Rapamycin-induced Gln3 Dephosphorylation Is Insufficient for Nuclear Localization: Sit4 AND PP2A PHOSPHATASES ARE REGULATED AND FUNCTION DIFFERENTLY

Jennifer J Tate,Isabelle Georis,André Feller,Evelyne Dubois,Terrance G Cooper

doi:10.1074/jbc.m806162200

Abstract

Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces cerevisiae. Gln3 is cytoplasmic in cells cultured with repressive nitrogen sources (Gln) and nuclear with derepressive ones (Pro) or after treating Gln-grown cells with the Tor inhibitor, rapamycin (Rap). In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which then dissociates from a Gln3-Ure2 complex and enters the nucleus. However, in contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that PP2A (another Tor pathway phosphatase)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells. It thus parallels nuclear Gln3 localization and NCR-sensitive transcription. However, because PP2A is not required for nuclear Gln3 localization in Pro, PP2A-dependent Gln3 dephosphorylation and nuclear localization are likely parallel responses to derepressive nitrogen sources. In contrast, Rap-induced nuclear Gln3 localization absolutely requires all four PP2A components (Pph21/22, Tpd3, Cdc55, and Rts1). In pph21Delta22Delta, tpd3Delta, or cdc55Delta cells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wild-type cells, indicating Rap-induced Gln3 dephosphorylation is insufficient to achieve nuclear localization. Finally, PP2A-dependent Gln3 dephosphorylation parallels conditions where Gln3 is mostly nuclear, while Sit4-dependent and Rap-induced dephosphorylation parallels those where Gln3 is mostly cytoplasmic, suggesting the effects of these phosphatases on Gln3 may occur in different cellular compartments.

Highlights

In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which dissociates from a Gln3-Ure2 complex and enters the nucleus. In contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that prominent roles of type 2A (PP2A)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells
Target of Rapamycin (Tor)2 is widely acknowledged as a global regulator whose actions control and integrate an array of cellular processes ranging from transcription to cell division [1,2,3,4]
PP2A Is Required For Rapamycininduced Nuclear Gln3-Myc13 Localization—Type 2A-related Sit4 phosphatase has been previously shown to participate in multiple downstream effects observed after treating cells with the Tor inhibitor, rapamycin [1, 14, 20, 35]

Summary

Introduction

Target of Rapamycin (Tor) is widely acknowledged as a global regulator whose actions control and integrate an array of cellular processes ranging from transcription to cell division [1,2,3,4]. When cells are growing in the presence of sufficient quantities of a good nitrogen source (e.g. glutamine), Gln is restricted to the cytoplasm; an outcome that requires the negative regulator, Ure2 This prevents Gln from binding to its target sites in the promoters of NCR-sensitive genes (10 –12). Tor Pathway Regulation of Gln and NCR-sensitive Transcription—A connection between the Tor pathway (1, 14 –25) and NCR-sensitive transcription was established quite unexpectedly by results from several transcriptome analyses in which Tor activity was inhibited by the clinically important drug, rapamcyin [15, 17,18,19] These and other studies laid the groundwork for development of an engaging molecular model explaining how NCR was achieved (Fig. 1A) [16]. Tor regulatory pathway at or above the level of events triggered by rapamycin treatment (Fig. 1A)

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