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 (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.
Highlights
One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1)
These data suggest the existence of an autoregulatory mechanism by which PP1α protein levels and activity remain relatively constant
We observed that endogenous PP1α protein levels decrease in response to increasing levels of induced 6HisHA-PP1α protein
Summary
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. 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. An abundant enzyme expressed in all cells, complex regulation of PP1 is thought to be essential for proper temporal and spatial regulation of PP1 catalytic activity towards individual substrates [2]. Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression, and an absolute requirement of this activity for mitotic exit [4–7]. There is evidence to suggest that phosphorylation of PP1 and its associated proteins by the cyclin-dependent kinases may regulate PP1 activity in a cell cycle stagedependent manner [8–10]. Current efforts are directed towards identification of cell cycle-dependent substrates for PP1, and how regulation of PP1 activity towards these substrates controls the cell division cycle
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