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.
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
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. Acomprehensive list of gene names for published known/predicted PP1 targeting subunits, adapted from Bollen et al. TIBS 2010), can be downloaded here.
What You See is What You Get:
Rapid Identification of GFP-PP1 Complexes Using Quantitative Proteomics
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 and characterize several novel PP1
targeting subunits, including RepoMan (targets PP1 to
chromatin, is essential for cell viability and plays an important
role in the maintenance of chromosome architecture during mitosis) and RRP1B (targets PP1 to pre-60S ribosomal subunits).
Our dual strategy has been optimized 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 commonly used affinity matrices. In addition, we continue to assess and improve cell fractionation methods for spatial interactome analyses.
Chamousset D, Mamane S, Boisvert FM, Trinkle-Mulcahy L .Efficient extraction of nucleolar proteins for interactome analyses .Proteomics 10:3045-50, 2010.
Chamousset, D., De Wever, V., Moorhead, G., Chen, Y., Boisvert, F.M., Lamond, A.I. and Trinkle-Mulcahy, L. RRP1B targets PP1 to mammalian cell nucleoli and is associated with pre-60S ribosomal subunits. Mol. Biol. Cell 21:4212-26, 2010.
Boulon S, Ahmad Y, Trinkle-Mulcahy L, Verheggen C, Cobley A, Gregor P, Bertrand E, Whitehorn M, Lamond A. Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics 9:861-79, 2010.
Trinkle-Mulcahy L., Boulon S., Lam Y.W., Urcia
R., Boisvert F.M., Vandermoere F., Morrice N.A., Swift S., Rothbauer
U., Leonhardt H. and Lamond A.I. Identifying specific protein interaction
partners using quantitative mass spectrometry and bead proteomes.
J Cell Biol. 183:223-39, 2008.
Trinkle-Mulcahy, L., Andersen, J., Lam, Y.W., Moorhead,
G., Mann, M. and Lamond, A.I. Repo-Man recruits PP1γ to chromatin
and is essential for cell viability. J. Cell Biol. 172:679-92,