Facile preparation of highly active casein kinase 1 using Escherichia coli constitutively expressing lambda phosphatase

Akizuki, Kazutoshi; Toyama, Taku; Sugiyama, Yasunori; Ishida, Atsuhiko; Kameshita, Isamu; Sueyoshi, Noriyuki

doi:10.1016/j.ab.2018.03.015

Cited by 8 publications

(4 citation statements)

References 38 publications

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“…Purified recombinant TcK2 was obtained in high yields and was in the dephosphorylated state as observed by mass analysis. This was expected as the protein was produced in Escherichia coli expressing lambda phosphatase ( 36 ). Both recombinant TcK2 protein constructs showed protein kinase activity and were able to phosphorylate recombinant mouse eIF2α at Ser52, as well as CREBTide and p70S6K peptides.…”

Section: Discussionmentioning

confidence: 94%

Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

Marcelino,

Fala,

da Silva

et al. 2023

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 94%

Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

Marcelino,

Fala,

da Silva

et al. 2023

Journal of Biological Chemistry

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“…Even after initial dephosphorylation with λ phosphatase, Hhp1-WT rapidly rephosphorylates C-terminal sites (fig. S2C) and catalytic domain sites ( 16 ) during the course of the experiment ( 4 , 17 , 20 , 26 , 27 ). Because Hhp1-6A and Hhp1-6N cannot, these mutants were even more active than dephosphorylated wild-type Hhp1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Substrate displacement of CK1 C-termini regulates kinase specificity

Cullati,

Akizuki,

Chen

et al. 2024

Sci. Adv.

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CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

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“…In case of auto-activating kinases different autophosphorylation levels can be overcome by preincubation with ATP prior to performing in vitro kinase assays with exogenous substrates. Unfortunately, a reliable pretreatment for auto-inactivating kinases, like CK1, does not exist and is limited to hardly reproducible procedures involving co-expression of or treatment with phosphatases [ 21 ]. Alternatively, autophosphorylation sites could be mutated or autophosphorylation domains truncated, however, resulting in artificially activated kinase proteins with disturbed sequence integrity [ 22 , 23 ].…”

Section: Introductionmentioning

confidence: 99%

Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example

et al. 2021

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Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC50 value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.

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Facile preparation of highly active casein kinase 1 using Escherichia coli constitutively expressing lambda phosphatase

Cited by 8 publications

References 38 publications

Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

Substrate displacement of CK1 C-termini regulates kinase specificity

Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example

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