Follow the Glowing PP1s

Abstract

Protein phosphatase 1 (PP1) exists in three isoforms encoded by three separate genes: PP1α, PPI β/δ, and PP1γ. Each of these interacts with targeting subunits and has a distinct subcellular localization. However, results with antibody labeling have yielded conflicting results. Therefore, Trinkle-Mulcahy et al. tracked the location of the PP1 isoforms by creating green fluorescent protein (GFP) fusion proteins. The fusion proteins were catalytically active and recognized by both GFP- and PP1-specific antibodies. All three proteins were found in the cytoplasm and nucleus, however, their nuclear distributions were different: PP1α was diffuse in the nucleus and excluded from the nucleoli, PP1γ was concentrated in the nucleoli, and PPI β/δ was found both in nucleoli and diffuse in the nucleoplasm. Further analysis of the PP1α and PP1γ showed that overexpression of GFP-labeled nuclear inhibitor of PP1 (NIPP1) caused the redirection of both PP1α and PP1γ to nuclear speckles. These results suggested that the PP1 isoforms exist in a dynamic equilibrium with their targeting proteins and that changing the amounts of the targeting subunits may be one mechanism for altering PP1 localization. Photobleaching experiments showed that the nuclear and cytoplasmic pools of PP1 are interchangeable and dynamic. Heterokaryon experiments showed that the relocalization of PP1 to nuclear speckles mediated by NIPP1 did not require new protein synthesis and that proteins could move from one nucleus to the other. Finally, fluorescence resonance energy transfer showed that PP1γ and NIPP1 interact directly in nuclear speckles, consistent with results using an NIPP1 mutant that did not bind PP1 and did not recruit PP1 to nuclear speckles. L. Trinkle-Mulcahy, J. E. Sleeman, A. I. Lamond, Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells. J. Cell Sci. 114 , 4219-4228 (2001). [Online Journal]