PNUTS (phosphatase nuclear targeting subunit) inhibits retinoblastoma-directed PP1 activity

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Abstract

Protein phosphatase type 1 catalytic subunit (PP1c) is a serine/threonine phosphatase involved in the dephosphorylation of many proteins in eukaryotic cells. It associates with several known targeting or regulatory subunits that directly regulate PP1c activity toward specific substrates. The recently identified Phosphatase Nuclear Targeting Subunit (PNUTS) binds to PP1c and inhibits PP1 activity toward phosphorylase a. One of the substrates of PP1c has been shown to be the cell cycle regulatory protein, Retinoblastoma (pRb). In this study, we show that PNUTS dissociates from PP1c under mildly hypoxic cell growth conditions that lead to an increase of PP1c activity toward pRb. We developed an assay that measures pRb-directed PP1c activity and show that a GST-PNUTS fusion protein inhibits phosphatase activity toward pRb when using PP1c from cell lysates, GST-PP1c, or purified PP1c. These studies suggest that PNUTS is involved in the regulation of PP1c activity toward pRb.

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Materials and methods

Cell growth conditions. CV-1P monkey kidney epithelial cells were grown in Dulbecco’s modified Eagle’s medium containing 10% newborn calf serum, 1% glutamine, and 1 mM sodium pyruvate (DMEM, Invitrogen). SKA ovarian carcinoma cells were grown in DMEM containing 10% fetal bovine serum. All cells were grown in a 37 °C humidified incubator containing 5% CO2 and maintained at or below 80–90% confluency.

Establishing hypoxic cultures. For hypoxia, cells were plated at 8×105 cells/dish (for CV-1P) and

Results and discussion

Exposure of CV1-P and SKA cells to mild hypoxia (1.0% oxygen) results in several changes in cell cycle regulatory molecules [11], [12], [13], one of which being a 40–50% increase in PP1c phosphatase activity toward the Retinoblastoma protein (pRb). The increase in PP1c activity is specific to pRb, as phosphorylase a phosphatase activity is unaffected by hypoxic conditions. In addition, the increase in PP1c-mediated pRb dephosphorylation is not a result of increased expression of PP1c [11].

Acknowledgements

We thank the Pace University Department of Biological Sciences and the Dyson School of Arts and Sciences for support of this project. We gratefully acknowledge Jerry DiSalvo for critical reading of the manuscript. E.U. and V.C.T. were supported by Student-Faculty Research Fellowships from the Eugene M. Lang Foundation.

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