Protein Kinase CK2 Phosphorylates the High Mobility Group Domain Protein SSRP1, Inducing the Recognition of UV-damaged DNA

Abstract

The structure-specific recognition protein SSRP1 plays a role in transcription and replication in the chromatin context. Mediated by its C-terminal high mobility group (HMG) box domain, SSRP1 binds DNA non-sequence specifically but recognizes certain DNA structures. Using acetic acid urea polyacrylamide gel electrophoresis and mass spectrometry, we have examined the phosphorylation of maize SSRP1 by protein kinase CK2␣. The kinase phosphorylated several amino acid residues in the C-terminal part of the SSRP1 protein.Two phosphorylation sites were mapped in the very Cterminal region next to the HMG box domain, and about seven sites are localized within the acidic domain. Circular dichroism showed that the phosphorylation of the two C-terminal sites by CK2␣ resulted in a structural change in the region of HMG box domain, because the negative peak of the CD spectrum at 222 nm was decreased by ϳ10%. In parallel, the phosphorylation induced the recognition of UV-damaged DNA, whereas the non-phosphorylated protein does not discriminate between UV-damaged DNA and control DNA. The affinity of CK2␣-phosphorylated SSRP1 for the DNA correlates with the degree of UV-induced DNA damage. Moreover, maize SSRP1 can restore the increased UV-sensitivity of a yeast strain lacking the NHP6A/B HMG domain proteins to levels of the control strain. Collectively, these findings indicate a role for SSRP1 in the UV response of eukaryotic cells.In eukaryotes, the packaging of genomic DNA with histones and other proteins into chromatin affects DNA-dependent processes, including transcription, recombination, replication, and repair. In many cases, chromatin represses these processes by restricting the access of DNA binding regulatory factors to their DNA target sites. Numerous nuclear activities have been identified that can overcome the repressive effects of chromatin. Post-translational modification of histones and ATP-dependent, chromatin-remodeling complexes are involved in altering the chromatin structure so that the DNA becomes more accessible to the DNA-binding proteins required for transcription initiation (1, 2). DNA repair and other DNA-dependent processes occurring in the chromatin context are also coupled to the structural properties of chromatin.DNA is frequently damaged by a variety of agents that are of endogenous or environmental origin. For many organisms, the most important environmental mutagen is the ultraviolet component of sunlight. The DNA lesions induced by UV light (mainly dipyrimidine photoproducts) are both mutagenic and cytotoxic. Therefore, constant removal and replacement of damaged nucleotide residues by DNA repair mechanisms is required to maintain the DNA as a carrier of stable genetic information (3, 4). DNA damage and repair processes are influenced to a great extent by the packaging of the DNA into chromatin. The repair machinery is therefore dependent on the assistance of ATP-dependent, chromatin-remodeling complexes (5-7).In human cells, a protein complex termed FACT (facilitates chromatin transcription)...

CK2 Phosphorylates SSRP1 and Inhibits Its DNA-binding Activity

“…Also, we found that the assembly of this ternary complex appears to be induced after UV irradiation of cells (7). Consistent with our findings is that CK2 was also shown to phosphorylate maize SSRP1 at the HMG region in vitro, and this phosphorylation seemed to induce the recognition of UV irradiation-damaged DNA by SSRP1 in vitro (21). Although SSRP1 possesses a number of CK2 consensus sites, it is still unclear which amino acids CK2 specifically phosphorylates and whether phosphorylation of these potential residues regulates the ability of SSRP1 to bind to DNA.…”

Heterochromatin and ND10 are cell-cycle regulated and phosphorylation-dependent alternate nuclear sites of the transcription repressor Daxx and SWI/SNF protein ATRX

“…Alternatively, SSRP1 might itself be phosphorylated (Krohn et al, 2003) to interact with ATRX-containing complex at heterochromatin. These associations, observable in vivo, suggested the formation of a multiprotein complex at the pericentromeric regions during or immediately after DNA replication.…”

Regulation of ΔFosB Stability by Phosphorylation

“…CK2 is a ubiquitous and constitutively active Ser/Thr kinase that has Ͼ300 purported substrates identified thus far and is implicated in multiple biological processes, including cell death and survival (Litchfield, 2003;Unger et al, 2004), cellular stress responses (Yanagawa et al, 1997;Kato et al, 2003), and DNA repair and chromatin remodeling (Barz et al, 2003;Krohn et al, 2003). More than one-third of the putative substrates of CK2 are proteins involved in the regulation of gene expression (Meggio and Pinna, 2003).…”