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Protein kinase CK2: a new target to overcome imatinib-resistance in chronic myeloid leukemia cells.
Full text linkChronic myeloid leukemia (CML), a malignant myeloproliferative disorder of hematopoietic system, is driven by the chromosomal translocation [t(9;22)(q34;q11)], yielding the Philadelphia chromosome and generating a fusion gene that encodes the Bcr-Abl protein, a constitutively active tyrosine kinase necessary and sufficient for the initiation, maintenance and progression of CML [Faderl S. et al., 1999]. Despite the great efficacy of the Bcr-Abl-specific inhibitor imatinib, which represents the gold-standard drug of choice for CML patients, resistance to this drug is recognized as a major problem in CML therapy failure [Bixby D. and Talpaz M., 2009]. In this context, this work focuses on the analysis of the protein kinase CK2, a ubiquitous, pleiotropic and constitutively active Ser/Thr kinase, composed of two catalytic (alpha and/or alpha prime) and two regulatory (beta) subunits. CK2 is abnormally elevated in a wide variety of tumors, where it does not induce directly the cancer but it is critically required to create a cellular environment favourable to the development of neoplasia, mainly through its anti-apoptotic and pro-survival role [Ruzzene M. and Pinna L.A., 2010].
The goal of the research is to shed light on the role of the protein kinase CK2 in chronic myeloid leukemia oncogenic signaling, using two different CML cell lines, LAMA84 and KCL22, either sensitive (S) or resistant (R) to imatinib.
In my laboratory it had been previously observed that resistant-LAMA84 CML cells, which are characterized by BCR-ABL1 gene amplification [Le Coutre P. et al., 2000], contain a two-fold higher amount of CK2 alpha and CK2 beta, but not CK2 alpha prime, subunits as compared to parental cells [Borgo C. et al., 2013]. Consistently, the quantification of the CK2 subunits demonstrates that CK2 protein is expressed at very high levels compared to total cellular proteins in LAMA84 cells. Subcellular fractionation analysis shows that most CK2 is located in the cytoplasmic fraction of R-LAMA84 cells, where it co-localizes with Bcr-Abl. CK2 and Bcr-Abl are members of the same multi-protein complex(es) only in R-LAMA84 cells, as demonstrated by their co-sedimentation in glycerol-gradients and co-immunoprecipitation. Interestingly, while cell treatment with imatinib does not affect the binding occurring between CK2 and Bcr-Abl, the CK2-specific inhibitor CX-4945 almost abrogates this interaction, suggesting that CK2 kinase activity plays a specific role in the binding.
In spite of the CK2 up-regulation occurring in imatinib-resistant LAMA84 cells, we also demonstrate that imatinib-resistant KCL22 cells express similar protein-level and activity of both CK2 and Bcr-Abl as compared to the sensitive counterpart. Moreover, CK2 co-immunoprecipitates with Bcr-Abl in both KCL22 cell variants. To assess whether CK2 might be a player in imatinib-resistant KCL22 cells, we investigated the complex Bcr-Abl oncogenic network dedicating particular attention to MAPK and PI3K/Akt/mTOR pathways, which have been frequently demonstrated to be up-regulated in cancer cells [Saini K.S. et al., 2013]. We found that resistant KCL22 cells are characterized by a strikingly higher phosphorylation extent of ERK1/2 T202/Y204, as previously reported by Colavita I. et al. (2010), Akt S473 and ribosomal protein S6 (rpS6) S240/4-235/6 as compared to sensitive cells. In R-KCL22 cells, the treatment with high concentrations of imatinib causes a substantial inhibition of ERK1/2 and Akt S473 phosphorylation, while, unexpectedly, it only partially affects the phosphorylation of rpS6, the common downstream effector of MAPK and PI3K/Akt/mTOR pathways. Interestingly, rpS6 phosphorylation is almost abrogated by CK2 down-regulation, as judged by cell treatment with CX-4945, which does not affect ERK1/2 and Akt activities, and by CK2 knocking down by siRNA. Consistent with the down-regulation of rpS6, protein involved in translation initiation, the treatment of R-KCL22 cells with CX-4945 reduces the protein synthesis efficacy of about 50% as compared to the control.
To further assess the contribution of CK2 to chronic myeloid leukemia, the effect of CK2-inhibition on cell viability was examined. CX-4945 significantly reduces the cell viability and induces apoptosis in both LAMA84 and KCL22 cell lines, either sensitive or resistant to imatinib. However, CX-4945 concentrations required to induce apoptosis in imatinib-resistant cells are lower than those effective in sensitive cells, suggesting that resistant cells become more dependent on CK2 for their survival. Interestingly, CX-4945 added in combination with imatinib promotes a synergistic reduction of cell viability in imatinib-resistant CML cell variant, partially rescuing the response to imatinib. In resistant KCL22 cells, we also show that CK2-inhibition sensitizes leukemic cells to the anticancer compounds U0126, an inhibitor of MAPK pathway, and rapamycin, the specific inhibitor of mTORC1 complex. Interestingly, the ternary association of CX-4945 with imatinib and U0126 represents the best effective combination of drugs to reduce the viability of R-KCL22 cells.
Taken together, our results identify CK2 as a pivotal player in CML imatinib-resistance and suggest that CK2 inhibitors might represent promising drugs for combined strategies to overcome CML imatinib-resistance
HOW PROTEIN KINASE CK2 IS INVOLVED IN IMATINIB-RESISTANT CHRONIC MYELOID LEUKEMIA CELLS?
No full textJ-SUBUNIT OF THE EUKARYOTIC INITIATION FACTOR 3 IS A NEW SUBSTRATE OF PROTEIN KINASE CK2
No full textINVOLVEMENT OF THE PROTEIN KINASE CK2 AND ITS INTERACTOME IN IMATINIB SENSITIVE AND RESISTANT CHRONIC MYELOID LEUKEMIA CELLS
No full textA journey through the cytoskeleton with protein kinase CK2
No full textSubstrate pleiotropicity, a very acidic phosphorylation consensus sequence, and an apparent uncontrolled activity, are the main features of CK2, a Ser/Thr protein kinase that is required for a plethora of cell functions. Not surprisingly, CK2 appears to affect cytoskeletal structures and correlated functions such as cell shape, mechanical integrity, cell movement and division. This review outlines our current knowledge of how CK2 regulates cytoskeletal structures, and discusses involved pathways and molecular mechanisms
Aberrant signalling by protein kinase CK2 in imatinib-resistant chronic myeloid leukaemia cells. Biochemical evidence and therapeutic perspectives
No full textThe protein kinase ck2 associates with and phosphorylates the eukaryotic initiation factor 3
No full textProtein kinase CK2 potentiates translation efficiency by phosphorylating eIF3j at Ser127
No full textAbstractIn eukaryotic protein synthesis the translation initiation factor 3 (eIF3) is a key player in the recruitment and assembly of the translation initiation machinery. Mammalian eIF3 consists of 13 subunits, including the loosely associated eIF3j subunit that plays a stabilizing role in the eIF3 complex formation and interaction with the 40S ribosomal subunit. By means of both co-immunoprecipitation and mass spectrometry analyses we demonstrate that the protein kinase CK2 interacts with and phosphorylates eIF3j at Ser127. Inhibition of CK2 activity by CX-4945 or down-regulation of the expression of CK2 catalytic subunit by siRNA cause the dissociation of j-subunit from the eIF3 complex as judged from glycerol gradient sedimentation. This finding proves that CK2-phosphorylation of eIF3j is a prerequisite for its association with the eIF3 complex. Expression of Ser127Ala-eIF3j mutant impairs both the interaction of mutated j-subunit with the other eIF3 subunits and the overall protein synthesis. Taken together our data demonstrate that CK2-phosphorylation of eIF3j at Ser127 promotes the assembly of the eIF3 complex, a crucial step in the activation of the translation initiation machinery
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