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  • br Other factors regulating DNA PKcs Recent


    Other factors regulating DNA-PKcs Recent work in the field has also focused on factors that regulate NHEJ and NHEJ factors, in particular proteins that modulate DNA-PKcs. In this section we will highlight a number of factors which have been found to regulate DNA-PKcs. DNA-PKcs phosphorylation at the Thr2609 cluster has also been implicated in DNA-PKcs association with the epidermal growth factor receptor (EGFR). EGFR is one of the key determinants of the radioresponse in cancer radiotherapy as elevation of EGFR frequently correlates with radioresistance in cancer Z-Ligustilide [92]. The underlying mechanism was revealed by the findings that EGFR and the downstream PI3K-Akt signaling pathway modulate DNA-PKcs activation and radiosensitivity [93], [94]. It was found that IR induces nuclear translocation of wild type EGFR but not mutants carrying oncogenic mutations at the tyrosine kinase domain, and that wild type EGFR binds to DNA-PKcs and promotes DSB repair [94]. A subsequent study further revealed that ATM-mediated DNA-PKcs Thr2609 phosphorylation is a critical prerequisite for EGFR function in radioresistance. Alanine substitution at Thr2609, but not at Ser2056, blocks the interaction between DNA-PKcs and EGFR and EGFR-mediated radioresistance [95]. Further, ATM deficiency or ATM kinase inhibition attenuates the EGFR dependent radioresponse. Akt kinase signaling has also been implicated to regulate DNA-PKcs activation and DSB repair [68], [93], [96]. Specific findings include nuclear Akt activation (as monitored by Ser473 phosphorylation) promotes its direct interaction with DNA-PKcs through the C-terminal domain of Akt. As a result, nuclear Akt activation facilitates DSB accumulation of DNA-PKcs and DSB repair and Akt kinase inhibition radiosensitizes DNA-PKcs and Ku80 proficient cells [68]. In addition, it was found that IR-mediated Akt activation requires erbB receptors and DNA-PKcs, whereas EGF-mediated Akt activation is independent of erbB receptors and DNA-PKcs [96]. It is seemingly that there are both similarity and distinction of IR and EGF signaling in the modulation of EGFR and Akt activities for DSB repair. Further investigations are needed to clarify the coordination of EGFR and Akt kinases in DNA-PKcs dependent DSB repair. The knowledge generated will be beneficial for the design and implementation of anti-EGFR, Akt, and DNA-PKcs strategies in cancer radiotherapy implication. Several lines of recent evidence also indicate that DNA-PKcs activation is affected by chromatin status and modulators such as β isoform of heterochromatin protein 1 (HP1). HP1 is known to bind to histone H3 trimethyl Lys9 (H3K9me3) for constitutive heterochromatin [97], [98], which affects many DNA activities including transcription, replication, and DSB repair [99]. In attempt to identify additional protein lysine methylations, a proteomic screening using the chromodomain (known to bind H3K9me3) of HP1β as the bait was developed and DNA-PKcs was identified as one of interacting proteins of HP1β [100]. Mass spectrometry analysis identified that DNA-PKcs methylation occurred at three lysine residues (K1150, K2746, K3248) upon DNA damage and that methylation is necessary to mediate DNA-PKcs interaction with HP1β [100]. K-to-R (non-methyl) mutation of DNA-PKcs or depletion of HP1β impairs DSB repair and radioresistance, indicating that DNA-PKcs methylation and/or its association with HP1β affects DSB repair. Although the detailed mechanism remains to be clarified, DNA-PKcs methylation is likely required for HP1β recruitment to DSB sites and/or facilitates the DSB repair process. In agreement, it was reported that HP1β is phosphorylated by Casein Kinase II (CK2) at Thr51 upon DSBs, and that HP1β phosphorylation decreases its affinity to H3K9me at heterochromatin and promotes HP1β mobilization to DSB sites [101]. CK2 kinase is a versatile kinase involved in many cellular regulations including chromatin modulation in DNA damage sensing and repair [102], [103]. Two recent studies further suggested that CK2 is required for DNA-PKcs activation upon DSBs as CK2 depletion disrupts DNA-PK complex formation at DSBs, attenuates IR-induced DNA-PKcs activation, and facilitates IR-induced cell killing [104], [105]. Taken together, these results strongly suggest that CK2 and HP1β dependent chromatin remodeling plays a prominent role in DNA-PKcs activation and DSB repair. It is likely that CK2-dependent HP1β phosphorylation and DSB translocation induces chromatin relaxation for the full activation of DNA-PKcs, although the detailed kinetic and sequential order of these events remains to be clarified. It is interesting to note that there is a direct interaction between Ku70 subunit and HP1γ isoform. Such interaction promotes HP1γ Ser83 phosphorylation which overlaps exclusively with relaxed euchromatin and active RNA PolII transcription [106]. It is plausible that Ku70 and HP1γ association might also participate in DNA-PKcs activation and DSB repair.