Cancer is a leading cause of death worldwide. Casein kinase 2 (CK2) is commonly dysregulated in cancer, impacting diverse molecular pathways. CK2 is a highly conserved serine/threonine kinase, constitutively active and ubiquitously expressed in eukaryotes. With over 500 known substrates and being estimated to be responsible for up to 10% of the human phosphoproteome, it is of significant importance. A broad spectrum of diverse types of cancer cells has been already shown to rely on disturbed CK2 levels for their survival. The hallmarks of cancer provide a rationale for understanding cancer’s common traits. They constitute the maintenance of proliferative signaling, evasion of growth suppressors, resisting cell death, enabling of replicative immortality, induction of angiogenesis, the activation of invasion and metastasis, as well as avoidance of immune destruction and dysregulation of cellular energetics. In this work, we have compiled evidence from the literature suggesting that CK2 modulates all hallmarks of cancer, thereby promoting oncogenesis and operating as a cancer driver by creating a cellular environment favorable to neoplasia.
DNA mismatch repair (MMR) deficiency plays an essential role in the development of colorectal cancer (CRC). We recently demonstrated in vitro that the serine/threonine casein kinase 2 alpha (CK2α) causes phosphorylation of the MMR protein MLH1 at position serine 477, which significantly inhibits the MMR. In the present study, CK2α-dependent MLH1 phosphorylation was analyzed in vivo. Using a cohort of 165 patients, we identified 88 CRCs showing significantly increased nuclear/cytoplasmic CK2α expression, 28 tumors with high nuclear CK2α expression and 49 cases showing a general low CK2α expression. Patients with high nuclear/cytoplasmic CK2α expression demonstrated significantly reduced 5-year survival outcome. By immunoprecipitation and Western blot analysis, we showed that high nuclear/cytoplasmic CK2α expression significantly correlates with increased MLH1 phosphorylation and enriched somatic tumor mutation rates. The CK2α mRNA levels tended to be enhanced in high nuclear/cytoplasmic and high nuclear CK2α-expressing tumors. Furthermore, we identified various SNPs in the promotor region of CK2α, which might cause differential CK2α expression. In summary, we demonstrated that high nuclear/cytoplasmic CK2α expression in CRCs correlates with enhanced MLH1 phosphorylation in vivo and seems to be causative for increased mutation rates, presumably induced by reduced MMR. These observations could provide important new therapeutic targets.
MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo.
MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player in the DNA mismatch repair (MMR) system and of great importance to correct incorporation errors that occur during DNA replication. Previously, we identified that posttranslational phosphorylation of MLH1 at amino acid position serine 477 can switch off MMR activity in vitro. We also found that mutation of serine 477 prevented posttranslational phosphorylation. Since MLH1 is involved in numerous MMR-independent cell processes, including the cell cycle control, we hypothesized that phosphorylation of MLH1 might alter the mediation of cell cycle-associated proteins and thus affect proliferation. To investigate the impact of phosphorylation of MLH1 on proliferation, an MTT assay was used. MutLα-deficient HEK293T cells were transiently cotransfected with pcDNA3.1+/MLH1 and pcDNA3.1+/PMS2 for the expression of the MutLα wildtype. For the expression of the non-phosphorylatable MutLα variant, cells were transiently cotransfected with pcDNA3.1+/MLH1S477A and pcDNA3.1+/PMS2. At 48 h after transfection, cells were treated with calyculin (50 nM), a serine-threonine-phosphatase inhibitor, to enhance the amount of phosphorylated MLH1. In parallel, cells were treated with orthovanadate (50 µM), a competitive inhibitor of protein-phosphotyrosine phosphatases, to exclude inhibitor side effects. DMSO was used as a negative control. After a cultivation period of 15 min to 3 h, cells were incubated with MTT reagent and proliferation was evaluated via an ELISA reader. In summary, significant differences of proliferation could be detected between the differently treated cells. Proliferation of calyculin-treated HEK293T cells overexpressing the non-phosphorylatable MutLα variant, however, was only weakly increased compared to cells overexpressing the MutLα wildtype. Due to the fact that calyculin and orthovanadate are able to influence a multitude of signaling pathways, the role of MLH1 phosphorylation cannot be conclusively answered here. Further experiments are necessary to clarify the function of phosphorylated MLH1 in proliferation.
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