Drosophila Rif1 is an essential gene and controls late developmental events by direct interaction with PP1-87B

Rif1, identified as a regulator of telomerase in yeast, is an evolutionarily conserved protein and functions in diverse processes including telomere length regulation, epigenetic gene regulation, anti-checkpoint activity, DNA repair and establishing timing of firing at replication origins. Previously we had identified that all Rif1 homologues have PP1 interacting SILK-RVxF motif. In the present study, we show that Drosophila Rif1 is essential for normal fly development and loss of dRif1 impairs temporal regulation of initiation of DNA replication. In multiple tissues dRif1 colocalizes with HP1, a protein known to orchestrate timing of replication in fly. dRif1 associates with chromosomes in a mitotic stage-dependent manner coinciding with dephosphorylation of histones. Ectopic expression of dRif1 causes enlarged larval imaginal discs and early pupal lethality which is completely reversed by co-expression of PP1 87B, the major protein phosphatase in Drosophila, indicating genetic and functional interaction. These findings suggest that dRif1 is an adaptor for PP1 and functions by recruiting PP1 to multiple sites on the chromosome.

Consensus PP1 Binding Motifs Regulate Transcriptional Corepression and Alternative RNA Splicing Activities of the Steroid Receptor Coregulators, p54nrb and PSF

Originally identified as essential pre-mRNA splicing factors, non-POU-domain-containing, octamer binding protein (p54nrb) and PTB-associated RNA splicing factor (PSF) are also steroid receptor corepressors. The mechanisms by which p54nrb and PSF regulate gene transcription remain unclear. Both p54nrb and PSF contain protein phosphatase 1 (PP1) consensus binding RVxF motifs, suggesting that PP1 may regulate phosphorylation status of p54nrb and PSF and thus their function in gene transcription. In this report, we demonstrated that PP1 forms a protein complex with both p54nrb and PSF. PP1 interacts directly with the RVxF motif only in p54nrb, but not in PSF. Association with PP1 results in dephosphorylation of both p54nrb and PSF in vivo and the loss of their transcriptional corepressor activities. Using the CD44 minigene as a reporter, we showed that PP1 regulates p54nrb and PSF alternative splicing activities that determine exon skipping vs. inclusion in the final mature RNA for translation. In addition, changes in transcriptional corepression and RNA splicing activities of p54nrb and PSF are correlated with alterations in protein interactions of p54nrb and PSF with transcriptional corepressors such as Sin3A and histone deacetylase 1, and RNA splicing factors such as U1A and U2AF. Furthermore, we demonstrated a novel function of the RVxF motif within PSF that enhances its corepression and RNA splicing activities independent of PP1. We conclude that the RVxF motifs play an important role in controlling the multifunctional properties of p54nrb and PSF in the regulation of gene transcription.

The translation initiation factor eIF2β is an interactor of protein phosphatase-1

It is reasonably well understood how the initiation of translation is controlled by reversible phosphorylation of the eukaryotic translation initiation factors eIF2α, eIF2Bϵ and eIF4E. Other initiation factors, including eIF2β, are also established phosphoproteins but the physiological impact of their phosphorylation is not known. Using a sequence homology search we found that the central region of eIF2β contains a putative PP1-(protein phosphatase-1) binding RVxF-motif. The predicted eIF2β-PP1 interaction was confirmed by PP1 binding and co-immunoprecipitation assays on cell lysates as well as with the purified components. Site-directed mutagenesis showed that eIF2β contains, in addition to an RVxF-motif, at least one other PP1-binding site in its C-terminal half. eIF2β functioned as an inhibitor for the dephosphorylation of glycogen phosphorylase and Ser51of eIF2α by PP1, but did not affect the dephosphorylation of Ser464 of eIF2Bϵ by this phosphatase. Strikingly, eIF2β emerged as an activator of its own dephosphorylation (Ser2, Ser67, Ser218) by associated PP1, since the substrate quality of eIF2β was decreased by the mere mutation of its RVxF-motif. These results make eIF2β an attractive candidate substrate for associated PP1 in vivo. The overexpression of wild-type eIF2β or eIF2β with a mutated RVxF-motif did not differentially affect the rate of translation, indicating that the binding of PP1 is not rate-limiting for translation under basal conditions.

Protein phosphatase 1 – targeted in many directions

Protein phosphatase 1 (PP1) is a major eukaryotic protein serine/threonine phosphatase that regulates an enormous variety of cellular functions through the interaction of its catalytic subunit (PP1c) with over fifty different established or putative regulatory subunits. Most of these target PP1c to specific subcellular locations and interact with a small hydrophobic groove on the surface of PP1c through a short conserved binding motif – the RVxF motif – which is often preceded by further basic residues. Weaker interactions may subsequently enhance binding and modulate PP1 activity/specificity in a variety of ways. Several putative targeting subunits do not possess an RVxF motif but nevertheless interact with the same region of PP1c. In addition, several ‘modulator’ proteins bind to PP1c but do not possess a domain targeting them to a specific location. Most are potent inhibitors of PP1c and possess at least two sites for interaction with PP1c, one of which is identical or similar to the RVxF motif.Regulation of PP1c in response to extracellular and intracellular signals occurs mostly through changes in the levels, conformation or phosphorylation status of targeting subunits. Understanding of the mode of action of PP1c complexes may facilitate development of drugs that target particular PP1c complexes and thereby modulate the phosphorylation state of a very limited subset of proteins.