Abstract
BackgroundChe-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in multiple myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding.MethodsSite-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three serine residues (Ser316, Ser320 and Ser321) in 308–325 aa region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser316, Ser320 and Ser321 were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by in vitro kinase, 2D gel electrophoresis assays and mass spectrometry analysis.ResultsHere, we confirmed that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser316, Ser320 and Ser321 and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation.ConclusionsChe-1 phosphorylation at Ser316, Ser320 and Ser321 by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability.
Highlights
Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress
We analysed the formation of heterochromatin foci by immunofluorescence in HCT116 cells, observing that Che-1 depleted cells exhibited a strong increase of Hoechst-positive heterochromatin foci (Fig. 1B)
We performed western blot (WB) analyses in several cell lines depleted or not for Che-1 expression, observing that the loss of Che-1 expression produced a strong reduction in both H3 and H4 histone acetylation (Fig. 1E and Supplementary Figure 1D)
Summary
Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. Che-1/AATF (Che-1) is a highly conserved nuclear protein over-expressed in several cancerous tissues, such as prostate, lung, colon, lymphomas as well as in multiple. Casein kinase II (CK2) is a ubiquitous highly pleiotropic serine/threonine protein kinase present in cells in a tetrameric form consisting of two catalytic subunits (CK2α and its isoform CK2α’) and two regulatory β subunits [19]. It is expressed in all tissues of eukaryotic organisms and localized in different cell compartments [20]. Several studies have demonstrated that this kinase can interact with and regulate many factors involved in chromatin remodelling [25, 26]
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