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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.

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Time course of induction followed by western blotting of whole cell lysates using antibody to PP1α. Equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours after doxycycline addition (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting using antibody specific for PP1α. Positions of endogenous PP1α and 6His-HA-PP1α are indicated by arrows to the left and right of the figure, respectively.
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Figure 8: Time course of induction followed by western blotting of whole cell lysates using antibody to PP1α. Equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours after doxycycline addition (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting using antibody specific for PP1α. Positions of endogenous PP1α and 6His-HA-PP1α are indicated by arrows to the left and right of the figure, respectively.

Mentions: One prediction would be that by increasing expression of the catalytic subunit of PP1α, overall PP1-specific activity within the cell would increase. To address this, we tested the phosphatase activity of LLWO2F whole-cell lysate using phosphorylase a as the substrate. To inhibit any endogenous PP2A activity, which can also dephosphorylate phosphorylase a and thus interfere with PP1 activity measurements, these assays were carried out in the presence of 5 nM okadaic acid, which does not inhibit PP1 activity [30]. Although reproducible, we observed only modest increases in PP1-specific activity found in lysates from induced cells compared to the uninduced controls (Figure 7). One possible explanation for this finding is that the overall level of PP1α within the cell remains relatively constant regardless of induction. Indeed, a time course of induction followed by western blotting of whole cell lysates using antibody to PP1α revealed diminution of the endogenous PP1α signal after 4 hr of induction, while the 6His-HA-PP1α signal increased (Figure 8). To further test this hypothesis, immunoprecipitation and western blotting experiments were carried out using PP1 isoform-specific antibodies. As shown in Figure 9, top panel, induced (approximate molecular weight of 40 kDa) and endogenous (approximate molecular weight of 37 kDa) PP1α are clearly resolved by SDS-PAGE. As predicted, both proteins are recognized by antibody specific for PP1α when performing immunoprecipitation or western blotting. Immunoprecipitation using PP1α antibody followed by western blotting using the same anti-PP1α reveals a reciprocal relationship in PP1α abundance following doxycycline induction; endogenous PP1α levels decrease in response to increasing levels of 6His-HA-PP1α (top panel, compare left two lanes). This difference in abundance is more striking in the whole-cell lysate lanes; endogenous PP1α fails to be detected in lysates prepared from induced cells (compare middle two lanes). Parallel experiments performed using the same lysate and antibody to hemagglutinin for immunoprecipitation and western blotting show the position of 6His-HA-PP1α and the fact that endogenous PP1α is not recognized by this antibody (bottom panel). Western blotting for additional isoforms of PP1 reveals that this reduction in PP1α following induction is isotype-specific; when 6His-HA-PP1α levels increase, concomitant with a decrease in PP1α levels, PP1δ and PP1γ1 levels remain relatively unchanged from those found in uninduced cell lysate (Figure 10). Taken together, these data suggest that an as yet undescribed negative-feedback or autoregulatory mechanism exists for PP1α which contributes towards maintaining a constant level of protein expression and enzyme activity.


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)

Time course of induction followed by western blotting of whole cell lysates using antibody to PP1α. Equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours after doxycycline addition (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting using antibody specific for PP1α. Positions of endogenous PP1α and 6His-HA-PP1α are indicated by arrows to the left and right of the figure, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC183861&req=5

Figure 8: Time course of induction followed by western blotting of whole cell lysates using antibody to PP1α. Equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours after doxycycline addition (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting using antibody specific for PP1α. Positions of endogenous PP1α and 6His-HA-PP1α are indicated by arrows to the left and right of the figure, respectively.
Mentions: One prediction would be that by increasing expression of the catalytic subunit of PP1α, overall PP1-specific activity within the cell would increase. To address this, we tested the phosphatase activity of LLWO2F whole-cell lysate using phosphorylase a as the substrate. To inhibit any endogenous PP2A activity, which can also dephosphorylate phosphorylase a and thus interfere with PP1 activity measurements, these assays were carried out in the presence of 5 nM okadaic acid, which does not inhibit PP1 activity [30]. Although reproducible, we observed only modest increases in PP1-specific activity found in lysates from induced cells compared to the uninduced controls (Figure 7). One possible explanation for this finding is that the overall level of PP1α within the cell remains relatively constant regardless of induction. Indeed, a time course of induction followed by western blotting of whole cell lysates using antibody to PP1α revealed diminution of the endogenous PP1α signal after 4 hr of induction, while the 6His-HA-PP1α signal increased (Figure 8). To further test this hypothesis, immunoprecipitation and western blotting experiments were carried out using PP1 isoform-specific antibodies. As shown in Figure 9, top panel, induced (approximate molecular weight of 40 kDa) and endogenous (approximate molecular weight of 37 kDa) PP1α are clearly resolved by SDS-PAGE. As predicted, both proteins are recognized by antibody specific for PP1α when performing immunoprecipitation or western blotting. Immunoprecipitation using PP1α antibody followed by western blotting using the same anti-PP1α reveals a reciprocal relationship in PP1α abundance following doxycycline induction; endogenous PP1α levels decrease in response to increasing levels of 6His-HA-PP1α (top panel, compare left two lanes). This difference in abundance is more striking in the whole-cell lysate lanes; endogenous PP1α fails to be detected in lysates prepared from induced cells (compare middle two lanes). Parallel experiments performed using the same lysate and antibody to hemagglutinin for immunoprecipitation and western blotting show the position of 6His-HA-PP1α and the fact that endogenous PP1α is not recognized by this antibody (bottom panel). Western blotting for additional isoforms of PP1 reveals that this reduction in PP1α following induction is isotype-specific; when 6His-HA-PP1α levels increase, concomitant with a decrease in PP1α levels, PP1δ and PP1γ1 levels remain relatively unchanged from those found in uninduced cell lysate (Figure 10). Taken together, these data suggest that an as yet undescribed negative-feedback or autoregulatory mechanism exists for PP1α which contributes towards maintaining a constant level of protein expression and enzyme activity.

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