Studies on intracellular protein-protein interactions using fluorescence resonance energy transfer (FRET) technology

Research project P 14653 FRET studies on protein-protein interactions in vivo Helmut RUIS 09.10.2000 A large number of signal transduction pathways play a role in the control of growth, division and survival of eukaryotic cells. In individual signal pathways as well as in the network-like interplay between such pathways, protein-protein interactions play a crucial role. Studying these in vivo in real time is one of the most challenging problems of molecular cell biology. Fluorescence resonance energy transfer (FRET) between proteins fused to.two different mutant derivatives (CFP and YFP) of the bacterial green fluorescent protein (GFP) allows to follow by fluorescence microscopy direct interactions between fusion proteins in living cells. The feasibility of this approach for budding yeast studies has recently been demonstrated by others. For some time, our group has studied signal transduction processes including the dynamic cellular localization of proteins involved in the general stress response of budding yeast (protein kinase A (PKA), Hog1 MAP kinase, the stress activated transcription factors Msn2 and Msn4). We are planning to continue such studies and to bring them to a new level of mechanistic insights by using the FRET methodology to detect, localize and follow kinetically specific protein-protein interactions playing a key role in this signal transduction network. In the project proposed here for funding we therefore intend to study: (1) The interaction between the regulatory PKA subunit Bcy1 with the Zds1 protein, which might function as a yeast analogue of higher eukaryote A kinase anchoring proteins (AKAPs). (2) The in vivo interactions of Zds1 with other cellular proteins. Zds1 appears to be a multifunctional protein. We hypothesize that one mechanistic basis for this is that, similar to mammalian AKAPs, it acts as a multivalent platform for a variety of signal transduction factors. W,ewill attempt to detect and to study such Zds1 interactions. 3) The interaction between the transcription factors Msn2 and Msn4 with the three yeast PKA Sozymes Tpkl, Tpk2 and Tpk3. Our previous results are consistent with the assumption that Msn2/4 phosphorylation plays a key role in the negative control of the generalstress response by PKA_ Particularly, we are interested in clarifying the cellular localization of such interactions and whether these are attenuated by stress factors, which might activate Msn2/4 by inhibiting its negative regulator PKA. (4) Stress signals might alternatively regulate Msn2/4 by activating (a) phosphoprotein phosphatase(s). We plan to identify (a) Msn2 phosphatase(s) by FRET studies and to investigate whether the interaction between Msn2 and phosphatase is controlled by stress. (5) Phosphatase-mutant GFP fusion protein constructs prepared for a FRET screen in connection with the investigation discussed in (4) shall be used to identify and to study systematically in vivo other interactions of phosphatases with yeast proteins. While this systematic study will only be carried out in a follow up project, we are planning to initiate it by studying putative interactions between the phosphatase calcineurin and Zds1. While there is genetic evidence for such an interaction in budding yeast, interactions between the mammalian AKAP79 and calcineurin have been demonstrated more directly and appear to be part of the AKAP signal transduction platform function referred to under (2). By such studies we hope to contribute to the understanding of an AKAP-like function of budding yeast Zds1.

Reversible modification of proteins is one of the fundamental regulatory mechanisms of cellular processes. We have been trying to study dynamic and mechanistic aspects of phosphorylation and dephosphorylation events related to stress and nutrient responses in the yeast Saccharomyces cerevisiae. Specifically, we have been focusing on a transcriptional activator that mediates most of transcriptional reprogramming during acute stress situations. Under optimal growth conditions this activator, Msn2, remains mostly cytoplasmic and functionally idle due to high activities of the cAMP-dependent protein kinase (PKA). PKA, a crucial signalling factor for many growth related processes in yeast, seems to directly target Msn2, and this phosphorylation seems to affect domains that control the intracellular distribution of Msn2. Stress and starvation signals antagonize these effects. Biochemical studies showed that apart from acute glucose starvation no other stressful conditions cause a decrease in Msn2 phosphorylation. In order to study whether protein phosphatases are stress signal carriers in the case of glucose starvation we identified a PP1 protein phosphatase complex as the main enzyme involved in the dephosphorylation of Msn2-PKA sites. Surprisingly, we found that PP1 related enzymes affect Msn2 modification by two different mechanisms. In combination with a still unidentified regulatory subunit the phosphatase will directly target Msn2. In the context of Reg1, a classic regulatory subunit of PP1 however, it will inhibit a second growth related kinase system that is able to modify a subset of sites recognized by PKA. Our studies tried to establish these signalling interactions not only by biochemical and genetic approaches but also through the development of microscopic techniques based on fluorescence resonance energy transfer (FRET). Despite promising developments of these techniques with regard to the signalling field in higher eukaryotes, unfortunately, similar approaches in yeast have generally not yielded results of useful quality.