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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

Coprecipitation of 6His-HA-PP1α with GST-PNUTS. Doxycycline-induced LLWO2F cell lysates were mixed with either GST or GST-PNUTS bound to glutathione-Sepharose beads, washed, and the bound proteins separated by SDS-PAGE and then immunoblotted with antibody to hemagglutinin. Position of 6His-HA-PP1α is indicated to the left of the panel, and is present only in the GST-PNUTS lane. The band above 6His-HA-PP1α, which is also present in the GST-alone lane, results from non-specific reactivity with the secondary antibody (horse-radish peroxidase-conjugated anti-IgG) used for chemiluminescent detection
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Figure 4: Coprecipitation of 6His-HA-PP1α with GST-PNUTS. Doxycycline-induced LLWO2F cell lysates were mixed with either GST or GST-PNUTS bound to glutathione-Sepharose beads, washed, and the bound proteins separated by SDS-PAGE and then immunoblotted with antibody to hemagglutinin. Position of 6His-HA-PP1α is indicated to the left of the panel, and is present only in the GST-PNUTS lane. The band above 6His-HA-PP1α, which is also present in the GST-alone lane, results from non-specific reactivity with the secondary antibody (horse-radish peroxidase-conjugated anti-IgG) used for chemiluminescent detection

Mentions: Targeting of PP1 to the nucleus is due in part to a PP1-associated nuclear targeting subunit (PNUTS; [27]). To further investigate the properties of inducible PP1α, we performed coprecipitation studies to determine if PNUTS can also be found associated with 6His-HA-PP1α. Towards this goal, a fusion protein of GST and PNUTS was tested for the ability to capture induced PP1α. Lysates from induced LLWO2F cells were combined with GST-alone- or GST-PNUTS-loaded glutathione Sepharose beads. Following separation by SDS-PAGE and western blotting, antibody to hemagglutinin was used to detect any associated 6His-HA-PP1α. Antibody reactivity would indicate a link from the GST epitope to the hemagglutinin epitope via a PNUTS to PP1α interaction. As shown in Figure 4, the hemagglutinin epitope was captured by the GST-PNUTS fusion protein, but not the GST-alone protein. These data support the idea that induced PP1α and the nuclear targeting PP1-associated protein PNUTS can form a complex. These results further support the view that induced PP1α behaves similarly if not identically to endogenous PP1α, and provides a possible mechanistic explanation for 6His-HA-PP1α localization to the nucleus.


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)

Coprecipitation of 6His-HA-PP1α with GST-PNUTS. Doxycycline-induced LLWO2F cell lysates were mixed with either GST or GST-PNUTS bound to glutathione-Sepharose beads, washed, and the bound proteins separated by SDS-PAGE and then immunoblotted with antibody to hemagglutinin. Position of 6His-HA-PP1α is indicated to the left of the panel, and is present only in the GST-PNUTS lane. The band above 6His-HA-PP1α, which is also present in the GST-alone lane, results from non-specific reactivity with the secondary antibody (horse-radish peroxidase-conjugated anti-IgG) used for chemiluminescent detection
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Coprecipitation of 6His-HA-PP1α with GST-PNUTS. Doxycycline-induced LLWO2F cell lysates were mixed with either GST or GST-PNUTS bound to glutathione-Sepharose beads, washed, and the bound proteins separated by SDS-PAGE and then immunoblotted with antibody to hemagglutinin. Position of 6His-HA-PP1α is indicated to the left of the panel, and is present only in the GST-PNUTS lane. The band above 6His-HA-PP1α, which is also present in the GST-alone lane, results from non-specific reactivity with the secondary antibody (horse-radish peroxidase-conjugated anti-IgG) used for chemiluminescent detection
Mentions: Targeting of PP1 to the nucleus is due in part to a PP1-associated nuclear targeting subunit (PNUTS; [27]). To further investigate the properties of inducible PP1α, we performed coprecipitation studies to determine if PNUTS can also be found associated with 6His-HA-PP1α. Towards this goal, a fusion protein of GST and PNUTS was tested for the ability to capture induced PP1α. Lysates from induced LLWO2F cells were combined with GST-alone- or GST-PNUTS-loaded glutathione Sepharose beads. Following separation by SDS-PAGE and western blotting, antibody to hemagglutinin was used to detect any associated 6His-HA-PP1α. Antibody reactivity would indicate a link from the GST epitope to the hemagglutinin epitope via a PNUTS to PP1α interaction. As shown in Figure 4, the hemagglutinin epitope was captured by the GST-PNUTS fusion protein, but not the GST-alone protein. These data support the idea that induced PP1α and the nuclear targeting PP1-associated protein PNUTS can form a complex. These results further support the view that induced PP1α behaves similarly if not identically to endogenous PP1α, and provides a possible mechanistic explanation for 6His-HA-PP1α localization to the nucleus.

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