Axel H. Schönthal

Department of Molecular Microbiology and Immunology, K. Norris Jr. Comprehensive Cancer Center, University of Southern California, 2011 Zonal Ave., HMR-405, Los Angeles, CA 90033

Received 8/25/98 Accepted 12/2/98

6. EXPERIMENTALLY INCREASED PP2A ACTIVITY

The down-regulation of phosphatase activity by the use of various natural product inhibitors has yielded lots of preliminary insight into phosphatase function, although this approach has been rather restricted due to the simultaneous effects on several different enzymes (see further above). A different avenue has been pursued with the use of the small tumor (T) antigens of polyomavirus or SV40. These proteins have been found to form stable complexes with PP2A and reduce its enzymatic activity (55, 100, 122), and therefore have served as rather specific tools to analyze in more detail the involvement of PP2A in signal transduction pathways. For example, introducing SV40 small T antigen into cells has helped establish a negative role for PP2A in the regulation of the mitogen activated protein (MAP) kinase pathway (78, 123). (The details of these interactions will be discussed elsewhere in this volume and thus will not be pursued here.)

The opposite experimental approach, the stably increased expression of selected protein phosphatases, would also be helpful to further study their role in signal transduction pathways. However, these types of studies have proven difficult to accomplish. Although it has been shown in mammalian cells that PP2A can be efficiently expressed after transfection of an expression vector containing the cDNA of the catalytic subunit of PP2A (124), there appears to be a potent autoregulatory mechanisms that keeps the overall amount of PP2A protein (catalytic subunit), as well as its enzymatic activity, at constant levels (125). As a consequence, it seems very difficult to establish cell lines with significantly increased overall activity of PP2A.

The observed autoregulatory control of PP2A expression may also provide an explanation for earlier seemingly contradictory findings by others who investigated PP2A expression. For example, in various mammalian cells and in fission yeast it has been shown that the level of PP2A protein remains constant throughout the cell cycle (126-128). In contrast, analyzing the amount of mRNA, others have demonstrated increased PP2A mRNA levels during the early stages of G1 in mammalian cells (129, 130). Moreover, Kakinoki et al. (131), by performing partial hepatectomy, presented evidence of almost constant levels of PP2A protein in regenerating liver, despite a 30-fold increase in PP2A mRNA. These observed discrepancies between elevated mRNA levels and rather constant protein levels can now be explained by the finding of a potent autoregulatory mechanism of PP2A synthesis that appears to work at the level of translation (125). In addition, in human keratinocytes a post-translational level of PP2A regulation was suggested (132).

In order to neutralize and overcome the autoregulatory feedback loop of PP2A expression, different experimental approaches may be useful. For example, microinjection studies have been used to introduce into cells various components of the phosphatase holoenzyme (121, 133). It is likely that in these experiments the large amount of microinjected protein is able to overwhelm any autoregulatory mechanism, at least for a short time, and induce the respective cellular responses. Experiments of this type, although limited by the small number of cells that can be used per experiment, have indeed provided valuable insights into phosphatase function (121, 134-136).

A further strategy to manipulate PP2A activity in cells was recently presented by Ruediger et al. (137). These authors generated an N-terminal mutant of the regulatory A subunit that was able to bind to the catalytic C subunit, but not to the regulatory B subunit. Expression of this A subunit mutant in cells resulted in an increase in the amount of PP2A core protein (A-C heterodimers) and a decrease in the amount of PP2A holoenzyme (B-A-C heterotrimers). Concomitantly, the relative activity of PP2A towards two different substrates, phosphorylase-a and a retinoblastoma (Rb) peptide, was altered. In the case of phosphorylase-a, PP2A activity was slightly stimulated, whereas with Rb peptide a significant inhibition of phosphatase activity was obtained (137). This differential effect of PP2A enzymatic activity generated consequences at the molecular and cellular level as well: transcription from the human immunodeficiency virus (HIV-1) long terminal repeat (LTR), as well as virus production, was inhibited in these cells (137). Thus, these results demonstrated that the manipulation of PP2A subunits, other than the catalytic C subunit, may be a useful experimental approach to manipulate PP2A expression and activity in living cells.