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

Cancer Cell International

John W Ludlow²  Emma Villa-Moruzzi³  Peter Klover⁴  Edward M Messing¹  Mark P Sowden⁵  Jay E Reeder¹ 

[1] Department of Urology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA;University of Rochester Cancer Center, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA;Department of Experimental Pathology, University of Pisa, Via Roma 55, Pisa, Italy;Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA;Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA

关键词: inducible protein expression;    phosphatase activity;    serine/threonine dephosphorylation;    retinoblastoma protein;    Type 1 protein phosphatase;   

Others  :  796078
DOI  :  10.1186/1475-2867-3-12

 received in 2003-04-28, accepted in 2003-07-23,  发布年份 2003

【 摘 要 】

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 (PP1α). A plasmid encoding a fusion protein of the catalytic subunit of PP1α 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-PP1α 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 PP1α, and was shown to associate with PNUTS, a PP1-nuclear targeting subunit. Like endogenous PP1α, immunocomplexed 6His-HA-PP1α is active toward phosphorylase a and the product of the retinoblastoma susceptibility gene, pRB. When forcibly overexpressing 6His-HA-PP1α, there is a concomitant decrease in endogenous PP1α levels.

Conclusions

These data suggest the existence of an autoregulatory mechanism by which PP1α 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 PP1α function in vivo are discussed.

【 授权许可】

   
2003 Reeder et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

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