Repo-Man (white) brings PP1γ onto chromatin at anaphase.

The Repo-Man brings a phosphatase to mitotic chromatin, as shown by Trinkle-Mulcahy et al. (page 679).

By countering kinase activities, protein phosphatase 1 (PP1) regulates many different cellular signaling pathways. PP1 comes in three flavors whose specificities rely mainly on their precise targeting. Trinkle-Mulcahy and colleagues sought to identify the proteins responsible for this targeting.

The authors began by looking for different localization patterns for the α and γ PP1 isoforms. PP1γ, they found, jumped onto chromosomes at anaphase. PP1α, on the other hand, was mainly excluded from mitotic chromatin.

This sharp difference allowed the group to find the protein that recruited PP1 to chromatin. Proteins that associated with PP1γ, but not PP1α, were identified by feeding cell lines that expressed either GFP, PP1α-GFP, or PP1γ-GFP with three different isotopes of arginine. They then isolated all the proteins that immunoprecipitated with anti-GFP antibodies. Using this approach, the authors could see which proteins came down with each PP1 isoform while rapidly discarding all nonspecific interactions.

One of the two proteins that interacted specifically with PP1γ, called Repo-Man, took the same anaphase leap onto chromosomes. A mutant version of Repo-Man that did not bind to the phosphatase blocked PP1γ's chromatin localization.

This mutant protein also caused widespread apoptosis, with the cells dying predominantly during interphase. The same effect was seen when Repo-Man levels were reduced by RNAi. Although the Repo-Man-PP1γ complex loads onto chromatin at anaphase, it remains there throughout interphase, when essential functions that require phosphatase activity (such as chromatin remodeling) take place.

The group suspects that Repo-Man may also regulate PP1γ activity once it is loaded onto chromatin, based on chromosome segregation defects observed in cells with high levels of Repo-Man. They now want to find the anaphase and interphase PP1γ substrates by pulling down proteins that interact with Repo-Man. Early results show a range of DNA-interacting proteins, as expected.