NOM1 Targets Protein Phosphatase I to the Nucleolus

Shanaka R Gunawardena,Brian L Ruis,Julia A Meyer,Meenal Kapoor,Kathleen F Conklin

doi:10.1074/jbc.m706708200

Shanaka R Gunawardena, Brian L Ruis + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m706708200

Copy DOI

Abstract

Protein phosphatase I (PP1) is an essential eukaryotic serine/threonine phosphatase required for many cellular processes, including cell division, signaling, and metabolism. In mammalian cells there are three major isoforms of the PP1 catalytic subunit (PP1alpha, PP1beta, and PP1gamma) that are over 90% identical. Despite this high degree of identity, the PP1 catalytic subunits show distinct localization patterns in interphase cells; PP1alpha is primarily nuclear and largely excluded from nucleoli, whereas PP1gamma and to a lesser extent PP1beta concentrate in the nucleoli. The subcellular localization and the substrate specificity of PP1 catalytic subunits are determined by their interaction with targeting subunits, most of which bind PP1 through a so-called "RVXF" sequence. Although PP1 targeting subunits have been identified that direct PP1 to a number of subcellular locations and/or substrates, no targeting subunit has been identified that localizes PP1 to the nucleolus. Identification of nucleolar PP1 targeting subunit(s) is important because all three PP1 isoforms are included in the nucleolar proteome, enzymatically active PP1 is present in nucleoli, and PP1gamma is highly concentrated in nucleoli of interphase cells. In this study, we identify NOM1 (nucleolar protein with MIF4G domain 1) as a PP1-interacting protein and further identify the NOM1 RVXF motif required for its binding to PP1. We also define the NOM1 nucleolar localization sequence. Finally, we demonstrate that NOM1 can target PP1 to the nucleolus and show that a specific NOM1 RVXF motif and the NOM1 nucleolar localization sequence are required for this targeting activity. We therefore conclude that NOM1 is a PP1 nucleolar targeting subunit, the first identified in eukaryotic cells.

Highlights

Elegant experiments by Trinkle-Mulcahy et al [6] have followed the location of PP1␥ during the cell cycle and demonstrate that it is highly concentrated in the nucleoli of interphase cells, it localizes at kinetochores early in mitosis and is recruited to mitotic chromatin during anaphase
Sequencing of candidate NOM1 interaction partners obtained in this screen identified three independent clones that encoded the catalytic subunit of protein phosphatase I␣ (PP1␣), making PP1␣ the most frequent cDNA identified
Trinkle-Mulcahy et al [6] have demonstrated that the location of PP1␥ changes during the cell cycle; it is highly concentrated in nucleoli of interphase cells, localizes at kinetochores early in mitosis, and is recruited to mitotic chromatin during anaphase

Summary

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid—NOM1 sequences that encode amino acids 247– 860 of the full-length protein were inserted into the yeast GBKT7 expression vector (Clontech) and the resultant plasmid introduced into the yeast AH109 strain. To generate NOM1 deletion constructs, primers were synthesized and used to amplify regions of NOM1 that encode amino acids 1–300 (NOM1-(1–300)), 1–350 (NOM1-(1–350)), 1–561 (NOM1(1–561)), and 247– 860 (NOM1-(247– 860)) Each of these fragments was inserted in-frame into the BamHI-XbaI site of the CSII-CDF-EF-3xFLAG vector. NOM1 inserts were introduced in-frame into this modified vector as BamHI-XbaI fragments to generate NOM1-mCherry fusion proteins with mCherry at the amino terminus. Human PP1␣ and PP1␥ cDNAs were generated by RT-PCR and cloned in-frame into the EcoRI site of the pcDNA6/HisC (Invitrogen) expression vector, which includes His and Xpress epitope tags at the amino terminus of sequences inserted into the multiple cloning site. Antibody conjugates were visualized using the ECL PlusTM Kit (GE Healthcare)

RESULTS

DISCUSSION

Conklin