Phosphorylation and activation of protein kinase CK2 by p34cdc2 are independent events.

Recombinant isolated beta-subunit of protein kinase CK2 is readily phosphorylated by p34cdc2/cyclin B kinase at Ser209 with favourable kinetic constants (Km = 1.7 microM, Vmax = 20 nmol.min-1.mg-1). Two synthetic peptides reproducing the 170-215 and the 206-215 C-terminal fragments of the beta-subunit are also phosphorylated though with tenfold higher Km values (19.5 and 28.0 microM, respectively). In contrast, both the beta-subunit associated with the alpha-subunit to give the heterotetrameric holoenzyme and the native CK2 are not appreciably phosphorylated by p34cdc2. These data suggest that the Ser209 beta-subunit phosphorylation observed in intact cells occurs prior to beta-subunit incorporation into the holoenzyme. The isolated CK2 alpha-subunit is not phosphorylated to any appreciable extent by p34cdc2 kinase. Its catalytic activity is nevertheless increased up to fivefold upon incubation with p34cdc2/cyclin B kinase complex. Such a stimulation of activity is comparable to that induced by the beta-subunit and it is paralleled by a 40% decrease of p34cdc2/cyclin B catalytic activity. Similar to beta-subunit, p34cdc2/cyclin B also protects the alpha-subunit against thermal inactivation. CK2 holoenzyme is also stimulated by p34cdc2/cyclin B, albeit less dramatically than the isolated alpha-subunit. Such an effect is also evident with CK2 holoenzyme reconstituted with a mutated beta-subunit lacking the p34cdc2 phosphorylation site and it is not accompanied by any appreciable phosphorylation of either the beta or the alpha-subunit. These data indicate that in vitro CK2 alpha-subunit interacts with and is activated by p34cdc2/cyclin B kinase by a mechanism that does not imply the phosphorylation of CK2

Breast cancer cells with acquired antiestrogen resistance are sensitized to cisplatin-induced cell death.

Antiestrogens are currently used for treating breast cancer patients who have estrogen receptor-positive tumors. However, patients with advanced disease will eventually develop resistance to the drugs. Therefore, compounds effective on antiestrogen-resistant tumors will be of great importance for future breast cancer treatment. In this study, we have investigated the effect of the chemotherapeutic compound cisplatin using a panel of antiestrogen-resistant breast cancer cell lines established from the human breast cancer cell line MCF-7. We show that the antiestrogen-resistant cells are significantly more sensitive to cisplatin-induced cell death than antiestrogen-sensitive MCF-7 cells and we show that cisplatin induces cell death by activating both the caspase and lysosomal death pathways. The antiestrogen-resistant cell lines express lower levels of antiapoptotic Bcl-2 protein compared with parental MCF-7 cells. Our data show that Bcl-2 can protect antiestrogen-resistant breast cancer cells from cisplatin-induced cell death, indicating that the reduced expression of Bcl-2 in the antiestrogen-resistant cells plays a role in sensitizing the cells to cisplatin treatment

Mapping the residues of protein kinase CK2 alpha subunit responsible for responsiveness to polyanionic inhibitors

Six mutants of protein kinase CK2 alpha subunit in which basic residues have been mutated into alanines were assayed for their capability to phosphorylate the peptide RRRADDSDDDDD. Two mutants (R228A and R278K279R280A) behaved more or less as alpha wild type and one (H160,166A) was nearly inactive, hampering the calculation of kinetic parameters. In contrast 3 mutants (K74-77A, K79R80K83A and R191,195K198A) phosphorylated the peptide with reduced efficiency accounted for by increased Km and decreased Vmax values. By using derivatives of the RRRADDSDDDDD peptide in which individual aspartyl residues were variably replaced by alanine(s) and two peptide substrates derived from I-2 (KYRIREQESSGEEDSDL and RRKDLHDDEEDEEMSETADGE) it was shown that mutations in the 191-198, 74-77 and 79-83 regions were the least detrimental whenever the acidic determinants were lacking at positions +1, +4/+5 and +3, respectively. These data support the conclusion that the basic residues present in the p+1 loop of CK2 alpha specifically recognize the acidic determinant adjacent to the C-terminal side of serine, while the specificity determinants located more down-stream are variably recognized by different residues of the unique basic cluster spanning between Lys74 and Lys83

UV light blocks EGFR signalling in human cancer cell lines

Udgivelsesdato: 2007-JanUV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both overexpress the EGF receptor, leads to arrest of the EGFR signaling pathway. The phosphorylation status of the receptor and the level of phosphorylated downstream signaling molecules i.e. AKT and the mitogen activated protein kinases (MAPKs) ERK1 and 2 is detected by Western blotting using phosphospecific antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin disorders, it might be possible to significantly reduce the proliferative potential of these cells making them good targets for laser-pulsed UV light treatment

UV light blocks EGFR signalling in human cancer cell lines

Udgivelsesdato: 2007-JanUV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both overexpress the EGF receptor, leads to arrest of the EGFR signaling pathway. The phosphorylation status of the receptor and the level of phosphorylated downstream signaling molecules i.e. AKT and the mitogen activated protein kinases (MAPKs) ERK1 and 2 is detected by Western blotting using phosphospecific antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin disorders, it might be possible to significantly reduce the proliferative potential of these cells making them good targets for laser-pulsed UV light treatment