Effects of CK2β subunit down-regulation on Akt signalling in HK-2 renal cells

Abstract

The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit β (shCK2β cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways.We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2β. However, in HK-2 cells the dependence on CK2β was confirmed by rescue experiments (CK2β re-expression in shCK2β HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2β downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2β was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2β HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3β S9, was increased. Differently to what observed in response to CK2β down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3β pS9 mirrored those induced by CK2β knockdown (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells.Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3β pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2β knock-down and CX-4945 treatment in HK-PLOS ONE | https://doi.

Okur-Chung Neurodevelopmental Syndrome-linked CK2α mutations have reduced kinase activity

“…In particular, T202/Y204 pERK1/2 sites have been shown to be to be affected indirectly by CK2 (Olsen et al 2012;Ritt et al 2007). We do not observe changes in T202/Y204 pERK1/2 site phosphorylation, confirming data by others which showed that knockdown or inhibition of CK2 did not alter pT202/pY204 ERK in Hela and renal HK-2 cells (Alcaraz et al 2020;Plotnikov et al 2011). Our data in patient cell lines suggest that typical signaling pathways in immortalized cells, may not be adequate for the assessment of CK2α activity in OCNDS-patient fibroblasts.…”

“…We tested pS129Akt, a substrate for CK2 in several immortalized cell lines. Neither was pS473 Akt affected, a site which was shown to be downregulated upon CK2 inhibition of knockdown in Jurkat and HK-2 renal cells (Alcaraz et al 2020;Di Maira et al 2005). This phosphosite was not reduced in patient fibroblasts, in line with findings in CK2α knockout embryos.…”

“…1C, D). We also tested for pS473 Akt which is phosphorylated by mTOR, downregulated by CK2 inhibitor CX4945 and in most cases correlates with the degree of pS129 Akt phosphorylation (Alcaraz et al 2020;Di Maira et al 2005;Sarbassov et al 2005). This site was also unaffected (Fig.…”

Genomic Fabric Remodeling in Metastatic Clear Cell Renal Cell Carcinoma (ccRCC): A New Paradigm and Proposal for a Personalized Gene Therapy Approach

“…VHL [12,13], VEGF (vascular endothelial growth factor [14]), CAIX (carbonic anhydrase IX [15]), PTEN [15], CR1 (complement C3b/C4b receptor 1 [16]), MERTK (MER proto-oncogene tyrosine kinase [17]), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha [18]), and AKT1 (AKT serine/threonine kinase 1 [19]) are among the most popular KC biomarkers. The PI3K/Akt pathway, one that is altered in several cancers, has been evaluated as a potential target for KC therapy [20][21][22].…”

Genomic Fabric Remodeling in Metastatic Clear Cell Renal Cell Carcinoma (ccRCC): A New Paradigm and Proposal for a Personalized Gene Therapy Approach

“…PTEN [15], CR1 (complement C3b/C4b receptor 1, [16]), MERTK (MER proto-oncogene tyrosine kinase [17]), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha [18]) and AKT1 (AKT serine/threonine kinase 1 [19]) are among the most popular KC biomarkers. The PI3K/Akt pathway, one that is altered in several cancers, has been evaluated as a potential target for therapies [20].…”