Sci. Aging Knowl. Environ., 2 June 2004
Vol. 2004, Issue 22, p. pe24
[DOI: 10.1126/sageke.2004.22.pe24]

PERSPECTIVES

Breaking in a New Function for Casein Kinase 2

Julio C. Morales, and Phillip B. Carpenter

The authors are in the Department of Biochemistry and Molecular Biology at the University of Texas-Houston Medical School, Houston, TX 77225, USA. E-mail: Phillip.B.Carpenter{at}uth.tmc.edu (P.B.C.)

http://sageke.sciencemag.org/cgi/content/full/2004/22/pe24

Key Words: DNA damage • DNA repair • single-stranded break repair • XRCC1

Introduction

Our chromosomes are constantly under assault from various DNA-damaging agents, such as ionizing radiation, ultraviolet (UV) light, and oxygen free radicals. These agents, as well as others, introduce breaks, gaps, and modifications into our DNA and thereby create abnormal DNA duplex structures that might lead to phenotypes associated with aging as well as cancer (1) (see Sinclair Perspective). Some immune cells, such as those from the B lineages, undergo developmentally programmed DNA damage and recombination events as a mechanism to diversify immune function (2). In the absence of DNA repair mechanisms that respond to these various forms of DNA damage, cells will generate increased levels of mutations and a corresponding increase in genomic instability. Defects in DNA repair have been linked to progeroid syndromes, which are characterized by the appearance of premature aging (see Fry Review and Monnat Review).

The factors and pathways underlying how cells respond to DNA-damaging agents have been the subject of numerous studies in organisms ranging from bacteria to humans. In higher eukaryotes, the cellular response to DNA damage includes the activation of pathways that influence processes as diverse as cell cycle control, DNA repair, transcription, and apoptosis (1). Because DNA damage might alter chromatin structure in the vicinity of the break or perturbed structure, the problem of DNA damage sensing and signaling appears to also be linked to chromatin biology (3) (see Vaquero Review).

A variety of data support the emerging principle that the nature of the cellular response to DNA damage depends on the type of damage inflicted on the genetic material. The stage of the cell cycle at which DNA damage occurs, or is processed at, also influences the choice of repair pathway taken. This finding suggests that different forms of DNA damage might be recognized by various "DNA damage sensors" that activate networks of signaling pathways, leading ultimately, in many cases, to DNA repair. Thus, UV-induced thymine-thymine dimer formation might trigger a set of responses that is intrinsically different from the pathways activated in response to either single-stranded DNA breaks (SSBs) or double-stranded DNA breaks (DSBs). For example, during the cellular response to DSBs, the phosphatidylinositol-like kinases ATM, ATR, and DNA-PK (see the subsection "The Ku-DNA-dependent protein kinase complex" in the Fry Review) phosphorylate a variety of downstream effector molecules, many of which accumulate at irradiation-induced foci (sites within the nucleus where DNA damage and repair are believed to occur) (1, 4). Mutations in the genes encoding these factors result in embryonic lethality (ATR), strong cancer predisposition (ATM), or a severe combined immunodeficiency phenotype (DNA-PK). These "master regulators" do not appear to be required for the cellular response to SSBs, a process that also triggers the formation of irradiation-induced foci. However, if SSBs are not repaired during S phase, they can be converted into DSBs, a process that would then be expected to activate the ATM/ATR response.

Phosphylation of XRCC1 by Casein Kinase 2 (CK2)

Although many proteins are phosphorylated during the response to DNA damage, the consequences of these modifications remain largely unknown at the mechanistic level. With this in mind, it was exciting to read a report by Caldecott and colleagues in the 2 April 2004 issue of Cell (5) that uses elegant biochemistry and genetics to clearly demonstrate that phosphorylation of the SSB repair protein XRCC1 by CK2 directly facilitates its DNA repair activities. Originally discovered in the laboratory of Eugene Kennedy (6), the identification of CK2 was the first description of a kinase and, as such, the onset of the protein phosphorylation field. Thus, the discovery of CK2 as a kinase that influences DNA repair indicates a new and exciting role for the world's oldest known kinase. CK2 has been well studied over the years in a variety of systems and has been implicated in numerous processes, including stress responses, apoptosis, and transcription [reviewed in (5)].

XRCC1 is believed to play a role in two forms of DNA repair: base excision repair and SSB repair (see figure 1 in the Sinclair Perspective for a description of XRCC1's role in base excision repair). XRCC1 was previously shown to interact with and stimulate the polynucleotide kinase (PNK) (7) and also to interact with DNA ligase III and DNA polymerase B (7), enzymes that are also involved in these repair processes. Because XRCC1 interacts with multiple proteins, it is believed to function as a scaffolding protein during DNA repair.

XRCC1 contains two "BRCT" motifs: protein-protein interaction modules that were first described in the Breast Cancer Susceptibility Gene 1 (BRCA1) protein and subsequently found in other proteins that function in various aspects of the maintenance of genomic stability. The spacer region between the two BRCT motifs in XRCC1 contains a cluster of eight potential CK2 phosphorylation sites. Caldecott and colleagues showed that this domain is phosphorylated in vitro by CK2; their analysis of tryptic fragments by mass spectrometry revealed that several of these sites, including Thr488, Ser518, Thr523, and Thr519, are phosphorylated in vivo. The researchers found that CK2-dependent phosphorylation of XRCC1 greatly enhances the interaction between CK2 and PNK in vitro. In particular, the authors demonstrated that the 5'-DNA kinase activity of PNK was greater in the presence of phosphorylated (versus mock phosphorylated) XRCC1. In addition, they showed that this interaction is mediated by the forkhead domain of PNK, a region known to interact with phosphorylated substrates (Fig. 1).



View larger version (6K):
[in this window]
[in a new window]
 
Fig. 1. CK2 and XRCC1 in SSB repair. (A) Schematic diagram of XRCC1. XRCC1 has eight CK2 phosphorylation sites in the linker region that comprises residues 403 to 538; four of the sites (shown as OH groups) are indicated in this diagram. (B) Phosphorylation of XRCC1 by CK2 results in SSB repair (SSBR) via activation of PNK. FHA, forkhead domain; ATP, adenosine triphosphate; ADP, adenosine diphosphate.

 
To examine the importance of CK2 phosphorylation in living cells, Caldecott and colleagues used an XRCC1 mutant Chinese hamster ovary cell line, EM9. These cells were stably transfected with a variety of constructs expressing different mutant forms of XRCC1, in which the phosphorylation sites (serine and threonine residues) in the linker region were converted to alanine. Mutation of these sites was found to substantially reduce the interaction between XRCC1 and PNK. Moreover, the interaction between these two proteins was observed to increase if they were isolated from cells that had been treated with the DNA-damaging agent hydrogen peroxide (H2O2), which causes the formation of SSBs. The authors further investigated whether the conserved CK2 phosphorylation sites in XRCC1 are required for the assembly of XRCC1 into nuclear foci at DNA strand breaks. Wild-type XRCC1, which normally localizes in a diffuse pattern in the nucleus, is recruited to discrete nuclear foci (which represent sites of DNA breakage) after treatment with H2O2. Mutant forms of XRCC1 that lack CK2 phosphorylation sites, however, are not recruited to such foci. Taken together, the data at hand suggest a new role for CK2 in the assembly of DNA repair foci at sites of SSBs, a potentially difficult issue to evaluate directly, given the cell lethality that is probably associated with CK2 mutations (5).

To address this issue, the authors instead used a specific inhibitor of CK2, 4,5,6,7-tetrabromo-2-azabenzamidazole (TBB), to examine XRCC1 foci formation in the absence of CK2 function. They also studied a mutant form of CK2 (containing a Val66->Ala66 substitution) that is resistant to TBB. Cell lines expressing wild-type CK2 or the Val66->Ala66 derivative were cultured in the presence of TBB. The presence of this inhibitor suppressed the formation of H2O2-induced XRCC1 foci when the wild-type CK2 protein, but not the TBB-resistant form, was expressed, indicating that CK2 function is required for the formation of these foci.

To further evaluate the role of phosphorylation by CK2 in DNA repair, Caldecott and colleagues used the comet assay (a gel electrophoresis-based method of detecting fragmented DNA) to measure SSB repair in EM9 cells expressing various mutant forms of XRCC1. XRCC1 is involved in a rapid repair pathway operating in interphase, as well as a second pathway that is specific to S/G2 phase and may operate at the sites of replication forks. After treatment of the various EM9 cells with H2O2, it was determined that the CK2 phosphorylation sites in XRCC1 are required for the rapid repair process in interphase.

Conclusion

The results of Caldecott and colleagues show that CK2-dependent phosphorylation of XRCC1 in a region between its two BRCT motifs promotes the interaction of XRCC1 and PNK, enhances PNK activity, and facilitates the repair of SSBs. Thus, these authors have found that CK2 controls DNA repair by modulating XRCC1 function. CK2 exists in various isoforms (8); whether the DNA repair activity of each isoform is distinct remains unknown. As discussed by the authors, the ability of CK2 to control SSB repair is restricted to XRCC1-mediated repair, because in EM9 cells that lack functional XRCC1, SSB repair occurs similarly in the presence or absence of the CK2 inhibitor TBB. Taken together, these findings point out a new role for CK2 in the DNA damage response and are of substantial interest, especially because unrepaired SSBs are potentially deleterious to the cell.

Given that CK2 functions in SSB repair processes, how might it impinge on other processes that involve DNA repair, such as those that generate genetic diversity in the immune system? In mammalian B cells, during the process of somatic hypermutation (SHM), the variable regions of immunoglobulins are specifically mutated in order to generate antibodies with high affinities. Such mutations are introduced by the activation-induced deaminase, an enzyme that converts cytosine to uracil to trigger various DNA repair processes. SHM is believed to proceed through SSB intermediates; whether proteins such as CK2 are involved in this specific repair pathway of the immune system remains to be determined.

Although CK2-dependent phosphorylation of XRCC1 is clearly important for the repair of SSBs, one wonders if and how CK2 itself is controlled, an issue that has yet to be resolved. Moreover, because numerous proteins involved in various types of events that signal DNA damage contain the CK2 consensus phosphorylation sites, the question of whether CK2 participates in forms of DNA repair other than SSB repair comes to mind. The apparent constitutive phosphorylation of XRCC1 (at least at a subset of the eight CK2 sites) raises the question of how XRCC1 activity is regulated, because unregulated nuclear focus formation and DNA repair activity might wreak cellular havoc. Given that XRCC1 appears to be constitutively phosphorylated by CK2 at least at a subset of sites, other interactions and/or modifications might control XRCC1 activities in DNA repair. In any event, the discovery that CK2 phosphorylates and directs XRCC1 repair activity shows that even the most well-studied proteins and enzymes have new tricks to teach us.


June 2, 2004
  1. B. B. Zhou, S. J. Elledge, The DNA damage response: putting checkpoints in perspective. Nature 408, 433-439 (2000).[CrossRef][Medline]
  2. K. D. Mills, D. O. Ferguson, F. W. Alt, The role of DNA breaks in genomic instability and tumorigenesis. Immunol. Rev. 194, 77-95 (2003).[CrossRef][Medline]
  3. C. L. Peterson, J. Cote, Cellular machineries for chromosomal DNA repair. Genes Dev. 18, 602-616 (2004).[Free Full Text]
  4. R. T. Abraham, Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15, 2177-2196 (2001).[Free Full Text]
  5. J. I. Loizou, S. F. El-Khamisy, A. Zlatanou, D. J. Moore, D. W. Chan, J. Qin, S. Sarno, F. Meggio, L. A. Pinna, K. W. Caldecott, The protein kinase CK2 facilitates repair of chromosomal DNA single-strand breaks. Cell 117, 17-28 (2004).[CrossRef][Medline]
  6. E. P. Kennedy, Sailing to Byzantium. Annu. Rev. Biochem. 61, 1-28 (1992). [CrossRef][Medline]
  7. C. J. Whitehouse, R. M. Taylor, A. Thistlethwaite, H. Zhang, F. Karimi-Busheri, D. D. Lasko, M. Weinfeld, K. W. Caldecott, XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair. Cell 104, 107-117 (2001).[CrossRef][Medline]
  8. D. W. Litchfield, Protein kinase C2: structure, regulation and role in cellular decisions of life and death. Biochem. J. 369, 1-15 (2003).[CrossRef][Medline]
Citation: J. C. Morales, P. B. Carpenter, Breaking in a New Function for Casein Kinase 2. Sci. Aging Knowl. Environ. 2004 (22), pe24 (2004).




THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
N6-Furfuryladenine is protective in Huntingtons disease models by signaling huntingtin phosphorylation.
L. E. Bowie, T. Maiuri, M. Alpaugh, M. Gabriel, N. Arbez, D. Galleguillos, C. L. K. Hung, S. Patel, J. Xia, N. T. Hertz, et al. (2018)
PNAS 115, E7081-E7090
   Abstract »    Full Text »    PDF »




Science of Aging Knowledge Environment. ISSN 1539-6150