RNAi screening for kinases and phosphatases identifies FoxO regulators

Abstract

Forkhead box class O (FoxO) transcription factors are key regulators of growth, metabolism, life span, and stress resistance. FoxOs integrate signals from different pathways and guide the cellular response to varying energy and stress conditions. FoxOs are modulated by several signaling pathways, e.g., the insulin-TOR signaling pathway and the stress induced JNK signaling pathway. Here, we report a genome wide RNAi screen of kinases and phosphatases aiming to find regulators of dFoxO activity in Drosophila S2 cells. By using a combination of transcriptional activity and localization assays we identified several enzymes that modulate dFoxO transcriptional activity, intracellular localization and/or protein stability. Importantly, several currently known dFoxO regulators were found in the screening, confirming the validity of our approach. In addition, several interesting new regulators were identified, including protein kinase C and glycogen synthase kinase 3␤, two proteins with important roles in insulin signaling. Furthermore, several mammalian orthologs of the proteins identified in Drosophila also regulate FOXO activity in mammalian cells. Our results contribute to a comprehensive understanding of FoxO regulatory processes.dFoxO ͉ insulin signaling ͉ PKC F orkhead box class O (FoxO) transcription factors are members of the forkhead box transcription factor superfamily, with orthologs in various species such as mammals (1, 2), C.elegans (3), Zebrafish (4) and Drosophila (5, 6). FoxO proteins possess a wide range of cellular functions ranging from the induction of apoptosis and cell cycle control, to the oxidative stress response and lifespan determination. In addition, FoxOs are well defined targets downstream of the conserved insulin/TOR and JNK signaling networks having an important role in the regulation of processes as diverse as cellular growth, stress resistance, and energy homeostasis (7,8). Consequently, FoxOs have several characterized bona fide target genes involved in metabolism and growth, such as g6pase (9), pepck (10), 4e-bp (5, 6), insulin receptor (6, 11), and myc (12).To date, several proteins are known to interact with FoxO transcription factors, regulating their intracellular localization and/or activity, and the number of newly identified regulators is rapidly increasing (13). One of the best documented is the AKT/ PKB -kinase, which phosphorylates FoxO in three conserved Ser/Thr residues, leading to FoxO cytoplasmic retention and transcriptional inactivation (14-17). In the cytoplasm, FoxO is ubiquitinylated and targeted for degradation (18,19). Upon growth factor depletion, FoxO is predominantly nuclear. Interestingly, FoxO transcriptional activity can also be modulated in the nucleus (20,21). Furthermore, some subpopulations of growing cells possess nuclear inactive FoxO, implying that additional layers of control exist in the nucleus (22). Given the variety of cellular functions where FoxOs are implicated, and the observation that FoxOs act as a converging point for many different signa...

ER stress potentiates insulin resistance through PERK-mediated FOXO phosphorylation

“…S2B). In an RNAi screen, depletion of Ire1 was reported to modestly reduce FOXO abundance but not to affect nuclear localization (Mattila et al 2008). We confirmed that depletion of Ire1 by >50% did not affect insulininduced relocalization of FOXO to the cytoplasm or FOXO mislocalization caused by depletion of Hrd3 (Supplemental Fig.…”

Synaptic Activity and Nuclear Calcium Signaling Protect Hippocampal Neurons from Death Signal-associated Nuclear Translocation of FoxO3a Induced by Extrasynaptic N-Methyl-d-aspartate Receptors

“…Several signaling molecules and pathways have been implicated in the regulation of the subcellular distribution of FoxO3a in various cell types (37)(38)(39)(40), among them JNK (41,42), the p38 MAP kinase (43), and protein phosphatase 2A (44). We found that pharmacological inhibition of JNK, p38 MAP kinase, or protein phosphatase 2A using the JNK-1 inhibitor peptide, SB203580, and okadaic acid, respectively, blocked glutamateinduced nuclear translocation of FoxO3a-GFP in hippocampal neurons (Fig.…”

Drosophila FoxO Regulates Organism Size and Stress Resistance through an Adenylate Cyclase

“…3, compare panels C and F). Parallel ac76e in situ stainings in the homozygous foxo 25 null mutant and its littermate control strain, EP-147 (20), demonstrated a clear reduction in the ac76e in situ signal in the foxo null flies both in gastric ceca and in the CA (Fig. 3, compare panels C and D).…”

“…The foxo null foxo 25 strain and its littermate control, EP-147, are described in reference 20 and DI-11-Gal4 in reference 2. To produce UAS-AC76E transgenic flies, the coding region of ac76e tagged with a three-hemagglutinin (HA)-tag sequence in its C terminus was cloned into the p[UAS] vector.…”

“…The antibodies used were anti-V5 (1:5,000; Invitrogen), anti-HA (1:3,000; Roche), anti-␣-tubulin (1:10,000; Sigma), anti-green fluorescent protein (GFP) (1:500; Santa Cruz Biotechnology), and secondary rabbit anti-mouse immunoglobulin G antibody conjugated with horseradish peroxidase (1:5,000; Upstate Biotechnology) and Alexa Fluor 568 -rabbit anti-mouse immunoglobulin G (1:1,000; Molecular Probes). RNA interference and qPCR were performed as described previously (25). The primers used to synthesize AC76E double-stranded RNA were GGTCAT GTCACACGCAATCGTCC and CACCCTGGCGATCCAGAGTGTG.…”