Dynamic targeting of protein phosphatase 1 (PP1) activity in vivo

点击翻译按钮获取中文摘要

Reversible protein phosphorylation is the major general mechanism regulating most physiological processes in eukaryotic cells, with protein kinases and protein phosphatases playing key roles in the control of cell proliferation, differentiation and a host of other critical events. A common theme in phosphatase regulation is a mechanism whereby localization of the enzyme determines its access to substrates. In the case of the ubiquitous serine/threonine protein phosphatase 1 (PP1), this is mediated by interaction of the catalytic subunit with a panel of regulatory proteins termed “targeting subunits” to generate a range of holoenzyme complexes with distinct subcellular roles. The evolution of this unique combinatorial regulatory mechanism (three PP1 isoforms plus mutually exclusive binding to a panel of >200 regulatory subunits to form heterodimer complexes with high substrate specificity) provides the opportunity to develop highly specific PP1 inhibitors, based on targeted disruption of single complexes. Although biochemical, bioinformatic, and proteomic approaches have identified a wide range of targeting subunits, the current list still cannot account for the large number of regulatory pathways in which PP1 is known to play a critical role and the continuing identification and characterization of novel targeting subunits is thus critical to our understanding of targeted phosphatase activity. Using a unique and powerful combination of live cell imaging with cell fractionation and quantitative interactome profiling we have mapped cell compartment- and isoform-specific targeting of PP1 to a range of functional multiprotein complexes, with roles in diverse processes including ribosome biogenesis, DNA damage repair and chromosome segregation. Along with providing the first comprehensive map of functional PP1 complexes throughout the cell, this is also the first clear indication that the isoforms play both distinct and overlapping roles. The mechanism and functional significance of isoform-specific targeting subunit binding remains unclear, as does the means by which levels and targeting of PP1 catalytic subunits are dynamically regulated to maintain cellular homeostasis. Preliminary results indicate regulation both at the level of transcription and by re-targeting of PP1 between pools of targeting subunits. Another key layer of information that needs to be added to our road map of targeted PP1 activity is the identification of its substrates within the molecular complexes to which it is targeted and we have developed two novel techniques to that end, based on phosphoproteomic analysis following perturbation of local PP1 levels and a catalytically compromised "substrate trap" mutant. Hypotheses and Research Objectives: The long-term objectives of my research program are to (i) define both the mechanism and significance of isoform-specific PP1 targeting and (ii) understand how the formation of PP1 holoenzyme complexes is coordinated within the cell. My hypothesis is that the targeting of PP1 catalytic subunits within the mammalian cell is a dynamic process that can be modulated as required to maintain cellular homeostasis. Specific short-term objectives are: I. Map the dynamic re-targeting of PP1 catalytic subunits and elucidate the underlying mechanism(s) II. Characterize large-scale complex associations and directly identify phosphatase substrates III. Identify the structural determinants of isoform-specific binding of PP1 targeting subunits I am confident that our multi-faceted and rigorous approach, combined with our dedication to technology development, will allow us to continue to make conceptual advances in the field of phosphatase research.