br The regulation of DNA PK activity

The regulation of DNA-PK activity How DNA-PK is activated or deactivated in response to DNA damage and other cellular stress, is still relatively unknown [38]. Previous studies have shown that two serine/threonine protein phosphatase PP5 and PP6 are involved in DNA-PK activity modulation [39], [40]. The Wabl group has shown PP5 interacts with DNA-PKcs and dephosphorylates with surprising specificity at least two functional sites, and TPPU with either hypo- or hyperphosphorylation of DNA-PKcs at these sites show increased radiation sensitivity [40]. Nevertheless, mass spectrometry analysis revealed that DNA-PK interacts with the protein phosphatase-6 (PP6) SAPS subunit PP6R1. PP6 is a heterotrimeric enzyme that consists of a catalytic subunit, plus one of three PP6 SAPS regulatory subunits and one of three ankyrin repeat subunits. Endogenous PP6R1 co-immunoprecipitated DNA-PK, and IR enhanced the amount of complex and promoted its import into the nucleus. In addition, siRNA knockdown of either PP6R1 or PP6 significantly decreased IR activation of DNA-PK, suggesting that PP6 activates DNA-PK by association and dephosphorylation [39]. In addition, Taccioli group has shown that part of DNA-PK complex is associated with cytoplasm membrane, and raft-resident proteins may separately recruit DNA-PK components to the cytoplasm membrane. Furthermore, an irradiation-induced differential protein phosphorylation pattern is dependent upon DNA-PKcs in lipid rafts [65]. Recently, the epidermal growth factor (EGF) receptor (EGFR) was reported to associate with DNA-PK too, and ionizing radiation, but not stimulation with EGF, triggers EGFR import into the nucleus and enhances the DNA-PKcs binding with EGFR. During the EGFR nuclear translocation process, the proteins Ku70/80 and the protein phosphatase 1 are also observed to transport into the nucleus. As a consequence, DNA-PK kinase activity is increased. Blockade of EGFR import by the anti-EGFR monoclonal antibody C225 abolished EGFR import into the nucleus and radiation-induced DNA-PK activation, inhibited DNA repair, and increased radiosensitivity of treated cells [72], [102]. Moreover, Houslay group also found the GTP exchange factor, exchange protein activated by cAMP (EPAC) coupled to Rap2 in the nucleus, and cAMP regulated the nuclear/cytoplasmic trafficking of DNA-PK through the EPAC and PKA pathways [103]. Intersecting regulatory inputs for cAMP employ EPAC to transduce positive effects, namely the Rap2-dependent nuclear exit and activation of DNA-PK, whereas protein kinase A (PKA) provides the negative input by antagonizing these actions [103]. These observations suggest modulation on DNA-PK nuclear translocation influences the DNA-PK activity too. Furthermore, Survivin is a member of the inhibitor of apoptosis family. IR induces a rapid nuclear accumulation of survivin and subsequent phosphorylation of the protein in the nucleus. Co-immunoprecipitation and immunofluorescence co-localization analyses revealed an interaction among survivin, Ku70, γ-H2AX, MDC1, and DNA-PKcs. SiRNA knock down of survivin resulted in an impaired DNA double strand break repair. Furthermore, siRNA knock down of surviving hampered S2056 autophosphorylation of DNA-PKcs and significantly decreased DNA-PKcs kinase activity [104].