ABSTRACT
p53-related protein kinase (PRPK), the human homologue of yeast Bud32, belonging to a small subfamily of atypical protein kinases, is inactive unless it is previously incubated with cell lysates. Here we show that such an activation of PRPK is mediated by another kinase, Akt/PKB, which phosphorylates PRPK at Ser250. We show that recombinant PRPK is phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody; that cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; and that the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK, but not PRPK dead mutant, and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signaling pathway.
Subject(s)
Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Catalysis , Enzyme Activation , Humans , Intracellular Signaling Peptides and Proteins , Jurkat Cells , Phosphorylation , Protein Kinases/genetics , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-akt/genetics , Serine/metabolism , Signal Transduction , Transduction, GeneticABSTRACT
Protein kinase CK2 is an ubiquitous and constitutively active kinase, which phosphorylates many cellular proteins and is implicated in the regulation of cell survival, proliferation and transformation. We investigated its possible involvement in the multidrug resistance phenotype (MDR) by analysing its level in two variants of CEM cells, namely S-CEM and R-CEM, normally sensitive or resistant to chemical apoptosis, respectively. We found that, while the CK2 regulatory subunit beta was equally expressed in the two cell variants, CK2alpha catalytic subunit was higher in R-CEM and this was accompanied by a higher phosphorylation of endogenous protein substrates. Pharmacological downregulation of CK2 activity by a panel of specific inhibitors, or knockdown of CK2alpha expression by RNA interference, were able to induce cell death in R-CEM. CK2 inhibitors could promote an increased uptake of chemotherapeutic drugs inside the cells and sensitize them to drug-induced apoptosis in a co-operative manner. CK2 blockade was also effective in inducing cell death of a different MDR line (U2OS). We therefore conclude that inhibition of CK2 can be considered as a promising tool to revert the MDR phenotype.
Subject(s)
Casein Kinase II/antagonists & inhibitors , Drug Resistance, Multiple , Drug Resistance, Neoplasm , T-Lymphocytes/pathology , Animals , Antibiotics, Antineoplastic/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/physiology , Blotting, Western , Bone Neoplasms/drug therapy , Bone Neoplasms/pathology , Casein Kinase II/genetics , Casein Kinase II/metabolism , Cell Survival/drug effects , Cells, Cultured , Doxorubicin/metabolism , Humans , Osteosarcoma/drug therapy , Osteosarcoma/pathology , Phosphorylation , RNA, Small Interfering/pharmacology , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/drug effects , Transfection , Vinblastine/pharmacologyABSTRACT
Treatment of Jurkat cells with specific inhibitors of protein kinase CK2 induces apoptosis. Here we provide evidence that the anti-apoptotic effect of CK2 can be at least partially mediated by upregulation of the Akt/PKB pathway. Such a conclusion is based on the following observations: (1) inhibition of CK2 by cell treatment with two structurally unrelated CK2 inhibitors induces downregulation of Akt/PKB, as judged from decreased phosphorylation of its physiological targets, and immunoprecipitate kinase assay; (2) similar results are observed upon reduction of CK2 catalytic subunit by the RNA-interference technique; (3) Akt/PKB Ser129 is phosphorylated by CK2 in vitro and in vivo; (4) such a phosphorylation of activated Akt/PKB correlates with a further increase in catalytic activity. These data disclose an unanticipated mechanism by which constitutive phosphorylation by CK2 may be required for maximal activation of Akt/PKB.