Maria Plana

Eukaryotic translation-initiation factor eIF2beta binds to protein kinase CK2: effects on CK2alpha activity.

eIF2 (eukaryotic translation-initiation factor 2) is a substrate and an interacting partner for CK2 (protein kinase CK2). Co-immuno-precipitation of CK2 with eIF2beta has now been observed in HeLa cells, overexpressing haemagglutinin-tagged human recombinant eIF2beta. A direct association between His6-tagged human recombinant forms of eIF2beta subunit and both the catalytic (CK2alpha) and the regulatory (CK2beta) subunits of CK2 has also been shown by using different techniques. Surface plasmon resonance analysis indicated a high affinity in the interaction between eIF2beta and CK2alpha, whereas the affinity for the association with CK2beta is much lower. Free CK2alpha is unable to phosphorylate eIF2beta, whereas up to 1.2 mol of phosphate/mol of eIF2beta was incorporated by the reconstituted CK2 holoenzyme. The N-terminal third part of eIF2beta is dispensable for binding to either CK2alpha or CK2beta, although it contains the phosphorylation sites for CK2. The remaining central/C-terminal part of eIF2beta is not phosphorylated by CK2, but is sufficient for binding to both CK2 subunits. The presence of eIF2beta inhibited CK2alpha activity on calmodulin and beta-casein, but it had a minor effect on that of the reconstituted CK2 holoenzyme. The truncated forms corresponding to the N-terminal or central/C-terminal regions of eIF2beta were much less inhibitory than the intact subunit. The results demonstrate that the ability to associate with CK2 subunits and to serve as a CK2 substrate are confined to different regions in eIF2beta and that it may act as an inhibitor on CK2alpha.

The N-terminal domain of the human eIF2β subunit and the CK2 phosphorylation sites are required for its function

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2alpha evidenced the direct involvement of this protein kinase in eIF2beta phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2beta or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2beta phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2beta also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2beta S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Delta2-138 N-terminal-truncated form of eIF2beta (eIF2beta-CT). Mutation at Ser2 and Ser67 did not affect eIF2beta integrating into the eIF2 trimer or being able to complex with eIF5 and CK2alpha. The eIF2beta-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2beta slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2beta S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2beta-CT form, being detectable at doses where eIF2beta and eIF2beta S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2beta for its function in mammals.

Phosphorylation of fibrinogen by casein kinase 1

Casein kinase 1 phosphorylated human fibrinogen, in a reaction that did not use GTP as phosphoryl donor and was neither stimulated by cyclic AMP or Ca2+, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. Maximal incorporation averaged 4 mol of phosphate per mol of fibrinogen, most of it in the largest CNBr-fragment of the alpha-chain. Phosphoamino acid analysis revealed that phosphorylation occurred only at seryl residues. The phosphorylation of fibrinogen by casein kinase 1 was reverted by alkaline phosphatase.

Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin

Protein kinase CK2 forms complexes with some protein substrates what may be relevant for the physiological control of this protein kinase. In previous studies in rat liver cytosol we had detected that the trimeric form of eukaryotic translation initiation factor 2 (eIF-2) co-eluted with protein kinase CK2. We have now observed that the ratio between eIF-2 and cytosolic CK2 contents in testis, liver and brain is quite similar, being eIF-2 levels about 5-fold higher than those of CK2. Furthermore eIF-2 was present in liver samples immunoprecipitated with anti-CK2α/α′ antibodies, confirming the existence of complexes containing both proteins. Nonetheless, these complexes would represent only a fraction of total cytosolic CK2 and eIF-2.

The carboxy‐terminal domain of Grp94 binds to protein kinase CK2α but not to CK2 holoenzyme

Surface plasmon resonance analysis shows that the carboxy‐terminal domain of Grp94 (Grp94‐CT, residues 518–803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2α) under non‐stressed conditions. A K D of 4×10−7 was determined for this binding. Heparin competed with Grp94‐CT for binding to CK2α. CK2β also inhibited the binding of Grp94‐CT to CK2α, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94‐CT. The use of CK2α mutants made it possible to map the Grp94‐CT binding site to the four lysine stretch (residues 74–77) present in helix C of CK2α. Grp94‐CT stimulated the activity of CK2α wild‐type but was ineffective on the CK2α K74–77A mutant.

Multiple forms of protein kinase CK2 present in leukemic cells: in vitro study of its origin by proteolysis.

Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20 S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunits in vitro. In both cases, CK2α′ was more resistant to these proteases than CK2α. When these proteases were assayed on the reconstituted (α2β2 holoenzyme, a 37 kDaα-band, analogous to that observed in AML extracts, was generated which was resistant to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies with CK2α deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2α. These results pointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomes evident when CK2 is overexpressed as in AML cells. (Mol Cell Biochem 191: 229–234, 1999)

Rat liver cytosol contains an inhibitor of the casein kinases 1 and 2 from the same source.

Cyclic AMP-independent casein kinases are present in a variety of mammalian tissues, including rabbit muscle [l-3] and reticulocytes [4], pig leucocytes [5], bovine adrenal cortex [6], rat liver nuclei [7] and cytosol [8,9]. Each one of these tissues contains two types of casein kinases which can be tentatively classified according to their molecular weights: type I has M, 34 000-42 000; type II has Mr 123 000-l 90 000. These two types of enzymes are also different in their kinetic properties. An important characteristic of both types of enzymes from rabbit muscle, pig leucocytes and rat liver cytosol is that they phosphorylate and inactivate I-form glycogen synthase in a cyclic AMPand Ca*+-independent manner. However, the effects promoted by the type I enzymes are greater than those promoted by the type II. Whether or not these kinases are regulated by intracellular modulators is still unknown. In [ lo,1 l] a heat-stable protein inhibitor of the G-type (type II) casein kinase has been described in adrenal cortex, which was inactive on the A-type (type I) enzyme from the same tissue. The presence of a protein inhibitor has also been reported in rat liver nuclei [ 121. The latter inhibits only the casein kinases of nuclear origin, being inactive on the cytosolic enzymes. Here we report a heat-labile inhibitor in rat liver cytosol, of both cytosolic casein kinase 1 (CK-1 or type I) and 2 (CK-2 or type II) from the same tissue. The inhibiting effect was not due to ATPase, protein phosphatase or protease activity. The possible role of this inhibitor in the physiological regulation of the cytosolic casein kinases is discussed.

KAP Degradation by Calpain Is Associated with CK2 Phosphorylation and Provides a Novel Mechanism for Cyclosporine A-Induced Proximal Tubule Injury

The use of cyclosporine A (CsA) is limited by its severe nephrotoxicity that includes reversible vasoconstrictor effects and proximal tubule cell injury, the latter associated whith chronic kidney disease progression. The mechanisms of CsA-induced tubular injury, mainly on the S3 segment, have not been completely elucidated. Kidney androgen-regulated protein (KAP) is exclusively expressed in kidney proximal tubule cells, interacts with the CsA-binding protein cyclophilin B and its expression diminishes in kidneys of CsA-treated mice. Since we reported that KAP protects against CsA toxicity in cultured proximal tubule cells, we hypothesized that low KAP levels found in kidneys of CsA-treated mice might correlate with proximal tubule cell injury. To test this hypothesis, we used KAP Tg mice developed in our laboratory and showed that these mice are more resistant to CsA-induced tubular injury than control littermates. Furthermore, we found that calpain, which was activated by CsA in cell cultures and kidney, is involved in KAP degradation and observed that phosphorylation of serine and threonine residues found in KAP PEST sequences by protein kinase CK2 enhances KAP degradation by calpain. Moreover, we also observed that CK2 inhibition protected against CsA-induced cytotoxicity. These findings point to a novel mechanism for CsA-induced kidney toxicity that might be useful in developing therapeutic strategies aimed at preventing tubular cell damage while maintaining the immunosuppressive effects of CsA.

Targeting Protein Kinase CK2: Evaluating CX-4945 Potential for GL261 Glioblastoma Therapy in Immunocompetent Mice

Glioblastoma (GBM) causes poor survival in patients even with aggressive treatment. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment but resistance always ensues. Protein kinase CK2 (CK2) contributes to tumour development and proliferation in cancer, and it is overexpressed in human GBM. Accordingly, targeting CK2 in GBM may benefit patients. Our goal has been to evaluate whether CK2 inhibitors (iCK2s) could increase survival in an immunocompetent preclinical GBM model. Cultured GL261 cells were treated with different iCK2s including CX-4945, and target effects evaluated in vitro. CX-4945 was found to decrease CK2 activity and Akt(S129) phosphorylation in GL261 cells. Longitudinal in vivo studies with CX-4945 alone or in combination with TMZ were performed in tumour-bearing mice. Increase in survival (p < 0.05) was found with combined CX-4945 and TMZ metronomic treatment (54.7 ± 11.9 days, n = 6) when compared to individual metronomic treatments (CX-4945: 24.5 ± 2.0 and TMZ: 38.7 ± 2.7, n = 6) and controls (22.5 ± 1.2, n = 6). Despite this, CX-4945 did not improve mice outcome when administered on every/alternate days, either alone or in combination with 3-cycle TMZ. The highest survival rate was obtained with the metronomic combined TMZ+CX-4945 every 6 days, pointing to the participation of the immune system or other ancillary mechanism in therapy response.

Modulators of rat liver cytosol casein kinases 1 and 2

Abstract Rat liver cytosol casein kinases 1 and 2 are similarly activated by spermine and inhibited by caffeine. On the contrary they are differently affected by heparin and basic proteins. Low concentrations of heparin inhibited selectively the phosphorylation of casein by casein kinase 2 whereas protamine and histones inhibited specifically casein kinase 1. On the contrary the basic proteins stimulated slightly the activity of casein kinase 2 and released its inhibition by heparin. Increasing the concentration of casein substrate reversed both the inhibition of casein kinase 1 by protamine as well as that of casein kinase 2 by heparin. The data suggest the existence of modulators having either similar or opposite effects on each type of casein kinase.