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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 knock-down (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-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3β pS9 was not reduced by CK2 blockade suggests that GSK3β phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3β pS9, and concomitantly decreased Snail1 levels (a GSK3β target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2β-downregulated cells, suggesting that reducing GSK3β pS9 could be a strategy to control Snail1 levels in any situation where CK2β is defective, as possibly occurring in cancer cells.

CK2 is a pleiotropic S/T protein kinase (formerly known as casein kinase 2) which is attracting increasing interest as therapeutic target, and the identification of its substrates is a crucial step in determining its involvement in different pathological conditions. We recently found that S131 of Akt2 (homologous to the well established CK2 target S129 of Akt1) is not phosphorylated by CK2 either in vitro or in vivo, although the consensus sequence recognized by CK2 (S/T-x-x-E/D/pS/pT) is conserved in it. Here, by exploiting synthetic peptides, in cell transfection experiments, and computational analysis, we show that a single sequence element, a T at position n+1, hampers phosphorylation, causing an α-helix structure organization which prevents the recognition of its own consensus by CK2. Our results highlight the role of negative determinants as crucial modulators of CK2 targeting and corroborate the concept that Akt1 and Akt2 display isoform specific features. Experiments with synthetic peptides suggest that Akt2 S131 could be phosphorylated by kinases of the Plk (Polo-like kinase) family, which are insensitive to the presence of the n+1 T. The low phylogenetic conservation of the Akt2 sequence around S131, as opposed to the extremely well-conserved Akt1 homologous sequence, would indicate a dominant positive role in the selective pressure only for the Akt1 phosphoacceptor site committed to undergo phosphorylation by CK2. By contrast, Akt2 S131 may mediate the response to specific physio/pathological conditions, being consequently shielded against basal CK2 targeting.

Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

Homeodomain-interacting protein kinase 2 (HIPK2) is a Ser/Thr kinase controlling cell proliferation and survival, whose investigation has been hampered by the lack of specific inhibitors able to dissect its cellular functions. SB203580, a p38 MAP kinase inhibitor, has been used as a tool to inhibit HIPK2 in cells, but here we show that its efficacy as HIPK2 inhibitor is negligible (IC₅₀>40 µM). In contrast by altering the scaffold of the promiscuous CK2 inhibitor TBI a new class of HIPK2 inhibitors has been generated. One of these, TBID, displays toward HIPK2 unprecedented efficacy (IC₅₀ = 0.33 µM) and selectivity (Gini coefficient 0.592 out of a panel of 76 kinases). The two other members of the HIPK family, HIPK1 and HIPK3, are also inhibited by TBID albeit less efficiently than HIPK2. The mode of action of TBID is competitive with respect to ATP, consistent with modelling. We also provide evidence that TBID is cell permeable by showing that HIPK2 activity is reduced in cells treated with TBID, although with an IC₅₀ two orders of magnitude higher (about 50 µM) than in vitro.

CK1δ, a member of the casein kinase 1 family, is involved in the regulation of various cellular processes and has been associated with the pathophysiology of neurodegenerative diseases and cancer. Therefore recently, interest in generating highly specific inhibitors for personalized therapy has increased enormously. However, the efficacy of newly developed inhibitors is affected by the phosphorylation state of CK1δ. Cellular kinases phosphorylating CK1δ within its C-terminal domain have been identified but still more information regarding the role of site-specific phosphorylation in modulating the activity of CK1δ is required. Here we show that Chk1 phosphorylates rat CK1δ at serine residues 328, 331, 370, and threonine residue 397 as well as the human CK1δ transcription variants 1 and 2. CK1δ mutant proteins bearing one, two or three mutations at these identified phosphorylation sites exhibited significant differences in their kinetic properties compared to wild-type CK1δ. Additionally, CK1δ co-precipitates with Chk1 from HT1080 cell extracts and activation of cellular Chk1 resulted in a significant decrease in cellular CK1δ kinase activity. Taken together, these data point towards a possible regulatory relationship between Chk1 and CK1δ.

CK2 is a pleiotropic protein kinase, which regulates many survival pathways and plays a global anti-apoptotic function. It is highly expressed in tumor cells, and is presently considered a promising therapeutic target. Among the many inhibitors available for this kinase, the recently developed CX-4945 and CX-5011 have proved to be very potent, selective and effective in inducing cell death in tumor cells; CX-4945 has recently entered clinical trials. However, no data are available on the efficacy of these compounds to overcome drug resistance, a major reasons of cancer therapy failure. Here we address this point, by studying their effects in several tumor cell lines, each available as variant R resistant to drug-induced apoptosis, and normal-sensitive variant S. We found that the inhibition of endogenous CK2 was very similar in S and R treated cells, with more than 50% CK2 activity reduction at sub-micromolar concentrations of CX-4945 and CX-5011. A consequent apoptotic response was induced both in S and R variants of each pairs. Moreover, the combined treatment of CX-4945 plus vinblastine was able to sensitize to vinblastine R cells that are otherwise almost insensitive to this conventional antitumor drug. Consistently, doxorubicin accumulation in multidrug resistant (MDR) cells was greatly increased by CX-4945.In summary, we demonstrated that all the R variants are sensitive to CX-4945 and CX-5011; since some of the treated R lines express the extrusion pump Pgp, often responsible of the MDR phenotype, we can also conclude that the two inhibitors can successfully overcome the MDR phenomenon.