t r i n k l e l a b . c o m

Protein Phosphorylation

The typical mammalian cell expresses thousands of proteins, over a third of which are thought to be "phosphoproteins". There is now overwhelming evidence that reversible phosphorylation of such proteins regulates most aspects of cell life. The process involves the addition of a negatively charged phosphate group to one or more of the amino acids that make up the protein. The enzymes involved are termed protein kinases (add phosphates) and protein phosphatases (remove phosphates), and the balance of their activities determines the so-called "phospho- state" of a protein. This in turn can affect the protein’s structure, location within the cell, interaction with other proteins and/or enzymatic activity.

Aberrant phosphorylation has been linked to many human diseases, including diabetes and cancer, and my research studies the targeting of phosphatase complexes within living cells to identify and understand phosphorylation-mediated processes that can give rise to such diseases in humans.

Genomic studies have provided a catalogue of protein kinases and phosphatases:

Protein Kinases	Protein Phosphatases
~518 total	~147 total
~90 phosphorylate Tyr residues	~107 dephosphorylate Tyr residues
~428 phosphorylate Ser/Thr residues	~40 phosphorylate Ser/Thr residues
>98% of phosphorylation events are on Ser/Thr residues, so how do the phosphatases keep up?

Targeted Phosphatases

Kinases are usually specific for only a few proteins, while phosphatases can dephosphorylate hundreds of proteins thoughout the cell. That's how 40 phosphatases can counter the action of over 400 kinases. Regulatory mechanisms are in place to ensure that phosphatases only dephosphorylate the right proteins at the right time. Some are hidden away until they're needed, while others are only active if they're modified in some way. A third method to regulate phosphatases involves forming complexes with other proteins that regulate their activity.

Phosphatases are targeted specifically to particular proteins at the right time and in the right place thanks to specific regulatory mechanisms. Some are regulated by localization (e.g. kept safely hidden until they're needed) and others by modification (e.g. active only when they're phosphorylated themselves). A third mechanism, however, is regulation by other proteins with which the phosphatase associates.

My research focuses on one such phosphatase, protein phosphatase 1 (PP1). PP1 is involved in a wide range of cellular processe, deriving both its intracellular localization and its substrate specificity from proteins with which it associates, termed "targeting subunits". Most of these targeting subunits bind to PP1 through an "RVXF" amino acid motif.

Analysis of the subcellular targeting of PP1 is complicated by the fact that it is expressed in mammalian cells as three closely related isoforms, α, β/δ and γ1, which are encoded by separate genes. These isoforms are more than 89% identical in amino acid sequence yet show distinct specificities for particular targeting subunits and hence distinct subcellular localization patterns

In order to study and compare the dynamic localization of these PP1 isoforms in living cells, I fused them to fluorescent reporter molecules and established cell llines that stably express these fluorescent PP1 fusion proteins. Comparing their localization patterns throughout the cell cycle and following cellular perturbations gives clues to the regulatory pathways in which they're involved and the proteins with which they associate.

What You See is What You Get: Rapid Identification of GFP-PP1 Complexes Using Quantitative Proteomics

By combining both imaging and proteomics in a “dual strategy” approach, the same GFP-tagged protein can be analyzed using fluorescence microscopy and then recovered by affinity purification from whole cell extracts or subcellular fractions for the rapid and reliable identification of its specific protein interaction partners. This is an extremely powerful method for identifying and characterizing new PP1complexes and hence new regulatory complexes within the cell.

This approach has already been used to identify several novel PP1 targeting subunits, including RepoMan, which targets PP1γ to chromatin, is essential for cell viability and plays an important role in the maintenance of chromosome architecture during mitosis. We are now using similar SILAC-based quantitative proteomics techniques to identify other proteins in Repo-Man/PP1 regulatory complex(es).

We have further optimized our dual strategy by the adoption of a very high affinity reagent for purifying GFP-tagged proteins (GFP binder) and the identification of proteins that routinely bind non-specifically to the affinity matrix (sepharose "bead proteome") and thus increase the background noise in an experiment. This list can be used as a specificity filter, to prioritize identified proteins for further analysis.