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• W1994668849 abstract "The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca2+/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2−/− mice, and ischemic neuronal injury was significantly reduced in the brains of sik2−/− mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1. The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca2+/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2−/− mice, and ischemic neuronal injury was significantly reduced in the brains of sik2−/− mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1. The degradation of SIK2 leads to the activation of TORC1-CREB cascade after ischemia CaMK I/IV is an upstream regulator of SIK2-TORC1 signaling Synaptic NMDA receptors lead to the activation of SIK2-TORC1 cascade SIK2 is a crucial regulator for cell fate and survival after neurotoxicity Ischemic brain injury involves a complex pathophysiological cascade that comprises many distinct pathological stressors, including hypoxia, oxidative stress, inflammation, and glutamate excitotoxicity (Dirnagl et al., 1999Dirnagl U. Iadecola C. Moskowitz M. Pathobiology of ischaemic stroke: an integrated view.Trends Neurosci. 1999; 22: 391-397Abstract Full Text Full Text PDF PubMed Scopus (3208) Google Scholar, Lo et al., 2003Lo E.H. Dalkara T. Moskowitz M.A. Mechanisms, challenges and opportunities in stroke.Nat. Rev. Neurosci. 2003; 4: 399-415Crossref PubMed Scopus (1439) Google Scholar). A number of molecules and compounds conferring resistance to these stresses have been identified; however, they have failed to be protective in clinical trials despite promising preclinical data (Ikonomidou and Turski, 2002Ikonomidou C. Turski L. Why did NMDA receptor antagonists fail clinical trials for stroke and traumatic brain injury?.Lancet Neurol. 2002; 1: 383-386Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, Lee et al., 1999Lee J.M. Zipfel G.J. Choi D.W. The changing landscape of ischaemic brain injury mechanisms.Nature. 1999; 399: A7-A14Crossref PubMed Scopus (1004) Google Scholar, Lo et al., 2003Lo E.H. Dalkara T. Moskowitz M.A. Mechanisms, challenges and opportunities in stroke.Nat. Rev. Neurosci. 2003; 4: 399-415Crossref PubMed Scopus (1439) Google Scholar). The accumulation of intracellular calcium (Ca2+) in neurons after ischemia is a major determinant of ischemic cell death (Lo et al., 2003Lo E.H. Dalkara T. Moskowitz M.A. Mechanisms, challenges and opportunities in stroke.Nat. Rev. Neurosci. 2003; 4: 399-415Crossref PubMed Scopus (1439) Google Scholar). Several recent studies have suggested that some of these limitations may be circumvented by targeting excitotoxic signaling pathways downstream of NMDA receptors (NMDARs) (Hardingham et al., 1999Hardingham G.E. Chawla S. Cruzalegui F.H. Bading H. Control of recruitment and transcription-activating function of CBP determines gene regulation by NMDA receptors and L-type calcium channels.Neuron. 1999; 22: 789-798Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, Hardingham et al., 2002Hardingham G.E. Fukunaga Y. Bading H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways.Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1353) Google Scholar, Taghibiglou et al., 2009Taghibiglou C. Martin H. Lai T. Cho T. Prasad S. Kojic L. Lu J. Liu Y. Lo E. Zhang S. et al.Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries.Nat. Med. 2009; 15: 1399-1406Crossref PubMed Scopus (108) Google Scholar). The activation of NMDARs has been linked to the modulation of a number of transcriptional factors, with either pro-survival or pro-death activity, suggesting that the alteration of transcription factor activity may crucially contribute to excitotoxic neuronal injuries (Taghibiglou et al., 2009Taghibiglou C. Martin H. Lai T. Cho T. Prasad S. Kojic L. Lu J. Liu Y. Lo E. Zhang S. et al.Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries.Nat. Med. 2009; 15: 1399-1406Crossref PubMed Scopus (108) Google Scholar). In particular, we and others have demonstrated that the transcription factor cAMP responsive element (CRE)-binding protein (CREB) protected the brain from ischemia mainly via its downstream neuroprotective genes (Hardingham et al., 2002Hardingham G.E. Fukunaga Y. Bading H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways.Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1353) Google Scholar, Mabuchi et al., 2001Mabuchi T. Kitagawa K. Kuwabara K. Takasawa K. Ohtsuki T. Xia Z. Storm D. Yanagihara T. Hori M. Matsumoto M. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo.J. Neurosci. 2001; 21: 9204-9213Crossref PubMed Google Scholar, Peng et al., 2006Peng P.L. Zhong X. Tu W. Soundarapandian M.M. Molner P. Zhu D. Lau L. Liu S. Liu F. Lu Y. ADAR2-dependent RNA editing of AMPA receptor subunit GluR2 determines vulnerability of neurons in forebrain ischemia.Neuron. 2006; 49: 719-733Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). NMDAR subtypes and localization have attracted much attention because synaptic and extrasynaptic NMDARs have been shown to exert distinct roles in excitotoxicity (Sattler et al., 2000Sattler R. Xiong Z. Lu W. MacDonald J. Tymianski M. Distinct roles of synaptic and extrasynaptic NMDA receptors in excitotoxicity.J. Neurosci. 2000; 20: 22-33Crossref PubMed Google Scholar). In particular, synaptic NMDARs, predominantly NR2A receptor subtypes, and extrasynaptic NMDARs, mainly NR2B subtypes, have opposite effects on CREB function, gene regulation, and neuronal survival (Hardingham et al., 2002Hardingham G.E. Fukunaga Y. Bading H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways.Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1353) Google Scholar, Peng et al., 2006Peng P.L. Zhong X. Tu W. Soundarapandian M.M. Molner P. Zhu D. Lau L. Liu S. Liu F. Lu Y. ADAR2-dependent RNA editing of AMPA receptor subunit GluR2 determines vulnerability of neurons in forebrain ischemia.Neuron. 2006; 49: 719-733Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, Liu et al., 2007Liu Y. Wong T. Aarts M. Rooyakkers A. Liu L. Lai T. Wu D. Lu J. Tymianski M. Craig A. Wang Y. NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo.J. Neurosci. 2007; 27: 2846-2857Crossref PubMed Scopus (640) Google Scholar). Moreover, CREB also plays a pivotal role in an ischemic tolerance phenomenon in which brief sublethal ischemic insults (or preconditioning) protect neurons against a subsequent severe ischemic injury (Kitagawa, 2007Kitagawa K. CREB and cAMP response element-mediated gene expression in the ischemic brain.FEBS J. 2007; 274: 3210-3217Crossref PubMed Scopus (187) Google Scholar, Mabuchi et al., 2001Mabuchi T. Kitagawa K. Kuwabara K. Takasawa K. Ohtsuki T. Xia Z. Storm D. Yanagihara T. Hori M. Matsumoto M. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo.J. Neurosci. 2001; 21: 9204-9213Crossref PubMed Google Scholar). CREB contributes to neuroprotection by inducing its target genes, such as brain-derived neurotrophic factor (BDNF), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Ppargc-1α: its product is known as PGC-1α), and B cell lymphoma 2 (Bcl-2) (Mabuchi et al., 2001Mabuchi T. Kitagawa K. Kuwabara K. Takasawa K. Ohtsuki T. Xia Z. Storm D. Yanagihara T. Hori M. Matsumoto M. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo.J. Neurosci. 2001; 21: 9204-9213Crossref PubMed Google Scholar, St-Pierre et al., 2006St-Pierre J. Drori S. Uldry M. Silvaggi J. Rhee J. Jäger S. Handschin C. Zheng K. Lin J. Yang W. et al.Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators.Cell. 2006; 127: 397-408Abstract Full Text Full Text PDF PubMed Scopus (1782) Google Scholar). In neurons, CREB-dependent gene expression has been implicated in complex and diverse processes, including development and plasticity (Lonze and Ginty, 2002Lonze B.E. Ginty D.D. Function and regulation of CREB family transcription factors in the nervous system.Neuron. 2002; 35: 605-623Abstract Full Text Full Text PDF PubMed Scopus (1744) Google Scholar). The activity of CREB is regulated by phosphorylation, and Ser133 was found to be its crucial phosphorylation site (Gonzalez and Montminy, 1989Gonzalez G.A. Montminy M.R. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2055) Google Scholar). Protein kinase A (PKA) is activated in the cAMP-signaling cascade and phosphorylates CREB at Ser133, enhancing its binding to the coactivators CREB-binding protein (CBP) and p300 (Vo and Goodman, 2001Vo N. Goodman R. CREB-binding protein and p300 in transcriptional regulation.J. Biol. Chem. 2001; 276: 13505-13508PubMed Scopus (650) Google Scholar). CBP/p300 then promote histone-chromatin remodeling through their intrinsic histone acetyltransferase activity and recruit basal transcription machinery to CRE-containing promoters (Goodman and Smolik, 2000Goodman R.H. Smolik S. CBP/p300 in cell growth, transformation, and development.Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). Ca2+/calmodulin-dependent protein kinase I/IV (CaMK I/IV) are important isoforms of CaMKs in neurons and play pivotal roles in cell survival. Indeed, CaMK IV is recognized as a key mediator of CREB-dependent cell survival in neurons because treatment with a CaMK inhibitor renders neurons vulnerable to ischemia concomitant with the loss of CREB phosphorylation at Ser133 (Mabuchi et al., 2001Mabuchi T. Kitagawa K. Kuwabara K. Takasawa K. Ohtsuki T. Xia Z. Storm D. Yanagihara T. Hori M. Matsumoto M. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo.J. Neurosci. 2001; 21: 9204-9213Crossref PubMed Google Scholar). However, phosphorylation of CREB at Ser133 alone is not sufficient to fully activate the expression of target genes in peripheral tissues and the central nervous system (CNS), suggesting that the initiation of transcription of CREB target genes is controlled by CREB phosphorylation at Ser133 and possibly by other mechanisms (Gau et al., 2002Gau D. Lemberger T. von Gall C. Kretz O. Le Minh N. Gass P. Schmid W. Schibler U. Korf H. Schütz G. Phosphorylation of CREB Ser142 regulates light-induced phase shifts of the circadian clock.Neuron. 2002; 34: 245-253Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Kornhauser et al., 2002Kornhauser J.M. Cowan C.W. Shaywitz A.J. Dolmetsch R.E. Griffith E.C. Hu L.S. Haddad C. Xia Z. Greenberg M.E. CREB transcriptional activity in neurons is regulated by multiple, calcium-specific phosphorylation events.Neuron. 2002; 34: 221-233Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). The discovery of a family of coactivators named transducer of regulated CREB activity (TORC, also known as CREB regulated transcriptional coactivator [CRTC], with three isoforms TORC1–3) provided new insights on CREB activation (Conkright et al., 2003Conkright M.D. Canettieri G. Screaton R. Guzman E. Miraglia L. Hogenesch J.B. Montminy M. TORCs: transducers of regulated CREB activity.Mol. Cell. 2003; 12: 413-423Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar, Iourgenko et al., 2003Iourgenko V. Zhang W. Mickanin C. Daly I. Jiang C. Hexham J. Orth A. Miraglia L. Meltzer J. Garza D. et al.Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells.Proc. Natl. Acad. Sci. USA. 2003; 100: 12147-12152Crossref PubMed Scopus (314) Google Scholar). Under nonstimulated conditions, TORC is phosphorylated and sequestered in the cytoplasm. Once dephosphorylated in response to Ca2+ and cAMP signals, it translocates to the nucleus (Bittinger et al., 2004Bittinger M.A. McWhinnie E. Meltzer J. Iourgenko V. Latario B. Liu X. Chen C.H. Song C. Garza D. Labow M. Activation of cAMP response element-mediated gene expression by regulated nuclear transport of TORC proteins.Curr. Biol. 2004; 14: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, Screaton et al., 2004Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates 3rd, J.R. Takemori H. et al.The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector.Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). In contrast to CBP/p300, TORC activates transcription by targeting the basic leucine-zipper (bZIP) domain of CREB in a phospho-Ser133-independent manner. TORC1 is abundantly expressed in the brain and plays an important role in hippocampal long-term potentiation at its late phase (Kovács et al., 2007Kovács K.A. Steullet P. Steinmann M. Do K.Q. Magistretti P.J. Halfon O. Cardinaux J.R. TORC1 is a calcium- and cAMP-sensitive coincidence detector involved in hippocampal long-term synaptic plasticity.Proc. Natl. Acad. Sci. USA. 2007; 104: 4700-4705Crossref PubMed Scopus (148) Google Scholar, Zhou et al., 2006Zhou Y. Wu H. Li S. Chen Q. Cheng X.W. Zheng J. Takemori H. Xiong Z.Q. Requirement of TORC1 for late-phase long-term potentiation in the hippocampus.PLoS ONE. 2006; 1: e16Crossref PubMed Scopus (93) Google Scholar). TORC2 is the most abundant TORC isoform in the liver and has been found to be involved in the gene expression of gluconeogenic programs and in the survival of pancreatic β cells (Koo et al., 2005Koo S.H. Flechner L. Qi L. Zhang X. Screaton R.A. Jeffries S. Hedrick S. Xu W. Boussouar F. Brindle P. et al.The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism.Nature. 2005; 437: 1109-1111Crossref PubMed Scopus (802) Google Scholar). Salt-inducible kinase (SIK) was identified as an enzyme induced in the adrenal glands of rats fed with a high-salt diet (Wang et al., 1999Wang Z. Takemori H. Halder S. Nonaka Y. Okamoto M. Cloning of a novel kinase (SIK) of the SNF1/AMPK family from high salt diet-treated rat adrenal.FEBS Lett. 1999; 453: 135-139Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). SIK isoforms (SIK1–3) belong to a family of AMP-activated protein kinases (AMPKs). SIK1 expression is induced by depolarization in the hippocampus and plays a role in the development of cortical neurons through the regulation of TORC1 (Feldman et al., 2000Feldman J.D. Vician L. Crispino M. Hoe W. Baudry M. Herschman H.R. The salt-inducible kinase, SIK, is induced by depolarization in brain.J. Neurochem. 2000; 74: 2227-2238Crossref PubMed Scopus (47) Google Scholar, Li et al., 2009Li S. Zhang C. Takemori H. Zhou Y. Xiong Z. TORC1 regulates activity-dependent CREB-target gene transcription and dendritic growth of developing cortical neurons.J. Neurosci. 2009; 29: 2334-2343Crossref PubMed Scopus (102) Google Scholar). However, it remains to be clarified whether the intracellular signaling of SIK-TORC is crucial for CREB-dependent neuronal survival, and if so, what acts as the upstream signaling cascade. In the present study we found high expression levels of SIK2 in neurons. The levels of SIK2 protein were lowered after ischemic injury and were accompanied by the dephosphorylation of TORC1. CaMK I/IV play an important role in the regulation of the SIK2 degradation by phosphorylating SIK2 at Thr484. To assess the importance of SIK2 during ischemic injury in vivo, we generated SIK2 knockout (sik2−/−) mice and found that they showed resistance to ischemic neuronal injury. One hour after OGD in the primary cortical cultures, CREB was phosphorylated, and the level of phosphorylation reached a maximum at 3–6 hr after reoxygenation, then declined to basal levels after 12 hr post-reoxygenation (Figure 1A and S1A available online). To measure CRE activity, neurons were infected with an adenovirus as a CRE reporter. We found a significant enhancement of CRE activity after 3–12 hr post-reoxygenation (Figure 1B). During OGD and early (1 hr) after reoxygenation, CREB phosphorylation was not followed by an increase in CRE activity, but late (12 hr) after reoxygenation, CRE activity remained high, regardless of the basal level of CREB phosphorylation. The results of a reporter assay using Gal4-fusion Ser133-disrupted CREB suggested the presence of the Ser133-independent activation of CREB following OGD (Figure S1B). To examine Ser133-independent regulation in cortical neurons, we performed CRE-reporter assays in the presence of cAMP agonists and a kinase inhibitor. Staurosporine at low dose (10 nM), a nonspecific kinase inhibitor, decreased the level of phospho-Ser133, but it induced the activity of a CRE reporter to comparable levels as the phospho-Ser133-inducer db-cAMP or forskolin (Figure S1C), suggesting the involvement of another type of CREB coactivator, TORC. It has been shown that CREB activity is blocked by the inhibition of calcineurin. Indeed, we found this to be true in neurons after OGD because the calcineurin inhibitor cyclosporine A (CsA) and FK 506 suppressed CRE activity (Figure 1C), despite the upregulation of CREB phosphorylation by FK 506 (Figure 1C). The mechanism by which calcineurin activates CREB could be the dephosphorylation-dependent nuclear entry of CREB-coactivator TORCs. To examine the activity of endogenous TORCs after OGD, the activity of Gal4-fusion full-length CREB (TORC activatable) and bZIP-less CREB (TORC silent) was monitored. The activity of the full-length CREB, but not bZIP-less CREB, was enhanced after OGD (Figure 1D), suggesting that TORCs may contribute to the Ser 133-independent activation of CREB after OGD reoxygenation. A high level of TORC1 protein and a moderate level of TORC2 protein were detected in primary cortical cultures (Figure S2A). Under basal conditions, endogenous TORC1 was predominantly localized in the cytoplasm of cortical neurons (Figure 2A , control). Although the phosphorylation levels of CREB at Ser133 were increased during OGD (Figure S1A), TORC1 remained in the cytoplasm before reoxygenation but quickly translocated into the nucleus after reoxygenation (Figure 2A). Indeed, GFP-TORC1 translocated into the nucleus in response to OGD (Figure S2B). Next, we examined the intracellular distribution of TORC1 in response to OGD by separately isolating nuclei from cytoplasmic compartments. OGD followed by reoxygenation induced the nuclear localization of TORC1 (Figure S2C). This process was accompanied by the dephosphorylation of TORC1 at Ser 167 (corresponding Ser residues: Ser171 [TORC2] and Ser163 [TORC3]) (Figure 2B), followed by the enhancement of its coactivator activity (Figure 2C). This reoxygenation-induced activation of TORC1 may be essential for CREB activation because the overexpression of a dominant-negative TORC1 (DN-TORC1, N-terminal 56 amino acids) strongly inhibited CRE activity after OGD (Figure 2D) and aggravated cell injury after OGD (Figure 2E). To elucidate the role of TORC1 in neuronal survival, we determined the relationship between CRE activity and cell death. We found that CRE activity in cortical neurons was enhanced by the overexpression of TORC1, and a constitutively active TORC1 (S167A) further upregulated CRE activity (Figure 2F). The overexpression of TORC1 or the TORC1S167A mutant resulted in a significant decrease of ischemic neuronal death (Figure 2G). The overexpression of TORC1 in cortical neurons induced the mRNA expression of CREB-dependent pro-survival genes, such as Ppargc-1α (PGC-1α) and BDNF (Figure 2H). In contrast, DN-TORC1 inhibited the upregulation of these genes after OGD (Figure S2D). Moreover, the OGD-induced reporter activity of Ppargc-1α and bdnf promoters was impaired by mutating their CREs (Figure S2E), suggesting that TORC1-CREB may actively determine neuronal survival after ischemia. TORC family coactivators are phosphorylated by SIK1, SIK2, and AMPK (Katoh et al., 2006Katoh Y. Takemori H. Lin X. Tamura M. Muraoka M. Satoh T. Tsuchiya Y. Min L. Doi J. Miyauchi A. et al.Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade.FEBS J. 2006; 273: 2730-2748Crossref PubMed Scopus (119) Google Scholar, Koo et al., 2005Koo S.H. Flechner L. Qi L. Zhang X. Screaton R.A. Jeffries S. Hedrick S. Xu W. Boussouar F. Brindle P. et al.The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism.Nature. 2005; 437: 1109-1111Crossref PubMed Scopus (802) Google Scholar, Screaton et al., 2004Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates 3rd, J.R. Takemori H. et al.The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector.Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, Takemori and Okamoto, 2008Takemori H. Okamoto M. Regulation of CREB-mediated gene expression by salt inducible kinase.J. Steroid Biochem. Mol. Biol. 2008; 108: 287-291Crossref PubMed Scopus (68) Google Scholar), and quantitative PCR analyses suggest a high level of SIK1 and SIK2 mRNA in the cortex (Figure S3A). We found that SIK2 protein was expressed in the hippocampus and cortex (Figures S3B and S3C) and was abundant in nonstimulated neurons (Figure S3D); however, SIK1 protein was not detected in these cells (data not shown) with a highly purified anti-SIK1 antibody (Uebi et al., 2010Uebi T. Tamura M. Horike N. Hashimoto Y. Takemori H. Phosphorylation of the CREB-specific coactivator TORC2 at Ser(307) regulates its intracellular localization in COS-7 cells and in the mouse liver.Am. J. Physiol. Endocrinol. Metab. 2010; 299: E413-E425Crossref PubMed Scopus (40) Google Scholar). Next, we examined the involvement of these kinases in the regulation of TORC1 after OGD in cortical neurons (Figure 3A ). The level of SIK1 remained low during and after OGD. The level of pAMPK increased during OGD but quickly returned to the basal level after reoxygenation. In contrast the level of SIK2 decreased in an early phase of reoxygenation (∼3 hr), and it was maintained at a low level until 24 hr post-reoxygenation, suggesting that this downregulation of SIK2 may be important for the activation of TORC1-CREB after reoxygenation. Therefore, we next determined the contribution of SIK2 to the regulation of TORC1 in cortical neurons. To elucidate the importance of SIK2, we tried to identify small compounds that could inhibit SIK2 activity more selectively than staurosporine. Fortunately, by the use of a small kinase-inhibitor library, we identified Compound C, a potent inhibitor of AMPK, as a SIK2 inhibitor (Figure S3E). The effective dose of Compound C against SIK2 in cultured cells was 10-fold lower than that against SIK1 or AMPK (Figures S3F and S3G). The IC50 of Compound C for the SIK2-dependent suppression of forskolin-induced CRE activity was approximately 0.3 μM, whereas the concentration required to inhibit the AMPK-mediated phosphorylation of acetyl-CoA carboxylase (ACC) was greater than 3 μM (Figure S3G). The dose response of Compound C suggested that 0.1–1.0 μM would enable us to distinguish its effect on SIK2 from its effects on SIK1 or AMPK. Indeed, 0.3–0.5 μM of Compound C upregulated CRE activity in cultured neurons after OGD (Figure S3H) and reduced neuronal death (Figure S3I). On the other hand, we demonstrated that Compound C, at the dose used for AMPK inhibition (>3 μM), was toxic to cortical neurons after OGD (Figure S3I). These findings suggested that SIK2 could have a greater effect on TORC1-CREB activity than SIK1 or AMPK. The overexpression of SIK2 and its constitutively active form (S587A) strongly inhibited CRE activity after OGD (Figure 3B), whereas the kinase-defective SIK2 (K49M) failed to suppress CRE activity. In agreement with the CRE-reporter assay, the overexpression of S587A increased cell death, whereas K49M decreased cell death after OGD (Figure 3C). Furthermore, the overexpression of the S587A mutant SIK2 resulted in a substantial amount of TORC1 in the cytoplasm after OGD (Figure 3D). The overexpression of SIK2 also suppressed the TORC1-dependent activation of CRE, and SIK-resistant TORC1 (S167A) blocked this suppression (Figure 3E). When SIK2 was knocked down using SIK2-specific microRNA (miRNA) (Figure 3F), CRE activity was relatively enhanced in the late phase after OGD (after 12 hr; Figure 3G). The knockdown of SIK2 also attenuated neuronal death after OGD (Figure 3H). Although overexpression of TORC1 did not confer an additional protective effect under SIK2 downregulation, the overexpression of DN-TORC1 abolished the protective effect of SIK2-specific miRNA (Figure 3H). These findings suggested that SIK2 plays an essential role in neuronal survival after OGD via a TORC1-dependent pathway. To determine which kinase cascades mediate the activation of CRE-dependent transcription, we pretreated cortical neurons with various kinase inhibitors and found that KN93, a CaMK II/IV inhibitor, blocked CRE-mediated transcription after OGD (Figure 4A ). Gal4-fusion TORC1 activity was also inhibited by KN93 (Figure 4B), and KN-93 also blocked the decrease in the levels of SIK2 protein after OGD (Figure S4A). To identify the specific isoform of CaMK that is implicated in TORC-CREB-dependent transcription, dominant-active forms of CaMKs (DA-CaMK I; dominant-active CaMK I [catalytic domain], DA-CaMK IIA [catalytic domain], and DA-CaMK IV [full-length protein without its auto-inhibitory domain]) were expressed in Gal4-fusion reporter systems (Figure 4C). The activity of TORC-responsive CREB and TORC-non-responsive CREB (Gal4-CREB bZIP-less) were upregulated by the overexpression of CaMK I and IV, but not by CaMK IIA. In addition to CREB, CaMK I and IV upregulate TORC1 activity (Figure 4C). Conversely, DN (dominant negative; kinase-dead form)-CaMK I (K49E) and DN-CaMK IV (K75E) blocked TORC1-mediated transcriptional activity after OGD (Figure 4D) and SIK2 degradation after OGD (Figure S4B). Also, the overexpression of DN-TORC1 resulted in a significant reduction in CRE activity that was enhanced by CaMK I or CaMK IV in cortical neurons under control conditions and after OGD (Figure S4C). This finding suggested that CaMK I and IV may be able to activate CREB (via phosphorylation at Ser133) and TORC. Endogenous CaMK IV is predominantly restricted to the nucleus, whereas overexpressed CaMK IV was localized in both the cytoplasm and nucleus (Figure S4D). To determine the role of endogenous CaMK IV, we confirmed its involvement in the regulation of OGD-induced TORC activation by means of RNA interference (RNAi) experiments. Treatment with rat CaMK IV-specific miRNA decreased the level of CaMK IV, not CaMKI (Figure S4E). We found that the knockdown of CaMK IV resulted in the inhibition of TORC1 activity (Figure S4F) and aggravated cell injury after OGD (Figure S4G). Furthermore, transfection of human CaMK IV, which is resistant to miRNA for rat CaMK IV, reversed the CaMK IV protein level and attenuated the cell injury by CaMK IV knockdown (Figures S4E and S4G). These results suggested that CaMK IV may upregulate CRE-mediated transcription in a CREB Ser133- and TORC1-dependent manner. Indeed, the overexpression of DA-CaMK IV significantly decreased neuronal injury after OGD (Figure 4E). On the basis of these findings, CaMK I and CaMK IV were identified as negative regulators of SIK2. CaMK I and IV are activated by binding elevated Ca2+ levels to calmodulin, Ca2+/calmodulin, and Ca2+ influx, principally through NMDARs, activated cytoplasmic, and nuclear CaMKs. However, it was reported that different subtypes of glutamate receptors have opposite actions on neuronal survival (Hardingham et al., 2002Hardingham G.E. Fukunaga Y. Bading H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and" @default.
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