Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase

Abstract

Phosphorylation and activation of ribosomal S6 protein kinase is an important link in the regulation of cell size by the target of rapamycin (TOR) protein kinase. A combination of selective inhibition and RNA interference were used to test the roles of members of the PP2A subfamily of protein phosphatases in dephosphorylation of Drosophila S6 kinase (dS6K). Treatment of Drosophila Schneider 2 cells with calyculin A, a selective inhibitor of PP2A-like phosphatases, resulted in a 7-fold increase in the basal level of dS6K phosphorylation at the TOR phosphorylation site (Thr398) and blocked dephosphorylation following inactivation of TOR by amino acid starvation or rapamycin treatment. Knockdown of the PP2A catalytic subunit increased basal dS6K phosphorylation and inhibited dephosphorylation induced by amino acid withdrawal. In contrast, depletion of the catalytic subunits of the other two members of the subfamily did not enhance dS6K phosphorylation. Knockdown of PP4 caused a 20% decrease in dS6K phosphorylation and knockdown of PP6 had no effect. Knockdown of the Drosophila B56-2 subunit resulted in enhanced dephosphorylation of dS6K following removal of amino acids. In contrast, knockdown of the homologs of the other PP2A regulatory subunits had no effects. Knockdown of the Drosophila homolog of the PP2A/PP4/PP6 interaction protein α4/Tap42 did not affect S6K phosphorylation, but did induce apoptosis. These results indicate that PP2A, but not other members of this subfamily, is likely to be a major S6K phosphatase in intact cells and is consistent with an important role for this phosphatase in the TOR pathway.

Introduction

The target of rapamycin (TOR) is a conserved protein kinase that lies at the hub of a signaling network responsible for sensing and integrating nutritional status, growth stimuli, and cell stress. Two of the best characterized targets of mammalian TOR, the ribosomal S6 protein kinases (S6K) and the eukaryotic initiation factor 4E binding proteins, are regulators of protein synthesis [1], [2], [3]. TOR-dependent activation of mammalian S6K increases protein synthesis by promoting assembly of the eukaryotic translation pre-initiation complex [4]. S6K is encoded by two genes in mammals, S6K1 and S6K2. Genetic experiments in mice showed that S6K1 is an essential mediator of the effects of TOR signaling on cell size and mass [5]. The single Drosophila homolog of S6K (dS6K) also plays a critical role in cell growth as flies lacking the gene have a delay in development, lower body weight, and smaller cells than wild-type flies [6].

Activation of mammalian S6K1 involves phosphorylation at multiple sites in response to nutrients and growth factors. Activation of S6K is initiated by phosphorylation of Thr389 within its hydrophobic motif by the TOR/raptor complex. Phosphorylation of Thr389 generates a docking site for phosphoinositide-dependent kinase 1 (PDK1), which phosphorylates Thr229 within the activation loop leading to activation of kinase activity. Inhibition of TOR activity with rapamycin leads to rapid dephosphorylation of these two sites [7]. The TOR and PDK1 phosphorylation sites (Thr238 and Thr398 in dS6K) are conserved in Drosophila S6K. S6K phosphorylation at Thr398 by Drosophila TOR (dTOR) is essential for kinase activation [8].

The protein phosphatases involved in the physiological dephosphorylation and inactivation of S6K have not been identified. Both mammalian S6K and dS6K are inactivated by dephosphorylation of the TOR site following amino acid starvation or inhibition of TOR with rapamycin [8], [9]. An initial link between the PP2A subfamily of protein phosphatases and TOR came from genetic experiments in yeast (reviewed in Di Como and Jiang [10]). The phosphatase 2A-associated protein of 42-kDa (Tap42) associates with the catalytic subunits of the yeast PP2A subfamily (PP2A, PP4, and PP6). Tap42 is a major effector of TOR signaling in yeast that interacts with the PP2A subfamily following phosphorylation by TOR [11]. Mammalian cells express a protein (α4/mTap42) that has 23% amino acid sequence identity with yeast Tap42. The α4/mTap42 protein interacts directly with the catalytic subunits of PP2A, PP4 and PP6 [12], [13], [14], [15] and modulates their enzyme activity [16]. The α4/mTap42 protein has also been reported to interact with mammalian S6K [17]. Although interaction of Tap42 with the PP2A subfamily has been conserved in higher eukaryotes, a role in TOR signaling has not been established. Unlike yeast Tap42, disruption of Drosophila Tap42 has no effect on cell growth [18].

Biochemical studies also support a role for PP2A subfamily phosphatases in TOR signaling in mammalian cells. In vitro fractionation and enzymatic characterization indicate that S6K is dephosphorylated by PP2A [19]. The catalytic subunit of PP2A can be isolated in a complex with S6K following cross-linking of soluble brain extracts [20] or by immunoprecipitation of S6K from Jurkat T cell lysates [21]. Dephosphorylation of S6K following amino acid starvation, rapamycin treatment, or cell stress is blocked by inhibitors with selectivity for the PP2A subfamily [21], [22]. However, these inhibitors cannot distinguish between the PP2A-like phosphatases [16], [23]. Consequently, the phosphatase that dephosphorylates S6K in vivo has not been identified.

PP2A, PP4, and PP6 comprise the type 2A subfamily within the serine/threonine phosphatase gene family. These enzymes play vital roles in multiple aspects of cellular signal transduction [24]. The term PP2A refers to a diverse group of phosphatases composed of a common catalytic subunit that forms oligomeric complexes with a variety of regulatory proteins. The most prevalent forms of PP2A contain a core dimer composed of the catalytic subunit and a scaffold protein termed the A subunit. The core dimer binds additional regulatory subunits that target PP2A to specific substrates [25], [26]. The mammalian PP4 catalytic subunit also interacts with scaffold and regulatory subunits [27], [28], while PP6 regulatory proteins have been identified in yeast [29]. Due to decreased complexity relative to mammalian cells, Drosophila has been a valuable system to study functions of individual catalytic and regulatory subunits within the PP2A subfamily [30], [31], [32]. The Drosophila genome contains homologs of the PP2A catalytic, scaffold, and regulatory subunits [30], [31], as well as homologs of PP4 [33], PP6 [34], and Tap42 [18].

To determine if one or more members of the PP2A subfamily or its regulatory proteins regulate S6K in vivo, a combined approach using pharmacological inhibition and RNA interference in Drosophila cells was used to assess their roles in controlling S6K phosphorylation at the TOR phosphorylation site. The results show that PP2A plays an important role in dephosphorylation of dS6K, while PP4, PP6, and dTap42 are not directly involved in regulating phosphorylation of this site.