CK2-mediated phosphorylation of Che-1/AATF is required for its pro-proliferative activity

Abstract

Background 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 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. Methods Site-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three-serine residue (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. Results Here, we confirm 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. Conclusions Che-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.

Che-1/miR-590-3p/TAZ axis sustains multiple myeloma disease.

“…Human Multiple myeloma (MM) cell lines Kms27, Kms18, RPMI8226 were cultured in Optimem (Thermo Fisher Scienti c) supplemented with 15% serum as already described 10 . Human HCT116, HeLa and BJ cell lines were cultured as already described 31,32 . EBV-immortalized lymphoblastoid cell line (LCL) was cultured in RPMI-1640 GlutaMAX supplemented with 15% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin 33 .…”