Small-Molecule Inhibitors of Ser/Thr Protein Phosphatases: Specificity, Use and Common Forms of Abuse

Swingle, Mark R.; Ni, Li; Honkanen, Richard E.

doi:10.1385/1-59745-267-x:23

Cited by 233 publications

(321 citation statements)

References 9 publications

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“…The two classes of enzymes can be distinguished on the basis of their differential sensitivity to the inhibitor okadaic acid (OKA)[31]. We treated prometaphase hRPE-1 cells with OKA before and after RO3306 addition and collected samples for immunoblotting (Figure 7(a)).…”

Section: Resultsmentioning

confidence: 99%

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

et al. 2018

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SAMHD1 is the major catabolic enzyme regulating the intracellular concentrations of DNA precursors (dNTPs). The S-phase kinase CDK2-cyclinA phosphorylates SAMHD1 at Thr-592. How this modification affects SAMHD1 function is highly debated. We investigated the role of endogenous SAMHD1 phosphorylation during the cell cycle. Thr-592 phosphorylation occurs first at the G1/S border and is removed during mitotic exit parallel with Thr-phosphorylations of most CDK1 targets. Differential sensitivity to the phosphatase inhibitor okadaic acid suggested different involvement of the PP1 and PP2 families dependent upon the time of the cell cycle. SAMHD1 turn-over indicates that Thr-592 phosphorylation does not cause rapid protein degradation. Furthermore, SAMHD1 influenced the size of the four dNTP pools independently of its phosphorylation. Our findings reveal that SAMHD1 is active during the entire cell cycle and performs an important regulatory role during S-phase by contributing with ribonucleotide reductase to maintain dNTP pool balance for proper DNA replication.

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Section: Resultsmentioning

confidence: 99%

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

et al. 2018

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“…A similar response was observed upon treatment with okadaic acid, a protein serine/threonine phosphatase inhibitor that is reported to fragment intermediate filaments (Blankson et al, 1995; Figure 7 and Table 1). However, this drug has many effects that are independent of the cytoskeleton (Swingle et al, 2007). In contrast, treatment with the microtubule depolymerizing agent colchicine had no significant effect on C T (Figure 7 and Table 1).…”

Section: Stretch Stimulates Rapid Membrane Turnover At the Apical Surmentioning

confidence: 96%

Distinct Apical and Basolateral Membrane Requirements for Stretch-induced Membrane Traffic at the Apical Surface of Bladder Umbrella Cells

Khandelwal²,

Apodaca

2009

MBoC

127

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Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K ؉ channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.

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“…2A). At concentrations in excess of their reported IC 50 s (Swingle et al 2007), inhibitors of PP1 (Inhibitor-2), PP1/PP2A/PP4/PP5 (okadaic acid and microcystin), and PP2B (cypermethrin) failed to activate 39 cleavage in place of CP.…”

Section: Resultsmentioning

confidence: 88%

Small molecule activators of pre-mRNA 3′ cleavage

Ryan

Khleborodova²,

Pan³

et al. 2009

RNA

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39 Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 39 cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavagesuppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 39 cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 39 cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 39 cleavage at 200 mM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 39 cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 39 cleavage.

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Small-Molecule Inhibitors of Ser/Thr Protein Phosphatases: Specificity, Use and Common Forms of Abuse

Cited by 233 publications

References 9 publications

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

Distinct Apical and Basolateral Membrane Requirements for Stretch-induced Membrane Traffic at the Apical Surface of Bladder Umbrella Cells

Small molecule activators of pre-mRNA 3′ cleavage

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