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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

RT-PCR of polysome and total RNA specific for endogenous PP1α and 6His-HA-PP1α. PCR products from polysome and total RNA from induced and uninduced cell lysates were prepared and analyzed as described in Materials and Methods. MW – molecular mass standard ladder.
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Figure 11: RT-PCR of polysome and total RNA specific for endogenous PP1α and 6His-HA-PP1α. PCR products from polysome and total RNA from induced and uninduced cell lysates were prepared and analyzed as described in Materials and Methods. MW – molecular mass standard ladder.

Mentions: To address the possible autoregulatory mechanism(s) of PP1α expression and activity, we analyzed the RNA levels of both endogenous PP1α and induced 6His-HA-PP1α. For uninduced cells, only endogenous PP1α RNA can be found associated with polysomes (Figure 11, second lane). While some 6His-HA-PP1α RNA can be detected in the total RNA, the majority PP1α RNA detected in the total preparation is endogenous (fourth lane). In contrast, it appears that for the doxycycline-induced cells (third lane), there is more 6His-HA-PP1α RNA associated with polysomes than endogenous PP1α. However, both messages appear to be present at the same level in the total RNA preparation (fifth lane). This apparent preference for 6His-HA-PP1α RNA association with polysomes suggest that autoregulation takes place, at least in part, at the translational level.


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)

RT-PCR of polysome and total RNA specific for endogenous PP1α and 6His-HA-PP1α. PCR products from polysome and total RNA from induced and uninduced cell lysates were prepared and analyzed as described in Materials and Methods. MW – molecular mass standard ladder.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: RT-PCR of polysome and total RNA specific for endogenous PP1α and 6His-HA-PP1α. PCR products from polysome and total RNA from induced and uninduced cell lysates were prepared and analyzed as described in Materials and Methods. MW – molecular mass standard ladder.
Mentions: To address the possible autoregulatory mechanism(s) of PP1α expression and activity, we analyzed the RNA levels of both endogenous PP1α and induced 6His-HA-PP1α. For uninduced cells, only endogenous PP1α RNA can be found associated with polysomes (Figure 11, second lane). While some 6His-HA-PP1α RNA can be detected in the total RNA, the majority PP1α RNA detected in the total preparation is endogenous (fourth lane). In contrast, it appears that for the doxycycline-induced cells (third lane), there is more 6His-HA-PP1α RNA associated with polysomes than endogenous PP1α. However, both messages appear to be present at the same level in the total RNA preparation (fifth lane). This apparent preference for 6His-HA-PP1α RNA association with polysomes suggest that autoregulation takes place, at least in part, at the translational level.

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