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  • gssg br Results and discussion br Conclusions We have develo


    Results and discussion
    Conclusions We have developed a method to produce wildtype and mutant Ric-8A proteins from E. coli that can be phosphorylated at the regulatory protein kinase CK2 phosphosites to near homogeneity. The phosphorylation status and activity of the purified Ric-8A proteins was verified by Phos-tag SDS-PAGE mobility shift assay, immunoblotting blotting with phosphosite-specific antibodies, mass spectrometry, and by Gα GTPγS binding activity assay. Due to the high purity and high degree of phosphorylation of Ric-8A proteins produced by this method, they may be ideal for protein structural studies. They may also find utility for the study of interactions between Ric-8A and G proteins, screening for Ric-8A modulators, and additional Ric-8A binding partners. Ric-8A proteins are also used traditionally as catalysts for the production of activated Gα-GTPγS for use in G protein gssg assays [16]. The dramatically enhanced activity of phosphorylated Ric-8A proteins produced by this method should prove advantageous for this use.
    Materials and methods
    Declarations of interest
    Author contributions
    Acknowledgements Intact mass analysis experiments were conducted in the University of Texas Health Science Center at San Antonio Institutional Mass Spectrometry Laboratory under the direction of Dr. Susan T. Weintraub, with support from NIH grant 1S10OD016417-01 (STW) for purchase of the mass spectrometer. The expert technical assistance of Sammy Pardo is gratefully acknowledged. Data interpretation was performed by Dr. Henriette A. Remmer at the University of Michigan Proteomics and Peptide Synthesis Core.
    Introduction Casein kinase 2 (CK2), a ubiquitous, highly pleiotropic and constitutively active serine/threonine protein kinase, is a heterotetramer composed of two catalytic subunits (α and /or α′) and two regulatory β subunits [1]. It is known that CK2 can phosphorylate more than 300 substrates, many of which are involved in signal transduction, gene expression and cell growth [2], [3]. Overexpression of CK2 has been documented in a great number of cancers such as lung cancer, breast carcinoma, prostate and kidney cancers, and elevated CK2 activity is found to be associated with tumorigenesis [4], [5]. Thus, CK2 has been regarded as a common denominator of diverse cancer cells and can represent a multi-purpose target for the treatment of different kinds of tumors. So far, there are two methods to reduce the function of CK2, one is to down-regulate the expression of CK2 protein by gene knockout such as siRNA, the other is to inhibit CK2 activity by small molecular inhibitors [6], [7]. However, the application of gene knockout technology is rather complicated and limited in clinic, and CK2 inhibitors could only inhibit the activity of the enzyme without degrading CK2 protein [8]. Ubiquitin-proteasome system (UPS), responsible for much of the regulated proteolysis in the cell, mainly contains ubiquitin (Ub), Ub-activating enzyme E1, Ub-conjugating enzymes E2s (UBCs), Ub-protein ligands E3s, and 26S proteasome [9]. E3 ligand is known as substrate-specific, which can catalyze transfer of ubiquitin from E2s to an amide linkage with the substrate or with a polyubiquitin chain already anchored to it, then the tagged substrate is degraded by the 26S proteasome [10]. Because the UPS represents a major system controlling many cellular processes, drugs suppressing the UPS can be efficacious in the treatment of cancers. So far, several small molecules like bortezomib and carfilzomib have been approved as proteasome inhibitors for clinical use [11], [12]. Proteolysis targeting chimeras (PROTACs), owning full advantage of the function of protein degradation by UPS, is a heterobifunctional compound with two recruiting ligands connected via a linker, in which one ligand is specific to the protein of interest while the other one specifically recruits E3 ligase [13], [14], [15]. Generally, an E3 ligase needs a specific recognition signal to recruit and ubiquitinate its natural substrate, however, PROTAC acts in a way that can effectively tether the E3 ligase to the substrate and result in the ubiquitination and subsequent proteasomal degradation of the target protein (Fig. 1) [16]. Typically, a small molecule protein inhibitor is usually selected as the ligand specific to the protein of interest, and so far thalidomide, lenalidomide, pomalidomide and their derivatives have been chosen as the recruiters of E3 ligase [17], [18], [19], [20]. At present, PROTAC has been widely applied to induce the degradation of various proteins, such as BRD4, Sirt2, and BCR-ABL [21], [22], [23].