Development of a Colorimetric and a Fluorescence Phosphatase-Inhibitor Assay Suitable for Drug Discovery Approaches

Sotoud, Hannieh; Gribbon, Philip; Ellinger, Bernhard; Reinshagen, Jeanette; Boknı́k, Peter; Kattner, Lars; El‐Armouche, Ali; Eschenhagen, Thomas

doi:10.1177/1087057113486000

Cited by 18 publications

(15 citation statements)

References 29 publications

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“…The necessity for such a prolonged incubation indicated that Rtr1 can dephosphorylate p NPP but with very low activity. Therefore, we switched to a more sensitive assay that uses the fluorescent compound DiFMUP, which is 500 times more sensitive than the p NPP assay (16) and has been used to assay the activity of K. lactis Rtr1 (10). …”

Section: Resultsmentioning

confidence: 99%

Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

et al. 2016

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Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

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Section: Resultsmentioning

confidence: 99%

Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

et al. 2016

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“…The K M for PRLs was determined separately (PRL-3 = 21 μM, PRL-2 and PRL-1 = 24 μM). The K M for PP1 (91 μM) and for PP2A (100 μM) was taken from the literature [ 38 , 39 ]. Protein concentrations are 50 nM for all PRLs and 2 mU for PP1 and 0.05 U for PP2A (see the Experimental Procedures ).…”

Section: Resultsmentioning

confidence: 99%

Procyanidins Negatively Affect the Activity of the Phosphatases of Regenerating Liver

et al. 2015

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Natural polyphenols like oligomeric catechins (procyanidins) derived from green tea and herbal medicines are interesting compounds for pharmaceutical research due to their ability to protect against carcinogenesis in animal models. It is nevertheless still unclear how intracellular pathways are modulated by polyphenols. Monomeric polyphenols were shown to affect the activity of some protein phosphatases (PPs). The three phosphatases of regenerating liver (PRLs) are close relatives and promising therapeutic targets in cancer. In the present study we show that several procyanidins inhibit the activity of all three members of the PRL family in the low micromolar range, whereas monomeric epicatechins show weak inhibitory activity. Increasing the number of catechin units in procyanidins to more than three does not further enhance the potency. Remarkably, the tested procyanidins showed selectivity in vitro when compared to other PPs, and over 10-fold selectivity toward PRL-1 over PRL-2 and PRL-3. As PRL overexpression induces cell migration compared to control cells, the effect of procyanidins on this phenotype was studied. Treatment with procyanidin C2 led to a decrease in cell migration of PRL-1- and PRL-3-overexpressing cells, suggesting the compound-dependent inhibition of PRL-promoted cell migration. Treatment with procyanidin B3 led to selective suppression of PRL-1 overexpressing cells, thereby corroborating the selectivity toward PRL-1- over PRL-3 in vitro. Together, our results show that procyanidins negatively affect PRL activity, suggesting that PRLs could be targets in the polypharmacology of natural polyphenols. Furthermore, they are interesting candidates for the development of PRL-1 inhibitors due to their low cellular toxicity and the selectivity within the PRL family.

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“…To determine whether it is the bulkiness of Bpa or the fact that it is an unnatural amino acid that accounts for the cellular stability it imparts to PDP3, we synthesized PDPs that contained the small amino acids d ‐alanine ( d al ), β‐alanine ( βal ), or bulky 2‐naphthylalanine ( Nal ). First, we tested if these PDPs disrupted in vitro the interaction of PP1 with the regulatory protein I2 to liberate PP1, such that it could dephosphorylate the fluorogenic substrate 6,8‐difluoro‐4‐methylumbelliferyl phosphate (DiFMUP) . The results show that all peptides maintain in vitro activity in a similar potency range, with PDP3 showing the highest EC 50 (Table and Figure S1 in the Supporting Information).…”

Section: Pdps Used For This Study and Their Ec50 Values For Deinhibitmentioning

confidence: 99%

Interrogating PP1 Activity in the MAPK Pathway with Optimized PP1‐Disrupting Peptides

et al. 2018

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Protein phosphatase‐1 (PP1)‐disrupting peptides (PDPs) are selective chemical modulators of PP1 that liberate the active PP1 catalytic subunit from regulatory proteins; thus allowing the dephosphorylation of nearby substrates. We have optimized the original cell‐active PDP3 for enhanced stability, and obtained insights into the chemical requirements for stabilizing this 23‐mer peptide for cellular applications. The optimized PDP‐ Nal was used to dissect the involvement of PP1 in the MAPK signaling cascade. Specifically, we have demonstrated that, in human osteosarcoma (U2OS) cells, phosphoMEK1/2 is a direct substrate of PP1, whereas dephosphorylation of phosphoERK1/2 is indirect and likely mediated through enhanced tyrosine phosphatase activity after PDP‐mediated PP1 activation. Thus, as liberators of PP1 activity, PDPs represent a valuable tool for identifying the substrates of PP1 and understanding its role in diverse signaling cascades.

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Development of a Colorimetric and a Fluorescence Phosphatase-Inhibitor Assay Suitable for Drug Discovery Approaches

Cited by 18 publications

References 29 publications

Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

Procyanidins Negatively Affect the Activity of the Phosphatases of Regenerating Liver

Interrogating PP1 Activity in the MAPK Pathway with Optimized PP1‐Disrupting Peptides

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