Differential Inhibition and Posttranslational Modification of Protein Phosphatase 1 and 2A in MCF7 Cells Treated with Calyculin-A, Okadaic Acid, and Tautomycin
“…Securin and cyclin B disappeared rapidly while the decline of pH3S10 and pThr-CDK substrates was OKA dose dependent (Figure 7(c)). Considering that in human cells OKA doses below 1 µM are reported to be ineffective against PP1 phosphatases [32,33], our data point to an involvement of phosphatase(s) of the PP1 superfamily in the dephosphorylation of SAMHD1 during mitosis.10.1080/15384101.2018.1480216-F0007Figure 7.Okadaic acid treatment maintains SAMHD1 phosphorylation in normal and transformed cells. Okadaic acid was added to hRPE-1 cells during synchronous mitotic exit (A-C), and to asynchronous populations of hRPE-1 (D) or THP-1 cells (E).…”
Section: Resultsmentioning
confidence: 76%
“…We took advantage of the 10–100 fold differential selectivity of the cell-permeable inhibitor okadaic acid (OKA) for PP2A over PP1 with active concentrations below 1 µM reported as specific for PP2A in human cell lines [32,33]. The requirement for ≥1 µM OKA to fully inhibit SAMHD1 dephosphorylation during mitotic exit suggests the involvement of the PP1 family of phosphatases.…”
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.
“…Securin and cyclin B disappeared rapidly while the decline of pH3S10 and pThr-CDK substrates was OKA dose dependent (Figure 7(c)). Considering that in human cells OKA doses below 1 µM are reported to be ineffective against PP1 phosphatases [32,33], our data point to an involvement of phosphatase(s) of the PP1 superfamily in the dephosphorylation of SAMHD1 during mitosis.10.1080/15384101.2018.1480216-F0007Figure 7.Okadaic acid treatment maintains SAMHD1 phosphorylation in normal and transformed cells. Okadaic acid was added to hRPE-1 cells during synchronous mitotic exit (A-C), and to asynchronous populations of hRPE-1 (D) or THP-1 cells (E).…”
Section: Resultsmentioning
confidence: 76%
“…We took advantage of the 10–100 fold differential selectivity of the cell-permeable inhibitor okadaic acid (OKA) for PP2A over PP1 with active concentrations below 1 µM reported as specific for PP2A in human cell lines [32,33]. The requirement for ≥1 µM OKA to fully inhibit SAMHD1 dephosphorylation during mitotic exit suggests the involvement of the PP1 family of phosphatases.…”
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.
“…Calyculin A is 7-to 9-fold more potent toward PP2A than PP1 in vitro [39] and, when used at concentrations of 50 nM or less, is selective for inhibition of the PP2A subfamily of protein phosphatases in intact cells [40,41]. S2 cells were treated with 50 nM calyculin A for 15 minutes and then starved for amino acids or treated with 20 nM rapamycin to inhibit TOR activity.…”
Section: Drosophila S6 Kinase Is Dephosphorylated By a Calyculin A-sementioning
Phosphorylation and activation of ribosomal S6 protein kinase is an important link in the regulation of cell size by the target of rapamycin (TOR) protein kinase. A combination of selective inhibition and RNA interference were used to test the roles of members of the PP2A subfamily of protein phosphatases in dephosphorylation of Drosophila S6 kinase (dS6K). Treatment of Drosophila Schneider 2 cells with calyculin A, a selective inhibitor of PP2A-like phosphatases, resulted in a 7-fold increase in the basal level of dS6K phosphorylation at the TOR phosphorylation site (Thr398) and blocked dephosphorylation following inactivation of TOR by amino acid starvation or rapamycin treatment. Knockdown of the PP2A catalytic subunit increased basal dS6K phosphorylation and inhibited dephosphorylation induced by amino acid withdrawal. In contrast, depletion of the catalytic subunits of the other two members of the subfamily did not enhance dS6K phosphorylation. Knockdown of PP4 caused a 20% decrease in dS6K phosphorylation and knockdown of PP6 had no effect. Knockdown of the Drosophila B56-2 subunit resulted in enhanced dephosphorylation of dS6K following removal of amino acids. In contrast, knockdown of the homologs of the other PP2A regulatory subunits had no effects. Knockdown of the Drosophila homolog of the PP2A/PP4/PP6 interaction protein α4/Tap42 did not affect S6K phosphorylation, but did induce apoptosis. These results indicate that PP2A, but not other members of this subfamily, is likely to be a major S6K phosphatase in intact cells and is consistent with an important role for this phosphatase in the TOR pathway.
“…Immunoprecipitation ± phosphatase ± assay PP2A catalytic subunit activity in immunoprecipitates was measured as release of 32 PO 4 from a peptide phosphorylated with protein kinase A and [g 32 P]ATP (Favre et al, 1994). Immunoprecipitations from transfected U2-OS or untransfected B104 cells were performed as described previously (Beijersbergen et al, 1994).…”
Section: Immunological Reagentsmentioning
confidence: 99%
“…The amount of released 32 PO 4 never exceeded 25% of the input counts. The peptide Leu-Arg-Arg-Ala-Ser-Val-Ala (Kemptide Val 6 -Ala 7 , Bachem) was phosphorylated in the presence of [g 32 P]ATP by the catalytic subunit of protein kinase A as described previously (Favre et al, 1994). Subsequently, the peptide was separated from un-incorporated [g 32 P]ATP on a Dowex 1 (Fluka AG) column with 30% acetic acid, freeze dried twice, and aliquoted at 25 mM in 50 mM Tris-HCl pH 7.5, 0.1 mM EDTA.…”
The proteins of the retinoblastoma family are potent inhibitors of cell cycle progression. It is well documented that their growth-inhibitory activity can be abolished by phosphorylation on serine and threonine residues by cyclin dependent kinases. In contrast, very little is known about the dephosphorylation of retinoblastoma-family proteins. We report here the isolation, by virtue of its ability to associate with p107, of a novel Protein Phosphatase 2A (PP2A) regulatory subunit, named PR59. PR59 shares sequence homology with a known regulatory subunit of PP2A, PR72, but di ers from PR72 in its expression pattern and its functional properties. We show that PR59 co-immunoprecipitates with the PP2A catalytic subunit, indicating that PR59 is a genuine component of PP2A holo-enzymes. In vivo, PR59 associates speci®cally with p107, but not with pRb. Elevated expression of PR59 results in dephosphorylation of p107, but not of pRb, and inhibits cell proliferation by causing cells to accumulate in G1. These data support a model in which the distinct PP2A regulatory subunits act to target the PP2A catalytic subunit to speci®c substrates and suggest a role for PP2A in regulation of p107.
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