Targeting PP1
During the 1980s, Philip Cohen’s group pioneered the concept of “targeting subunits”for PP1.
These are regulatory proteins that bind the catalytic subunit to provide subcellular localization and substrate specificity.
This regulatory mechanism explains how a handful of phosphatases can counteract, with high specificity, the activity of >400 kinases.
These regulatory proteins were denatured and removed during classical biochemical purification methods, which is why they were missed originally.
The first two PP1 targeting that were identified were the abundant G subunit (binds glycogen, mediates de-phosphorylation of bound substrates such as glycogen synthase and glycogen phosphorylase kinase) and M subunit (binds myosin, mediates dephosphorylation of the regulatory light chain ).
Both were subsequently shown to be subject to phosphorylation-mediated regulation themselves (by multiple kinases), which alters the phosphatase activity of their respective complexes
Current paradigm: PP1 is dynamically distributed throughout the cell in a wide range of regulatory complexes
This is an elegant, "LEGO-like" approach to build in specificity....
....but it complicates the use of inhibitors targeted at the catalytic site of the enzyme (a common approach for research- and therapeutic-based inhibition of kinase activity).
To design targeted inhibitors for PP1, we must identify and functionally characterize the specific holoenzyme complexes that mediate specific dephosphorylation events in the cell.
Mapping the subcellular distribution of PP1 holoenzyme complexes
Our lab was the first to stably express fluorophore-tagged PP1 (GFP; green fluorescent protein) for analysis of its distribution throughout the cell cycle and in response to various perturbations. This provided the first demonstration of its dynamic targeting in live cells, which can be overridden by overexpression of any particular targeting subunit (resulting in recruitment of excess PP1 activity to the relevant substrates) or by over expression of a dominant-negative mutant of the targeting subunit that cannot bind PP1 (resulting in recruitment of less PP1 activity to the relevant substrates).
One further complication: PP1 is expressed in human cells as 3 closely related isoforms (~90% identical) encoded by separate genes. They differ primarily at their N- and C-termini.
The 3 isoforms (alpha, beta and gamma) show both overlapping and distinct associations with the pool of available targeting subunits, which govern their specific (and dynamic) subcellular localization patterns.
This fluorophore-based approach also allowed us to develop complementary quantitative proteomic methods to capture the tagged PP1 isoforms (using a high affinity GFP nanobody reagent) from various cell types under a range of different conditions, for snapshot mapping of their interactomes (by affinity-purification/mass spectrometry) at specific times and in specific subcellular locations.
Challenge:
The PP1 interactome is complex, representing holoenzyme complexes (>200 putative targeting subunits identified to date), substrates, partners in multi protein complexes, scaffolds, etc.
To reduce complexity, we fractionate cells (to independently map cytoplasmic and nuclear complexes) and we screen all hits for PP1 binding motifs to identify targeting subunits.
Step 1: Map the interactomes of the PP1 isoforms to identify targeting subunit "hubs"
Step 3: Determine the phosphorylation events regulated by the PP1 complex
Step 2: Map the interactomes of individual targeting subunits with high resolution to get clues to the cellular function(s) of the PP1 complex
Step 4: Determine the functional relevance of the PP1-mediated dephosphorylation events in specific cellular pathways.
Current Lab Projects
We have ongoing projects that are functionally characterizing the contribution of specific PP1 complexes to a wide range of cellular pathways, including nuclear organization, ribosome biogenesis, DNA damage sensing and repair, stress response and regulation of protein expression. These projects are aimed at developing novel therapeutic approaches targeted at PP1-mediated events that are dysregulated in cancer and in neurodegenerative diseases.