Hallberg 9603733 While a large number of serine/threonine protein kinases have been described, the number of serine/threonine protein phosphatases that have been characterized is, by contrast, far smaller. It is probably unlikely that the removal of phosphates from particular proteins is not as highly regulated as their addition, so this apparent inequality in modifying and "de-modifying" enzymes is an interesting problem. With regard to the 2A subset of protein phosphatases (PP2A), an enzyme whose activity has been causally linked to the regulation of DNA replication, RNA transcription, signal transduction, and intermediary metabolism, it has recently been recognized that: 1) the active enzyme is a heterotrimer composed of a catalytic (C), structural (A), and regulatory (B) subunit, and 2) the regulatory (B) subunit can be one of several totally different proteins. It is now known that the kind of B subunit assembled into the trimer can affect both the localization of the enzyme within the cell as well as changing its substrate specificity. Thus, apparently by regulating the "quality" of its PP2A trimers, the cell can endow PP2A catalytic subunits with a variety of much narrower substrate targets. In order to better understand how this enzyme regulates particular cellular processes, we need to understand the "population dynamics" of the different cellular forms of PP2A and to define their in vivo targets. A combined molecular genetic and biochemical approach to identifying PP2A functional and structural dynamics is readily pursuable using the baker's yeast, Saccharomyces cerevisiae. The genes encoding two, and possibly three, C subunits, two B subunits, and an A subunit from S. cerevisiae have been cloned and characterized. Specific phenotypes of strains expressing mutant alleles of a number of these different genes have shown that PP2A function is required for the control of specific steps in the cell cycle, bud site selection and bud growth, and regulation of expression of the general stres s response. This laboratory has begun developing yeast strains unable to correctly express one or more components of PP2A or to express altered forms of such components and then examining the phenotypic consequences of such alterations and also assess the state of assembly of PP2A trimers in the cell. Immunoprecipitation procedures and non-denaturing polyacrylamide gel electrophoresis combined with western analyses will make it possible to follow the dynamics of assembly and disassembly of virtually all the now known PP2A components during different growth and cell cycle stages. In addition, by immunofluorescence microscopy, it will be possible to localize within the cell where specific forms of PP2A may accumulate. As different forms of PP2A have now been shown to be critical in the regulation of cell division, the studies described should add to our understanding of the mechanisms by which a cell controls the timing of when or when not to divide, a critical capacity that differentiates normal from uncontrolled growth .
