approaches to understanding protein kinase signaling networks

Stephenson,

Higgins

2023

Front. Pharmacol.

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Protein kinases play vital roles in controlling cell behavior, and an array of kinase inhibitors are used successfully for treatment of disease. Typical drug development pipelines involve biological studies to validate a protein kinase target, followed by the identification of small molecules that effectively inhibit this target in cells, animal models, and patients. However, it is clear that protein kinases operate within complex signaling networks. These networks increase the resilience of signaling pathways, which can render cells relatively insensitive to inhibition of a single kinase, and provide the potential for pathway rewiring, which can result in resistance to therapy. It is therefore vital to understand the properties of kinase signaling networks in health and disease so that we can design effective multi-targeted drugs or combinations of drugs. Here, we outline how pharmacological and chemo-genetic approaches can contribute to such knowledge, despite the known low selectivity of many kinase inhibitors. We discuss how detailed profiling of target engagement by kinase inhibitors can underpin these studies; how chemical probes can be used to uncover kinase-substrate relationships, and how these tools can be used to gain insight into the configuration and function of kinase signaling networks.

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Kinase‐Catalyzed Crosslinking and Immunoprecipitation (K‐CLIP) to Explore Kinase‐Substrate Pairs

Cited by 6 publications

References 40 publications

Affinity-Based Kinase-Catalyzed Crosslinking to Study Kinase–Substrate Pairs

Affinity-Based Kinase-Catalyzed Crosslinking to Study Kinase–Substrate Pairs

Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase

Pharmacological approaches to understanding protein kinase signaling networks

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