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  • The classification of CK substrates

    2020-03-25

    The classification of CK2 substrates based on active enzyme subunit composition provides a framework for understanding the regulatory function of CK2β subunit. However, few experiments exist that functionally explore such classification [17], [18], [19], [20], [21], [22], [23]; this reflects practical limitations of elucidating kinase specificity. In this regard, high-throughput methods provide an alternative to traditional techniques for phosphosites identification (e.g., ELISA and phospho-specific antibodies). For instance, mass spectrometry-driven approaches figure at the most promising high-throughput techniques with a subsequent increase in the use of in silico high-throughput methods for data interpretation. Currently, in silico methodologies enclose motif-based identification of phosphorylation sites, structural information integration, integration of phosphorylation site structural context, phospho-clusters modeling, integration of Protein–Protein Interaction Network (PPIN) information, and multi-organisms prediction [24], [25]. Here, we propose four linear patterns for identifying class-III CK2 sites. These patterns were constructed manually based on literature and database information describing well known class-III substrates.
    Materials and methods
    Results and discussion
    Conclusions The four designed linear patterns aimed to assist CK2 beta-dependent substrates prediction. The discrimination power of these patterns relies on the recognition of a basic cluster at a suitable distance from the phosphoacceptor site in the substrate. Since the patterns utilized the CK2 consensus sequence for matching substrates we performed the prediction of class-III substrates on experimentally determined CK2 substrates to reduce noise. As a result we obtained a list of 327 predicted class-III substrates, 467 sites. The functional classification of these substrates indicated a role of beta-dependent regulation in viral infection and biological processes and pathways such as apoptosis, DNA repair and RNA metabolism. It also suggested that the human substrates are primarily nuclear located with a number of them also found in cytoplasm. A cautious interpretation of these results is needed since this analysis derives from an in silico predictive approach. Thus, future experiments are required to validate the results of the prediction and the functional analysis.
    Author contributions
    Introduction Casein kinase 2 (CK2) is a tetrameric protein kinase composed of two catalytic α-subunits (α and α′) and two regulatory β-subunits (Pinna & Allende, 2009). CK2 regulates many cellular functions that are important for Bezafibrate development (Lou et al., 2008) and cellular homeostasis (Blanquet, 2000; Brunet et al., 2015). Despite the initial lack of substrate following its discovery, CK2 was subsequently shown to phosphorylate numerous substrates, including other protein kinases, thus acting as a “master regulator” among protein kinases (Meggio & Pinna, 2003; Trembley et al., 2010) using both ATP and GTP (Litchfield, 2003). Upregulation of CK2 activity is associated with many diseases, such as cancers (Sarno et al., 2002), cardiac hypertrophy (Eom et al., 2011; Hauck et al., 2008), and ischemic injury (Hu & Wieloch, 1993; Ka et al., 2015). CK2 is expressed in brain and is associated with cellular injury (Ka et al., 2015), and indeed CK2 has been reported to be a neuroprotectant by directly modulating NADPH oxidase activity during cerebral ischemia (Kim et al., 2009). CK2 is also expressed in glial cells such as oligodendrocytes (Huillard et al., 2010a) and a brief and moderate AMPA receptor activation in rat oligodendrocyte cell cultures triggers CK2 activity to mediate injury (Canedo-Antelo et al., 2014; Canedo-Antelo et al., 2015). Consequently, CK2 inhibitors applied before AMPA receptor activation alleviated AMPA-mediated excitotoxic oligodendrocyte death (Canedo-Antelo et al., 2015). Stroke typically affects both the neurons in brain gray matter as well as glial cells and axons in brain white matter (WM). WM constitutes half of human brain volume and injury to axons largely translates into functional loss observed after a stroke. Because axons are natural extensions of neuronal cell bodies, it is often misconceived that similar mechanisms mediate injury to both portions of the cellular structure. This perception has been fortified by the fact that the rodent brain contains only ~10% WM and because commonly-used stroke models in rodents spare the corpus callosum, which is the main WM tract. Therefore, ischemic brain injury in rodent models overwhelmingly reflects the outcome of neuronal (gray matter) injury. WM is composed of axons, oligodendrocytes, microglia, and astrocytes (Baltan, 2009; Fields, 2008). Thus, WM is composed of a complex cellular environment in which glial cell-cell interactions intricately maintain axon function. As a result, mechanisms of WM injury significantly differ from gray matter. Consequently, protective interventions in neurons may become ineffective at promoting recovery of, or even become injurious to, WM. Therefore, an ideal stroke therapeutic must be directed towards both neuronal and axon-glia protection. The role of kinases remains unexplored in WM function and injury mechanisms; hence an intriguing question remains as to whether or not CK2, which has been previously shown to act as a master regulator and a neuroprotectant (Hu & Wieloch, 1993; Ka et al., 2015), can protect WM against ischemia. The use of optic nerve as a pure WM tract provides a unique platform to test WM-specific injury mechanisms in isolation (Baltan, 2012a; Baltan et al., 2008a; Baltan et al., 2011a; Murphy et al., 2013; Stahon et al., 2016; Stys et al., 1991; Tekkok et al., 2007). To our knowledge, this is the first demonstration that inhibition of a protein kinase before or after injury is protective of WM structure and function. Using specific small molecule inhibitors that cross the blood-brain barrier (BBB) (Cheng et al., 2012; Sallam et al., 2008; Vita et al., 2005; Zheng et al., 2013), we show that CK2 inhibition recruits both the AKT and CDK5 signaling pathways to confer protection to both young and aging WM structure and function when applied before injury, while AKT signaling specifically contributes to post-ischemic protection. We therefore suggest that CK2 inhibitors, which are currently in phase I–II clinical trials for cancer therapy, could be repurposed to provide a novel therapeutic target for ischemic stroke patients. The role of CK2 signaling in WM injury will also be applicable to other neurodegenerative conditions that are characterized by axonal injury, such as traumatic brain injury, Alzheimer\'s disease, spinal cord injury, and multiple sclerosis.