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Inducible expression of catalytically active type 1 serine/threonine protein phosphatase in a human carcinoma cell line.

Reeder JE, Sowden MP, Messing EM, Klover P, Villa-Moruzzi E, Ludlow JW - Cancer Cell Int. (2003)

Bottom Line: Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation.When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels.Implications of these findings for the study of PP1alpha function in vivo are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA. jludlow@vestatherapeutics.com

ABSTRACT
BACKGROUND: One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity. RESULTS: We have established a cell line for inducing protein expression of the type 1, alpha-isotype, serine/threonine protein phosphatase (PP1alpha). A plasmid encoding a fusion protein of the catalytic subunit of PP1alpha with a 6-histidine peptide (6His) and a peptide from hemagluttinin (HA) was transfected into the UMUC3 transitional cell carcinoma cell line, previously transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo. A stable cell line designated LLWO2F was established by selection with hygromycin B. 6His-HA-PP1alpha protein appeared in cell lysates within two hours following addition of doxycycline to the culture medium. This protein localizes to the nucleus as does endogenous PP1alpha, and was shown to associate with PNUTS, a PP1-nuclear targeting subunit. Like endogenous PP1alpha, immunocomplexed 6His-HA-PP1alpha is active toward phosphorylase a and the product of the retinoblastoma susceptibility gene, pRB. When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels. CONCLUSIONS: These data suggest the existence of an autoregulatory mechanism by which PP1alpha protein levels and activity remain relatively constant. RT-PCR analyses of isolated polysome fractions support the notion that this putative autoregulatory mechanism is exerted, at least in part, at the translational level. Implications of these findings for the study of PP1alpha function in vivo are discussed.

No MeSH data available.


Related in: MedlinePlus

Dephosphorylation of phosphorylase a by anti-hemagglutinin immunoprecipitates from uninduced (Control) and induced (Doxycycline) LLW02 cells. Activity was assayed by the release of [32P] H3PO4 from radiolabeled phosphorylase a (1–2 × 105 cpm/nmol). 1 unit of activity releases 1 nmol of phosphate/min at 30°C.
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Figure 5: Dephosphorylation of phosphorylase a by anti-hemagglutinin immunoprecipitates from uninduced (Control) and induced (Doxycycline) LLW02 cells. Activity was assayed by the release of [32P] H3PO4 from radiolabeled phosphorylase a (1–2 × 105 cpm/nmol). 1 unit of activity releases 1 nmol of phosphate/min at 30°C.

Mentions: Two different approaches were taken to address the biochemical activity of inducible PP1α. First, immunocomplexed 6His-HA-PP1α was tested for its ability to dephosphorylate phosphorylase a. Phosphorylated on a single serine residue, this substrate is often used to monitor the biochemical activity of PP1 in vitro [28]. As shown in Figure 5, greater phosphatase activity is detected in immunoprecipitates from induced LLWO2F cell lysates compared to uninduced. This is consistent with the notion that inducible PP1α is enzymatically active. Our second approach involved testing immunopurified 6His-HA-PP1α for its ability to dephosphorylate the growth suppressor protein pRB. Having previously reported on the ability of immunocomplexed endogenous PP1 to dephosphorylate pRB [20], we followed the same experimental approach. Figure 6 shows a significant decrease in the 32P radiolabel intensity for pRB following incubation with the anti-hemagglutinin immunoprecipitate compared to the normal mouse IgG control immunoprecipitate. In addition, okadaic acid, a potent inhibitor of PP1 activity [29], significantly inhibits pRB dephosphorylation in this reaction. Taken together, these data indicate that inducible PP1α is biochemically active.


Inducible expression of catalytically active type 1 serine/threonine protein phosphatase in a human carcinoma cell line.

Reeder JE, Sowden MP, Messing EM, Klover P, Villa-Moruzzi E, Ludlow JW - Cancer Cell Int. (2003)

Dephosphorylation of phosphorylase a by anti-hemagglutinin immunoprecipitates from uninduced (Control) and induced (Doxycycline) LLW02 cells. Activity was assayed by the release of [32P] H3PO4 from radiolabeled phosphorylase a (1–2 × 105 cpm/nmol). 1 unit of activity releases 1 nmol of phosphate/min at 30°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183861&req=5

Figure 5: Dephosphorylation of phosphorylase a by anti-hemagglutinin immunoprecipitates from uninduced (Control) and induced (Doxycycline) LLW02 cells. Activity was assayed by the release of [32P] H3PO4 from radiolabeled phosphorylase a (1–2 × 105 cpm/nmol). 1 unit of activity releases 1 nmol of phosphate/min at 30°C.
Mentions: Two different approaches were taken to address the biochemical activity of inducible PP1α. First, immunocomplexed 6His-HA-PP1α was tested for its ability to dephosphorylate phosphorylase a. Phosphorylated on a single serine residue, this substrate is often used to monitor the biochemical activity of PP1 in vitro [28]. As shown in Figure 5, greater phosphatase activity is detected in immunoprecipitates from induced LLWO2F cell lysates compared to uninduced. This is consistent with the notion that inducible PP1α is enzymatically active. Our second approach involved testing immunopurified 6His-HA-PP1α for its ability to dephosphorylate the growth suppressor protein pRB. Having previously reported on the ability of immunocomplexed endogenous PP1 to dephosphorylate pRB [20], we followed the same experimental approach. Figure 6 shows a significant decrease in the 32P radiolabel intensity for pRB following incubation with the anti-hemagglutinin immunoprecipitate compared to the normal mouse IgG control immunoprecipitate. In addition, okadaic acid, a potent inhibitor of PP1 activity [29], significantly inhibits pRB dephosphorylation in this reaction. Taken together, these data indicate that inducible PP1α is biochemically active.

Bottom Line: Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation.When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels.Implications of these findings for the study of PP1alpha function in vivo are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA. jludlow@vestatherapeutics.com

ABSTRACT
BACKGROUND: One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity. RESULTS: We have established a cell line for inducing protein expression of the type 1, alpha-isotype, serine/threonine protein phosphatase (PP1alpha). A plasmid encoding a fusion protein of the catalytic subunit of PP1alpha with a 6-histidine peptide (6His) and a peptide from hemagluttinin (HA) was transfected into the UMUC3 transitional cell carcinoma cell line, previously transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo. A stable cell line designated LLWO2F was established by selection with hygromycin B. 6His-HA-PP1alpha protein appeared in cell lysates within two hours following addition of doxycycline to the culture medium. This protein localizes to the nucleus as does endogenous PP1alpha, and was shown to associate with PNUTS, a PP1-nuclear targeting subunit. Like endogenous PP1alpha, immunocomplexed 6His-HA-PP1alpha is active toward phosphorylase a and the product of the retinoblastoma susceptibility gene, pRB. When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels. CONCLUSIONS: These data suggest the existence of an autoregulatory mechanism by which PP1alpha protein levels and activity remain relatively constant. RT-PCR analyses of isolated polysome fractions support the notion that this putative autoregulatory mechanism is exerted, at least in part, at the translational level. Implications of these findings for the study of PP1alpha function in vivo are discussed.

No MeSH data available.


Related in: MedlinePlus