Calcium (Ca2+) signaling activated in response to membrane depolarization regulates neuronal maturation, connectivity and plasticity, but the role of store-operated Ca2+ entry (SOCE) in neurons is poorly understood. Here, we report that SOCE is regulated by membrane potential in cerebellar granule neurons (CGNs) in the opposite direction of voltage-gated Ca2+ channels; maximal activation of SOCE occurs in resting conditions where this Ca2+ influx promotes the degradation of transcription factor Sp4, a regulator of neuronal morphogenesis and function. We report that lowering [K+]EXT caused depletion of intracellular Ca2+ stores and induced a Ca2+ influx with characteristics of SOCE. Under these conditions, we also observed the relocalization of the ER Ca2+ sensor STIM1. We found that compounds that block SOCE prevented the ubiquitination and degradation of Sp4 in low extracellular potassium ([K+]EXT). Further, we show that STIM1 knockdown blocked degradation of Sp4 while expression of a constitutively active STIM1 decreased Sp4 protein abundance under depolarization conditions. Our findings provide evidence for the dynamic regulation and downstream signaling effect of SOCE in neurons and suggest a new pathway for Ca2+ signaling to shape neuronal gene expression.
Background p21-activated kinase 6 (PAK6) is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family. We investigated the role of PAK6 in radiation-induced cell death in human prostate cancer cells. Methods We used a short hairpin RNA (shRNA) strategy to stably knock down PAK6 in PC3 and DU145 cells. Radiation sensitivities were compared in PAK6 stably knockdown cells versus the scrambled shRNA-expressing control cells. Results PAK6 mRNA and protein levels in PC3 and DU145 cells were upregulated upon exposure to 6 Gy of radiation. After irradiation, an increased percentage of apoptotic cells and cleaved caspase-3 levels were demonstrated in combination with a decrease in cell viability and a reduction in clonogenic survival in PAK6-knockdown cells. In addition, transfection with PAK6 shRNA blocked cells in a more radiosensitive G2-M phase and increased levels of DNA double-strand breaks. We further explored the potential mechanisms by which PAK6 mediates resistance to radiation-induced apoptosis. Inhibition of PAK6 caused a decrease in Ser112 phosphorylation of BAD, a proapoptotic member of the Bcl-2 family, which led to enhanced binding of BAD to Bcl-2 and Bcl-XL and release of cytochrome c culminating into caspase activation and cell apoptosis. Conclusions The combination of PAK6 inhibition and irradiation resulted in significantly decreased survival of prostate cancer cells. The underlying mechanisms by which targeting PAK6 may improve radiation response seem to be multifaceted, and involve alterations in cell cycle distribution and impaired DNA double-strand break repair as well as relieved BAD phosphorylation.
The regulation of transcription factor function in response to neuronal activity is important for development and function of the nervous system. The transcription factor Sp4 regulates the developmental patterning of dendrites, contributes to complex processes including learning and memory, and has been linked to psychiatric disorders such as schizophrenia and bipolar disorder. Despite its many roles in the nervous system, the molecular mechanisms regulating Sp4 activity are poorly understood. Here, we report a site of phosphorylation on Sp4 at serine 770 that is decreased in response to membrane depolarization. Inhibition of the voltage-dependent NMDA receptor increased Sp4 phosphorylation. Conversely, stimulation with NMDA reduced the levels of Sp4 phosphorylation, and this was dependent on the protein phosphatase 1/2A. A phosphomimetic substitution at S770 impaired the Sp4-dependent maturation of cerebellar granule neuron primary dendrites, whereas a non-phosphorylatable Sp4 mutant behaved like wild type. These data reveal that transcription factor Sp4 is regulated by NMDA receptor-dependent activation of a protein phosphatase 1/2A signaling pathway. Our findings also suggest that the regulated control of Sp4 activity is an important mechanism governing the developmental patterning of dendrites. Keywords: cerebellar granule neurons, dendrite, NMDA receptor, phosphorylation, PP1/PP2A, transcription factor Sp4. The regulated activity of transcription factors plays crucial roles in the development and maintenance of connections in the nervous system. Neuronal activity, in the form of membrane depolarization, is known to influence transcription factor activity (Greer and Greenberg 2008). Membrane depolarization activates voltage-gated calcium channels such as the NMDA receptor to initiate signaling cascades, including the activation of downstream kinases and phosphatases, to alter transcription factor function (Morishita et al. 2001;Genoux et al. 2002;Cohen and Greenberg 2008;Hardingham 2009;Paoletti et al. 2013). Thus, many of the profound effects of the NMDA receptor on neuronal development, viability, and plasticity are mediated, in part, through the regulated posttranslational modification of transcription factors.Received November 4, 2013; revised manuscript received January 6, 2014; accepted January 9, 2014.Address correspondence and reprint requests to Grace Gill, Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. E-mail: Grace.Gill@tufts.eduAbbreviations used: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; CG, cerebellar granule; FLAG-Sp4, N-terminal FLAG-tagged Sp4; GFP, green fluorescent protein; LC/MS/MS, liquid chromatography-tandem mass spectrometry; OA, okadaic acid; PAGE, polyacrylamide gel electrophoresis; PP1/PP2A, protein phosphatase 1/2A; pSp4, Sp4 phosphorylated at S770; S770A res, RNAi-resistant FLAG-Sp4 with a serine to alanine point mutation at amino acid 770; S770D res, RNA...
Glutamatergic signaling through N-methyl-D-aspartate receptors (NMDARs) is important for neuronal development and plasticity and is often dysregulated in psychiatric disorders. Mice mutant for the transcription factor Sp4 have reduced levels of NMDAR subunit 1 (NR1) protein, but not mRNA, and exhibit behavioral and memory deficits (Zhou et al., 2010). In developing cerebellar granule neurons (CGNs), Sp4 controls dendrite patterning (Ramos et al., 2007). Sp4 target genes that regulate dendrite pruning or NR1 levels are not known. Here we report that Sp4 activates transcription of Nervous Wreck 2 (Nwk2; also known as Fchsd1) and, further, that Nwk2, an F-BAR-domain containing protein, mediates Sp4-dependent regulation of dendrite patterning and cell surface expression of NR1. Knockdown of Nwk2 in CGNs increased primary dendrite number, phenocopying Sp4 knockdown, and exogenous expression of Nwk2 in Sp4-depleted neurons rescued dendrite number. We observed that acute Sp4 depletion reduced levels of surface, but not total, NR1, and this was rescued by Nwk2 expression. Furthermore, expression of Nr1 suppressed the increase in dendrite number in Sp4- or Nwk2-depleted neurons. We previously reported that Sp4 protein levels were reduced in cerebellum of subjects with bipolar disorder (BD) (Pinacho et al., 2011). Here we report that Nwk2 mRNA and NR1 protein levels were also reduced in postmortem cerebellum of BD subjects. Our data suggest a role for Sp4-regulated Nwk2 in NMDAR trafficking and identify an Sp4-Nwk2-NMDAR1 pathway that regulates neuronal morphogenesis during development and may be disrupted in bipolar disorder.
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