CK2 in pancreatic alpha and beta cells.
The pancreas is the pancreas located transversely in the upper abdomen and has both exocrine and endocrine functions. The exocrine function includes secretion of a digestive secretion consisting of HCO3- and digestive enzymes into the small intestine. The endocrine, hormone-producing portion of the organ is located in the so-called islets of Langerhans inside the organ. These specialized cells produce glucagon (α cells), insulin (β cells), pancreatic polypeptide (γ cells) or somatostatin (δ cells). Glucagon-producing α cells and insulin-producing β cells are responsible for maintaining a constant blood glucose concentration in the blood (blood glucose homeostasis) regardless of nutritional status. Insulin secreted by the β cells ensures that blood glucose levels are lowered after a carbohydrate-rich meal; glucagon secreted by the α-cells ensures that blood glucose levels are increased during periods of dietary restriction. The finely tuned interaction of these two antagonistically acting hormones results in an adjustment of the blood glucose level to physiological values of about 5 mM or 90 mg/100 ml. When glucose homeostasis is disturbed by an absolute (type 1) or relative (type 2) insulin deficiency, the metabolic disease diabetes mellitus ("diabetes") develops, affecting about 6 million people in Germany.
The synthesis and secretion of insulin is regulated not only by glucose concentration, but in interaction with various transcription factors and hormones. A very crucial transcription factor for the regulation of insulin expression is the homeobox protein PDX-1. PDX-1 is not at the top of the signaling chain, but is itself regulated by transcription factors. These include the transcription factors of the USF family, which influence the transcription of PDX-1 by binding to so-called E-box sequences in the DNA.
Fig.: Modified after: Felicia W. Pagliuca, Douglas A. Melton: How to make a functional β-cell. Development 2013 140: 2472-2483; doi: 10.1242/dev.093187
We have been able to identify both PDX-1 and USF as interaction partners and substrates of CK2. PDX-1 provides a substrate for the protein kinase CK2 both in vitro and in vivo. Site-directed mutagenesis has successfully identified the serine residue at position 232 and the threonine residue at positon 231 as CK2 phosphorylation sites. Phosphorylation at these sites affects both transcriptional activity with respect to the insulin promoter and PDX-1-dependent insulin secretion. In addition, phosphorylation by CK2 destabilizes the PDX-1 protein and sensitizes it to proteasomal degradation, likely via the E3 ubiquitin ligase-adaptor protein PCIF1 (Klein et al., 2017). However, phosphorylation by CK2 does not affect the subcellular localization of PDX-1 (Meng et al., 2010a, Meng et al., 2010b). Data on PDX-1- CK2 interaction are summarized in the following figure.
Fig.: Network of interactions between protein kinase CK2 and PDX-1 in pancreatic β cell with their possible consequences.
The upstream stimulatory factors USF1 and 2, transcription factors from the bHLH leucine zipper family, which bind to so-called E-box motifs on the DNA, are involved in the regulation of PDX-1 transcription. USF complexes have a repressive effect on the expression of PDX-1.
Our group in cooperation with AG Kietzmann, Oulu, Finland, has found USF2 as an interaction partner of the regulatory β subunit of CK2 in a yeast two-hybrid analysis. Here, CK2 does not accept USF2 as a substrate, but USF1 does, which mostly initiates transactivation events in complex with USF2. From our data on the USF-CK2-PDX-1 interaction, we were able to derive the following hypothesis (see figure).
Fig.: Influence of CK2 on the expression of PDX-1.
PDX-1 is a transcription factor that is not expressed in adult pancreatic α-cells. Exogenous overexpression of this protein results in severe restriction of the formation and secretion of the blood glucose-raising hormone glucagon. By downregulating CK2 activity in α-cells, we have seen in initial experiments that PDX-1 is stabilized in these cells and exerts a repressive effect on glucagon expression. Intensive studies on this research project are currently underway.
Protein kinase CK2 thus appears to be able to mutually regulate the expression and secretion of the two antagonistic pancreatic hormones, insulin and glucagon. It is thus involved in a meaningful physiological concept that can ensure an effective adjustment of glucose homeostasis.
Fig.: Schematic of the effect of CK2 on pancreatic α and β cell functions.
All in all, CK2 seems to be involved in the complicated network of metabolic regulation, exerting rather a negative regulatory effect on the functionality of pancreatic β-cells and a positive regulatory effect on the functionality of pancreatic α-cells. Inhibition of CK2 activity may therefore represent an option for the treatment of type 2 diabetes mellitus, in which not only insufficient insulin production but also hyperglucagonemia is often described.
Publications of the group
Meng R, Götz C, Montenarh M. The role of protein kinase CK2 in the regulation of the insulin production of pancreatic islets. Biochem Biophys Res Commun. (2010a) 401:203-6.
Meng R, Al-Quobaili F, Müller I, Götz C, Thiel G, Montenarh M. CK2 phosphorylation of Pdx-1 regulates its transcription factor activity. Cell Mol Life Sci. (2010b) 67:2481-9.
Welker S., Götz C., Servas C., Laschke M.W., Menger M.D., Montenarh M. Glucose regulates protein kinase CK2 in pancreatic β-cells and its interaction with PDX-1. Int J Biochem Cell Biol. 45:2786-95 (2013).
Lupp S., Götz C., Khadouma S., Horbach T., Dimova EY., Bohrer A.M., Kietzmann T., and Montenarh M. The upstream stimulatory factor USF1 is regulated by protein kinase CK2 phosphorylation. Cell Signal. 26:2809-2817 (2014).
Spohrer S., Dimova E.Y., Kietzmann T., Montenarh M., and Götz C. The nuclear fraction of protein kinase CK2 binds to the upstream stimulatory factors (USFs) in the absence of DNA. Cell Signal. 28: 23-31 (2016).
Klein S., Meng R., Montenarh M., and Götz C. The phosphorylation of PDX-1 by protein kinase CK2 Is crucial for its stability. Pharmaceuticals 2017, 10 (1), 2.
Spohrer S, Groß R, Nalbach L, Schwind L, Stumpf H, Menger MD, Ampofo EA, Montenarh M and Götz C. Functional interplay between the transcription factors USF1 and PDX-1 and protein kinase CK2 in pancreatic -cells. Scientific Reports (2017), 7: 16367.
Ampofo E, Nalbach L, Menger MD, Montenarh M, Götz C. Protein Kinase CK2-A Putative Target for the Therapy of Diabetes Mellitus? Int J Mol Sci. 2019 20(18).
Scheuer R, Philipp SE, Becker A, Nalbach L, Ampofo A, Montenarh M and Götz C. Protein Kinase CK2 Controls CaV2.1-Dependent Calcium Currents and Insulin Release in Pancreatic β-Cells. Int. J. Mol. Sci. 2020, 21, 4668;
Becker A, Götz C, Montenarh M, Philipp SE. Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1. Int J Mol Sci. (2021), 22(23):13133.
Pack M, Götz C, Wrublewsky S, Montenarh M SGC-CK2-1 Is an Efficient Inducer of Insulin Production and Secretion in Pancreatic β-Cells. Pharmaceutics. (2021), 14(1):19.
Pack M, Gulde TN, Völcker MV, Boewe AS, Wrublewsky S, Ampofo E, Montenarh M, Götz C. Protein Kinase CK2 Contributes to Glucose Homeostasis by Targeting Fructose-1,6-Bisphosphatase 1. Int J Mol Sci 2022 Dec 27;24(1):428.
Inhibitors of the protein kinase CK2
My research group has been working on protein kinase CK2 for several years. Over the last few years, we have been able to identify many interaction partners and new substrates of protein kinase CK2. These include proteins that are important for cell cycle regulation, proliferation, and such basal processes as RNA splicing. Based on these findings, inhibition of CK2 should have serious effects on cell growth.
CK2 activity is increased in cancer cells and supports their proliferation. Inhibition of this enzyme thus represents a potential therapeutic approach in tumor therapy and indeed two inhibitors, CX4945 and CIGB-300, are in clinical use for the treatment of tumors.
Through a collaboration with the AG Prof. Dr. Joachim Jose, Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, we have access to newly produced inhibitors for the protein kinase CK2, which are tested in our laboratory for their suitability as cell membrane-permeable agents and for their specificity and selectivity towards CK2. The kinase inhibitor TF ((Z)-6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)-methylene-dibenzo[b,d]furan3(2H)-one) represents a highly potent inhibitor of human protein kinase CK2.
Fig.: Structure of TF ((Z)-6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)-methylene-dibenzo[b,d]furan3(2H)-one) (Gratz A, Kuckländer U, Bollig R, Götz C, Jose J. Identification of novel CK2 inhibitors with a benzofuran scaffold by novel non-radiometric in vitro assays. Mol Cell Biochem. 356: 83-90 (2011).
In cell culture experiments with various tumor cell lines as well as normal cells, TF was found to decrease cellular CK2 activity, implying that TF can cross the cellular membrane and inhibit CK2 in its enzymatic activity.
The reduction in cellular CK2 activity was accompanied by a reduction in cell viability, with tumor cells being much more sensitive than normal cells. Only in the tumor cells was apoptosis of the cells detectable on the basis of activation of caspases and cleavage of PARP; normal cells were unaffected.
An inhibitor that spares normal cells as much as possible and impairs or even kills tumor cells in their growth is ideal for the development of therapeutic strategies against cancer. TF is currently being tested in a tumor model. Initial results from a tumor model in mice look promising (see Fig.).
Fig.: Tumor sizes in a tumor model with untreated (control) and with TF-treated animals after 14 (start of treatment), 21 and 28 days of treatment.
Publications of the group
Gratz A, Kuckländer U, Bollig R, Götz C, Jose J. Identification of novel CK2 inhibitors with a benzofuran scaffold by novel non-radiometric in vitro assays. Mol Cell Biochem. 356: 83-90 (2011).
Götz, C., Gratz, A., Kucklaender, U., and Jose, J. TF - a novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP. BBA – General subjects 1820: 970-977 (2012).
Schnitzler A., Gratz A., Bollacke A., Weyrich M., Kuckländer U., Wünsch B., Götz C., Niefind K. and Jose J. A π-halogen bond of dibenzofuranones with the gatekeeper Phe113 in human protein kinase CK2 leads to potent tight binding inhibitors. Pharmaceuticals (2018), 11, 23.
