Regulation of subcellular localization of protein kinase A and Msn2 in the control of cell growth and the transcriptional stress response of budding yeast

In nature, a unicellular organism like the yeast Saccharomyces cerevisiae lives in an environment that is often and rapidly changing in terms of quality and quantity of nutrients available and of other environmental factors favourable or unfavourable for growth. Under such conditions, unicellular organisms able to adjust quickly and reversibly to both favourable and unfavourable conditions will have a high selective advantage. It is not surprising therefore that unicellular organisms presently living in nature have developed molecular mechanisms dealing with this problem, which, in principle, are also conserved to some extent in different cell types of the more complex multicellular organisms. Using the budding yeast Saccharomyces cerevisiae as a model organism, we and others have previously been interested in cellular factors playing a role in regulating responses of yeast cells to their environment. The cAMP-dependent protein kinase A of S. cerevisiae plays a major role in its efficient response to both conditions favourable for cell growth and cell division and to stress conditions. This project is an attempt to use primarily information gained in our own recent investigations about protein kinase A to study the molecular details of its action and the factors involved in the processes studied. By this approach, we hope to improve our understanding of how yeast cells cope successfully with their rapidly changing natural environment. Such studies should also contribute more indirectly to our understanding of the complexities of responses of higher eukaryotic cells to their environment. More specifically, we have shown already that the protein kinase A regulatory Bcyl subunits are localized in the nucleus of rapidly growing yeast cells, while they become delocalized during growth on poor quality carbon sources and during stationary phase. Proper nuclear localization is necessary for rapid re-entry of stationary phase cells into a logarithmic growth phase. In this context we are planning to investigate in more detail the molecular mechanisms controlling the dynamic localization of protein kinase A catalytic and regulatory subunits and the functions of this dynamic localization. Particularly we are planning to study whether Bcyl-interacting proteins and Bcyl modifications are involved in the localization of the regulatory Bcyl subunit and to study the functions of specific protein kinase A localization in cellular growth control. Furthermore it is known for some time that protein kinase A is a powerful negative regulator of the budding yeast stress response. We have been able to show that this negative control occurs at least partly by regulating the activity of the stress-activated transcription factor Msn.2. We have succeeded in showing that stress translocates Msn.2 from the cytoplasm to the nucleus, and that this translocation is inhibited and/or reversed by protein kinase A activity. In this context, we would like to investigate in the framework of the present project details of the molecular mechanisms involved in the control of Msn2 localization by PKA. Specifically, we plan to investigate whether protein kinase A interacts directly with Msn2. Having obtained this key information the molecular mechanisms involved in the regulation of nuclear import and nuclear export of Msn.2 by protein kinase A will be studied.

Yeast cells must sense important environmental parameters and process this information to develop their best survival strategies. Nutrient supply and physical stresses normally determine how much a cell will invest into growth and proliferation versus protection against and repair of cellular damage. A signalling system based on the cAMP dependent protein kinase (PKA) has been shown to play a crucial role in this decision. Among other things, PKA heavily influences the genomic expression profile as high activities prevent the expression of a large stress of protection genes. In contrast, low activities allow the induction of these genes. Under exponential growth conditions the main target of PKA is a pair of functionally redundant transcription activators, called Msn2 and Msn4. By inducing stress related genes, these two zinc finger proteins coordinate a common response to a large variety of stress conditions. As shown by us Msn2 and Msn4 function as integrating devices for PKA signals and stress signals. Finding a mechanistic description of the signal integration process was one major goal of the project. A second aim was to design better ways of monitoring PKA activities within the living cells in order to gain an understanding of to which environmental conditions this kinase is responding. Observations we were particularly interested in were related to the intracellular localizations of the main regulatory players: Bcy1, the inhibitory subunit of PKA, one of its catalytic subunits and the presumed target, Msn2. All localization experiments were based on microscopic observations with GFP tagged versions of the proteins. Results showed that Bcy1 is a mostly nuclear protein whereas Tpk1, the catalytic subunit appears more evenly distributed. However, during cAMP depletion one can find Tpk1 concentrated in the nucleus together with Bcy1. Bcy1 only changes its localization under prolonged growth on poor carbohydrate sources. We subsequently identified one protein that seems to serve as a scaffolding platform for Bcy1 in the cytoplasm and analysed their interactions in more detail. Further progress was slightly hampered due to the complicated growth conditions that have to be used in this system. We decided to turn our main attention to the control of Msn2. Environmental conditions regulate Msn2 function at several mechanistically independent steps. Our studies showed that PKA activity has a major impact on at least three cellular mechanisms that regulate Msn2 function. High PKA activity blocks nuclear import, advances nuclear export and inhibits DNA binding of Msn2, respectively. Looking at the Msn2 nuclear import domain, we were able to deduce that acute glucose depletion is the only environmental condition in logarithmically growing yeast that is directly transduced via PKA. All other stresses must target Msn2 by different signalling systems that just antagonize PKA induced modifications on Msn2. Such stresses do not affect nuclear accumulation by regulating the import signal but only by regulating nuclear export or retention. One system we have studied in this context is the high osmolarity response system that uses the MAP kinase Hog1 as a transducer. PKA mediated inhibition of DNA binding is likely to be caused by phosphorylation of the zinc finger. Using a mutational approach we were able to construct a hyperactive allele of Msn2 that has lost its regulation by PKA. This allele causes attenuation of growth and proliferation and might thus lead to the future identification of factors involved in growth decisions.