CK2-mediated phosphorylation of Hsp90β as a novel mechanism of rifampin-induced MDR1 expression

which phosphorylates Hsp90β. Consequently, PXR increases and P-gp expression is induced. Conclusion: A mechanism for inducing P-gp expression by rifampin exposure is newly identified. ABSTRACT The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as

retention protein (CCRP), additional P-gp inducing pathways are gradually being discovered (11)(12)(13). Using a phosphoproteomic approach, we focused on Hsp90β, a highly conserved 90-kDa molecular chaperone/heat shock protein, as a key regulator of P-gp expression. Hsp90β is phosphorylated by casein kinase 2 (CK2) and forms a complex with PXR in the cytoplasm (14,15). In the study described herein, we reveal that this pathway can be induced by rifampin.
CK2 is a nuclear matrix-associated, ubiquitously expressed, and evolutionally conserved serine/threonine protein kinase consisting of two α or α' catalytic and two β regulatory subunits (16). CK2 phosphorylates a wide range of cellular proteins and is involved in a variety of biological functions, such as cell cycle progression (17), signal transduction (18), circadian rhythms (19), and gene expression (20). Importantly, CK2 levels are elevated in tumors compared to normal tissue, where overexpression of its catalytic subunit leads to the development of T-cell lymphoma and mammary tumorigenesis (21,22), indicating the significance of CK2 in proliferation, as well as in the formation of neoplasias (23 specific Pro-QD stain, 2D profiles of phosphoproteins were compared between those exposed to DMSO and rifampin (Fig. 1A).
Eight phosphoproteins whose intensities were significantly different between the two treatments were identified using trypsin digest and nano-LC/MS/MS of spots (data not shown).
Of these eight proteins, Hsp90β showed the highest differential staining intensity between treatment groups ( Fig. 1A and 1B). Hsp90β protein levels were also confirmed using Western blot analysis by comparing Hsp90β expression before and after IMAC elution.  Rifampin treatment resulted in the upregulation of PXR protein, but not mRNA (Fig. 5C) (Fig. 6). In contrast, CK2 and CK2α levels increased following rifampin treatment (Fig. 6).

CK2α and [ 3 H]-rifampin binding was then confirmed in vitro. [ 3 H]-Emodin, a known CK2
binding partner (30), served as a positive control (Fig. 7A, B). The amount of binding increased depending on the concentrations of both drugs, and was inhibited with the addition of non-tritium-labeled drugs and TBB.
Inhibition concentrations of rifampin, emodin, and TBB were determined using a pre-binding assay (data not shown). Binding affinity of [ 3 H]-rifampin to A160/A175 mutated CK2α compared with wild type CK2α. Affinity to mutated CK2α was lower than that of wild type CK2α (Fig. 7C). Figure 8 shows  showed hydrogen bond with ligand (Fig.8C).
In silico mutation with Ala showed an increase of binding energy (His160Ala: 2.95 Kcal/mol, Asp175Ala: 2.80 Kcal/mol) indicating a decrease in binding affinity.

Hsp90β location confirming -PXR and
Hsp90β quantities were compared at the cytoplasm and nucleus according to the rifampin treatment (Fig. 9). PXR was increased at both the cytoplasm and nucleus after rifampin treatment. However, the increase was much higher at the nucleus than at the cytoplasm. Hsp90β was same with or without rifampin treatment at cytoplasm. No signal of Hsp90β was found in nucleus.
Graphic presentation -The contents of this study is summarized in Fig. 10. As already reported in several papers, each step of Figures   10B, C, and D is again confirmed for solidifying our hypothesis of the rifampininduced MDR1 overexpression mechanism.
Rifampin activity of binding to CK2 and activating CK2 (Fig. 10A) has been added to this paper. To date, the mechanism of rifampininduced MDR1 overexpression was only known for binding between rifampin and PXR.
However, the mechanisms shown in Figs. 10B, C, and D is caused by Fig. 10A and all of these steps constitute a new mechanism (Fig. 10E) of rifampin induced MDR1 overexpression.

DISCUSSION
The induction of P-gp expression has long been acknowledged for decreasing the bioavailability of drugs co-administered with rifampin (31). Rifampin acts as a ligand and direct activator of nuclear receptor PXR, and its role in P-gp induction has been unknown until now. PXR is believed to be retained in the cytoplasm by binding to Hsp90 and CCRP (15).
Upon activation by xenobiotics, such as rifampin, PXR dissociates and translocates into the nucleus where it interacts with RXR to induce the transcriptional activation of several genes involved in drug metabolism (12,32,33). The ability of rifampin to independently activate CK2 has yet to be described. This pathway was evaluated in two experiments in this study. In a direct binding assay, the V max and Km values could not be determined due to the experimental conditions. To reach saturation status, the drug dose would have to increase or the protein quantity decrease. When the protein concentration was lower than the minimum recommended by the manufacturer, the experiment failed. Therefore, the binding assay was performed in a non-saturated state.
Since CK2 shows activity against more than 300 substrates based on a 2003 report (35) and is associated with several pathways, CK2 inhibitors have been used to improve the efficacy of many drugs. To date, combination therapy of CK2 inhibitors with anticancer agents has been investigated based on the known roles of CK2, including in apoptosis, the PI3K-Akt-mTOR pathway, angiogenesis, NF-κB, Wnt signaling, the epithelialmesenchymal transition, DNA damage repair, and Hsp90 machinery (36). Hsp90 inhibitors are also being investigated as a combination partner with anticancer drugs. A recent report described that combination therapy with an     Green, CK2; cyan, binding site pocket; yellow, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB); magenta, rifampin.