br Materials and methods br Results

Materials and methods
Results and discussion
Conclusions In this study, we have investigated the possibility that hierarchical phosphorylation by CK2 may play a role in cellular signal transduction. Hierarchical phosphorylation by CK2 requires multiple consensus determinants with specific spacing, and can be as enzymatically efficient as canonical phosphorylation. Consensus motifs are found in a variety of human proteins, several of which are known to be multiply phosphorylated in Glutathione (GSH/GSSG/Total) Fluorometric Assay Kit sale at the corresponding sites. Potential substrates of CK2 hierarchical phosphorylation were significantly enriched among regulators of several crucial processes, including gene expression, development, and small GTPase signaling. Taken together, our results provide strong in vitro evidence that hierarchical phosphorylation by CK2 may have several important roles in cellular signaling pathways. The following are the supplementary data related to this article.
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Acknowledgments We wish to thank the members of the Litchfield and Gingras labs for their helpful discussion. This work was supported by funding from the Canadian Institutes of Health Research (CIHR) to D.W.L. (MOP 37854) and A.-C.G. (MOP 84314). N.S. was supported by a scholarship from the Canadian Cancer Society Research Institute (with D.W.L.) and is currently supported by a postdoctoral fellowship from the CIHR (with A.-C.G.). M.G. and J.P.T. were supported by scholarships from the CIHR and the Ontario Graduate Scholarship Program.
Introduction Stroke is the second leading cause of death and the third leading cause of disability worldwide. Ischemic stroke accounts for approximately 87% of stroke cases, contributing to the major portion of death and post-stroke disability in patients (Mozaffarian et al., 2016). The ischemic territory of stroke consists of the core and penumbra, which are respectively characterized by acute and delayed cell death (Lipton, 1999; Lo, 2008; Wu et al., 2017). Thus, protecting against delayed neuronal death, mainly apoptosis, in penumbra is a major strategy to improve post-stroke recovery. Previous studies indicate that endoplasmic reticulum stress (ERS) and subsequently triggered unfolded protein response (UPR) may play a pivotal role in ischemia/reperfusion (I/R) injury-induced neuronal apoptosis in ischemic stroke (Wu et al., 2017; Xin et al., 2014; Nakka et al., 2010). During ERS, 78 kDa glucose-regulated protein (GRP78) is dissociated from two ER membrane proteins, inositol-requiring enzyme 1 (IRE1) and phosphorylation of protein kinase-like ER kinase (PERK), and one Golgi membrane protein, activating transcription factor 6 (ATF6), which subsequently activates IRE1-, PERK- and ATF6-mediated three major branches of UPR. IRE1, ATF6 and PERK activation further increases the transcription of GRP78, which promotes protein folding and quality control to cope with unfolded protein accumulation, restoring ER homeostasis and avoiding apoptosis (Xin et al., 2014; Hetz, 2012; Lee, 2014). Thus, GRP78 is a widely used as an indicator for the onset for ER stress (Hetz, 2012; Lee, 2014). Moreover, PERK mediated eIF2-ATF4-CHOP activation contributes to cellular apoptosis under ER stress. Phosphorylated PERK activates eukaryotic translation initiation factor 2 (eIF2) by increasing eIF2 phosphorylation, which potentiates the translation of activating transcription factor 4 (ATF4). Increased ATF4 promotes the transcription of CCAAT/enhancer binding protein homologous protein (CHOP), a pro-apoptotic transcription factor, facilitating apoptosis (Xin et al., 2014; Hetz, 2012). However, the regulation of eIF2-ATF4-CHOP in ischemic stroke and underlying mechanisms are poorly understood. A number of studies showed that casein kinase 2 (CK2) is involved in ERS/UPR and apoptosis (Hessenauer et al., 2011; Hosoi et al., 2012; Schneider et al., 2012; Intemann et al., 2014). CK2, a selective serine/threonine kinase, consists of two α subunits (CK2α) and two β subunits (CK2β), forming a tetramer. CK2α is the catalytic subunit (St-Denis and Litchfield, 2009). Increased CK2 facilitates cell survival, while inhibition of CK2 promotes apoptosis in non-neuronal cells (Hosoi et al., 2012; Manni et al., 2012; Buontempo et al., 2016). Recent studies reported that CK2 is more abundant in the brain and closely associated with neuronal survival, neuritogenesis, synaptic transmission and synaptic plasticity (Kim et al., 2009; Wang and Schachner, 2015). Moreover, the activity of CK2 was significantly reduced in ischemic stroke model rats, and cerebral I/R injury significantly decreased the expression of CK2 (Lee et al., 2004; Kim et al., 2012; Zhou et al., 2016). Previous studies showed that CK2 could regulate PERK, ATF4 and CHOP, which is involved in apoptosis in cancer cells (Schneider et al., 2012; Götz and Montenarh, 2013). In addition, cerebral I/R-induced neuronal apoptosis is a key mechanism of ischemic stroke pathogenesis and ERS plays a pivotal role in I/R-induced apoptosis (Xin et al., 2014). Above evidence suggested that CK2 might be involved in cerebral I/R-induced neuronal apoptosis via eIF2-ATF4-CHOP pathway. However, the role of CK2 in I/R injury-induced neuronal apoptosis has not been explored.