CK2 Phosphorylates SSRP1 and Inhibits Its DNA-binding Activity

摘要

We have previously shown that CK2 associates with the human high-mobility group protein SSRP1 and that this association increases in response to UV irradiation. CK2 also phosphorylates SSRP1 in vitro. Here we extend this work by investigating CK2 regulation of SSRP1 function through phosphorylation. Phosphorylation of SSRP1 by CK2 inhibited the nonspecific DNA-binding activity of SSRP1 and FACT (facilitating chromatin-mediated transcription) complex in vitro. Using a serine/threonine-scanning Auto-spot peptide array coupled with a filter-based kinase assay with synthetic peptides as substrates, we identified serines 510, 657, and 688 as phosphorylation targets of CK2 in vitro. Mutagenesis of the three serines revealed that serine 510 was more important for the regulation of SSRP1 DNA-binding activity. Furthermore, we found that SSRP1 was phosphorylated in cells in response to UV (but not γ) irradiation. These results suggest that CK2 regulates the DNA-binding ability of SSRP1 and that this regulation may be responsive to specific cell stresses. We have previously shown that CK2 associates with the human high-mobility group protein SSRP1 and that this association increases in response to UV irradiation. CK2 also phosphorylates SSRP1 in vitro. Here we extend this work by investigating CK2 regulation of SSRP1 function through phosphorylation. Phosphorylation of SSRP1 by CK2 inhibited the nonspecific DNA-binding activity of SSRP1 and FACT (facilitating chromatin-mediated transcription) complex in vitro. Using a serine/threonine-scanning Auto-spot peptide array coupled with a filter-based kinase assay with synthetic peptides as substrates, we identified serines 510, 657, and 688 as phosphorylation targets of CK2 in vitro. Mutagenesis of the three serines revealed that serine 510 was more important for the regulation of SSRP1 DNA-binding activity. Furthermore, we found that SSRP1 was phosphorylated in cells in response to UV (but not γ) irradiation. These results suggest that CK2 regulates the DNA-binding ability of SSRP1 and that this regulation may be responsive to specific cell stresses. CK2 is a ubiquitous and evolutionarily conserved kinase. This enzyme is a heterotetrameric protein complex consisting of two regulatory β subunits (28 kDa) and two catalytic α (42 kDa) or α′ (38 kDa) subunits with the stoichiometry of either α2β2, α′2β2, or αα′β2 as the holoenzyme (1Gietz R.D. Graham K.C. Litchfield D.W. J. Biol. Chem. 1995; 270: 13017-13021Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Genetic studies in yeast (2Padmanabha R. Chen-Wu J.L. Hanna D.E. Glover C.V. Mol. Cell. Biol. 1990; 10: 4089-4099Crossref PubMed Scopus (306) Google Scholar) and in mice (3Buchou T. Vernet M. Blond O. Jensen H.H. Pointu H. Olsen B.B. Cochet C. Issinger O.G. Boldyreff B. Mol. Cell. Biol. 2003; 23: 908-915Crossref PubMed Scopus (224) Google Scholar) demonstrate that this enzyme is essential for viability and animal embryogenesis. Biochemical and functional analyses of this enzyme reveal that it can phosphorylate a broad spectrum of protein substrates in vitro and regulate a variety of cellular functions including transcription in the nucleus. Consistent with its regulatory role in the nucleus, CK2 associates with a number of chromatin and nuclear matrix proteins in the cell (4Tawfic S. Davis A.T. Faust R.A. Gapany M. Ahmed K. J. Cell. Biochem. 1997; 64: 499-504Crossref PubMed Scopus (20) Google Scholar, 5Guo C. Davis A.T. Ahmed K. J. Biol. Chem. 1998; 273: 13675-13680Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 6Guo C. Yu S. Davis A.T. Ahmed K. Cancer Res. 1999; 59: 1146-1151PubMed Google Scholar). One of the mammalian CK2-interacting nuclear proteins is the previously identified DNA-binding protein called SSRP1 (structure-specific recognition protein 1) (7Keller D.M. Lu H. J. Biol. Chem. 2002; 277: 50206-50213Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). SSRP1 (named T160 in mice