Abstract
A number of cancers are characterized by elevated expression of CK2 (formerly casein kinase II), which has been implicated as a key component in cell proliferation and transformation. Two lines of evidence, (a) deregulated expression of CK2 and (b) CK2beta ubiquitination and degradation of these in a proteasome-dependent manner prompted further investigation of the regulation of CK2beta protein stability. We demonstrate that mutating six surface-exposed lysine residues to arginine (6KR) to interfere with ubiquitin attachment can stabilize CK2beta. Examination of 6KR expression in cells revealed increased stability over time and increased its steady-state expression level compared with CK2beta. In cells, 6KR was no longer sensitive to proteasome inhibition but maintained an elevated expression level. In our studies, 6KR functioned as a normal CK2 regulatory subunit, because it participated in CK2beta dimerization, associated with catalytic subunits, was autophosphorylated, and formed active, stable CK2 tetramers. The physiological role of CK2beta stabilization was investigated in cell proliferation assays, which showed a significant decrease in proliferation in cells expressing 6KR compared with CK2beta. Overall, our results indicate that a stabilized form of CK2beta can be used to inhibit cell proliferation.
Highlights
Fundamental cellular processes such as proliferation and survival involve regulation by CK2,3 a serine/threonine protein kinase that is ubiquitously expressed in eukaryotic cells [1]
Studies investigating CK2 tetramer assembly determined that a dimer of CK2 subunits forms the core of the enzyme, and the catalytic subunits subsequently bind to the CK2 core [21,22,23,24,25,26]
CK2 is normally expressed at a higher level than the catalytic subunits of CK2, allowing some CK2 to be incorporated into CK2 tetramers and stabilized, whereas the excess CK2 is rapidly degraded with a half-life of less than 1 h [32]
Summary
Plasmid Constructs—The previously described [24] HA-CK2 construct was inserted into the HindIII/XhoI sites of the pcDNA3.1(ϩ) plasmid (Invitrogen). Immunoprecipitations and Binding Assays—Immunoprecipitations and binding assays were performed by transiently transfecting cells as indicated and preparing cell lysates as described above. Immunoprecipitations were preformed on equivalent amounts of total protein using protein-A-Sepharose and anti-HA(12CA5), anti-CK2, anti-CK2␣, or anti-CK2␣Ј antibodies as indicated and incubated for 1 h at 4 °C with rotation. For binding assays cell lysates with equivalent amounts of total protein were incubated with 100 l of nickel-Sepharose bead slurry (prepared according to the manufacturer’s instructions) for 1 h at room temperature with rotation to isolate histidine-tagged CK2 proteins. Proteins were eluted by incubating the beads with 35 l of 2ϫ Laemmli sample buffer for 5 min. Lysates were prepared in 500 l of cell lysis buffer, and 150 g of total protein was used for each immunoprecipitation. To visualize labeled proteins, eluted samples were run on an SDS-PAGE gel, fixed in 50% methanol, 10% glacial acetic acid, soaked in Enhance solution (PerkinElmer Life Sciences), soaked in cold ddH2O, dried and exposed to autoradiography film for 20 h or more
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