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A Brain-Specific SGK1 Splice Isoform Regulates Expression of ASIC1 in Neurons

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A Brain-Specific SGK1 Splice Isoform Regulates Expression of ASIC1 in Neurons A brain-specific SGK1 splice isoform regulates expression of ASIC1 in neurons Maria F. Arteaga, Tatjana Coric, Christoph Straub, and Cecilia M. Canessa* Department of Cellular and Molecular Physiology, Yale University, 333 Cedar Street, New Haven, CT 06520 Communicated by Steven C. Hebert, Yale University School of Medicine, New Haven, CT, January 30, 2008 (received for review October 23, 2007) Neurodegenerative diseases and noxious stimuli to the brain two additional full-length human and mouse SGK1 cDNAs. These enhance transcription of serum- and glucocorticoid-induced cDNAs differ from the canonical one at the 5Ј end. The gene spans kinase-1 (SGK1). Here, we report that the SGK1 gene encodes a Ϸ118 kb, with the additional exons located far upstream of the brain-specific additional isoform, SGK1.1, which exhibits distinct initially identified 5Ј end. These exons are differentially spliced to regulation, properties, and functional effects. SGK1.1 decreases give rise to three different transcripts designated here as SGK1, expression of the acid-sensing ion channel-1 (ASIC1); thereby, SGK1.1, and SGK1.2. Fig. 1A shows a schematic representation of SGK1.1 may limit neuronal injury associated to activation of ASIC1 the exon–intron organization of the mouse SGK1 gene. The N in ischemia. Given that neurons express at least two splice iso- termini of the three splice isoforms are encoded by different exons, forms, SGK1 and SGK1.1, driven by distinct promoters, any changes whereas they share an identical catalytic domain and C-terminal in SGK1 transcript level must be examined to define the isoform hydrophobic motif (exons 6–16). induced by each stimulus or neurological disorder. The region upstream of exon 1 contains signature sequences consistent with a TATA box Ϸ1 kb and 0.7 kb from the initiation alternative promoter ͉ Proton-activated channel ͉ site. A different promoter in intron 4 controls transcription of the serum- and glucocorticoid-induced kinase canonical SGK1 isoform. This is the only promoter that has been characterized experimentally (21); it contains a TATA box near the GK1 is a S/T protein kinase expressed in many mammalian start site of transcription and a glucocorticoid-responsive element Stissues. It was originally identified as a glucocorticoid (1) and (GRE), consistent with the observation that glucocorticoids in- cell-volume-responsive gene (2). The most extensively studied crease mRNA abundance of the canonical isoform in most tissues. target of SGK1 is the epithelial sodium channel, ENaC, which plays No GRE was identified in the 10 kb upstream of exons 1 or 4. a crucial role in the regulation of body sodium (3). The phenotype Tissue distribution and relative abundance of SGK1.1 isoform of SGK1-null mice is a partial deficit in renal sodium reabsorption were examined by quantitative (q)RT-PCR of mouse tissues. whereby the mice are prone to volume depletion when exposed SGK1.1 mRNA was detected exclusively in brain; in all other tissues to a low-salt diet (4). There is substantial evidence that SGK1 examined expression was negligible (Fig. 1C Left). Similar experi- also works in the signaling pathways increasing cell survival and ments conducted with SGK1.2 primers showed very low expression in all tissues; therefore, we did not pursue further studies with this apoptosis in vertebrates (5) and in the nematode Caenorhabditis isoform. In brain, expression of SGK1.1 transcript was Ϸ1/10 of the elegans (6). level of SGK1 (Fig. 1C Right). The functional role of SGK1 in the mammalian nervous system Transcriptional regulation of the SGK1.1 isoform was examined has been explored by numerous studies that have reported increases by qRT-PCR in differentiated neuronal mouse B1E-115 cells in SGK1 transcript induced by diverse stimuli and conditions. High exposed to dexamethasone or to depolarization (by increasing the levels of SGK1 mRNA have been observed in ischemia (7), injury ϩ concentration of K ), a method that simulates neuronal activity. (8), in various animal models of Parkinson’s disease (9, 10), Depolarization increased SGK1.1 transcript, whereas dexametha- amyotrophic lateral sclerosis (11), Rett syndrome (12, 13), Hun- sone did not induce a significant change in mRNA levels of any tington’s disease (14), and in the dorsal horn of the spinal cord after isoform (Fig. 1D). induction of inflammation in the corresponding innervated periph- Distribution of SGK1.1 in brain structures was analyzed by in situ eral tissues (15). hybridization using a probe specific for the SGK1.1 splice isoform. Recent studies have started to probe the functional effects of Low magnification of a brain section shows staining of all regions elevated SGK1 expression in neurons of the central nervous system. of hippocampus, dentate gyrus, and cerebral cortex layers. A The current evidence points to a role of SGK1 in activity-dependent cerebellum section shows staining of Purkinje cells and granular facilitation of learning and memory formation (16, 17), consolida- layer (Fig. 1E). Comparison of the SGK1.1 isoform with the tion of long-term memory (18), facilitation of expression of long- distribution of SGK1 by in situ hybridization published online by the NEUROSCIENCE term potentiation in hippocampal neurons (19), and modulation of Allen Institute for Brain Science (www.brain-map.org) indicates synaptic plasticity in the dorsal horn of the spinal cord (15). overlap of expression of SGK1.1 and SGK1 in most areas of the The capacity of SGK1 to modulate expression of ion channels mouse central nervous system. and transporters at the plasma membrane of many cell types (20) also provides a means to alter membrane excitability in neurons. SGK1.1 Is the Most Abundant Protein Isoform in Brain. The relative This prompted us to further investigate SGK1 in the nervous abundance of SGK1 and SGK1.1 proteins was determined by system. Here, we report a splice isoform, SGK1.1, exclusively expressed in the nervous system. We describe the distinct features, transcriptional regulation, and functional effects of SGK1.1 in Author contributions: C.M.C. designed research; M.F.A., T.C., C.S., and C.M.C. performed neurons. research; C.M.C. contributed new reagents/analytic tools; M.F.A. and C.M.C. analyzed data; and M.F.A. and C.M.C. wrote the paper. Results The authors declare no conflict of interest. SGK1 Splice Isoforms and Distribution in Mouse Brain. Hitherto, it was Freely available online through the PNAS open access option. thought that the SGK1 gene spanned Ϸ6 kb, a segment of genomic *To whom correspondence should be addressed. E-mail: [email protected]. DNA that contains the promoter and all of the exons of the This article contains supporting information online at www.pnas.org/cgi/content/full/ reference sequence transcript of SGK1. However, the GenBank 0800958105/DC1. database (National Center for Biotechnology Information) reports © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800958105 PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 ͉ 4459–4464 Downloaded by guest on September 27, 2021 Fig. 1. Schematic of the SGK1 gene, differential expression, and distribution of isoforms. (A) The SGK1 gene expands Ϸ100 kb. White and blank boxes indicate 5Ј and 3Ј untranslated regions and exons; line represents introns. Arrows and arrowheads indicate positions of qPCR primers and the in situ hybridization probe specific for the SGK1.1 isoform. The gene contains at least three isoforms with corresponding promoters located upstream of exon 1, intron 3, and intron 4 for transcription of SGK1.1, SGK1.2, and SGK1 (canonical isoform), respectively. The SGK1 promoter contains a GRE. (B) Amino acid sequence of the N termini of SGK1 isoforms; intron–exon boundaries are indicated with arrows. Polybasic motif (ϩ) with large hydrophobic residues (⌬) in the N terminus of SGK1.1 is shown above the protein. (C) qRT-PCR of mouse tissues normalized to the value of SGK1.1 in brain Ϯ SD. Comparison of SGK1 and SGK1.1 in mouse brain. (D) Expression of SGK1 and SGK1.1 transcripts examined by qRT-PCR of N1E-115 cells treated with dexamethasone or increasing external Kϩ concentration to 50 mM. Each bar is the mean of six experiments normalized to GAPDH Ϯ SD. (E) In situ hybridization of mouse brain and cerebellum with SGK1.1-specific antisense and sense probes. e1 antisense and e2 sense probe on brain and e3 antisense and e4 sense probe on cerebellum are shown. quantitative Western blot analysis of mouse tissues. In previous homogenates of brain, heart, and lung, we identified SGK1 (49/45 work, we demonstrated that the abundance of SGK1 protein is kDa) and SGK1.1 (60 kDa) only in brain (Fig. 2A). lower than expected from the level of its own transcript. This The relative high protein abundance of SGK1.1 in brain when disparity is due to rapid degradation by the ubiquitin/proteasomal compared with its low mRNA level suggests that it might be more system (22). We used a transgenic mouse strain with insertion of a stable than the canonical SGK1. We confirmed by pulse–chase Ͼ bacterial artificial chromosome (BAC) containing the whole-mouse experiments that the half-life of SGK1.1 is longer (t1/2 180 min) SGK1 gene (Ϸ200 kb) modified by the addition of three HA than the one previously determined for SGK1 (t1/2 of 28 min) (Fig. epitopes at the C terminus of the coding region (23). In tissue 2B). The high stability of SGK1.1 protein is due to the absence of the proteasomal degradation signal in the N terminus of SGK1. SGK1.1 Resides at the Plasma Membrane by Binding to PtdIns(4,5)P2. Cellular localization of SGK1.1 was examined by immunofluores- cence of CHO cells cotransfected with SGK1.1-V5 and PH-GFP, a fusion of GFP and the pleckstrin-homology domain of phospho- lipase C␦, which selectively binds PtdIns(4,5)P2.Fig.3A shows colocalization of the two proteins at the plasma membrane.
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