Pre treatment with U0126 in c83 2C cells abol ished MiTF quality control phosphorylation, as well as its subsequent degradation. A similar result was also observed in Malme 3 M melanoma cells pre treated with U0126. These data suggest that phosphorylation of MiTF by Erk1/2 was necessary for its degradation. It was previously reported that the c Kit signal trig gered dual phosphorylation of MiTF, one at serine 73 by Erk2 and the other on serine 409 by Erk1/2 down stream kinase p90 RSK 1. To examine whether UVC also exhibited a similar effect on MiTF through p90 RSK 1, we pre treated c83 2C cells with RSK 1 inhibitor SL0101 before UVC radiation, MiTF degradation was still observed, suggesting that p90 RSK 1 phos phorylation of MiTF was not a critical event under this condition, and Erk1/2 was the major kinase for UVC triggered MiTF phosphorylation and degradation.

Phosphorylation on serine 73 is responsible for proteasome mediated MiTF degradation To confirm that MiTF degradation is mediated by pro teasome pathway, c83 2C cells were treated Inhibitors,Modulators,Libraries with MG132, a proteasome inhibitor and then exposed to UVC. MiTF exhibited an unchanged Inhibitors,Modulators,Libraries expression under these conditions. Next we expressed MiTF WT and MiTF S73A in MiTF negative A375 melanoma cells, and examined their accumulation after UVC. As shown in Fig 3B, MiTF WT showed on western blot as a doublet band, MiTF S73A, on the other hand, exhibited a single band that corresponded to the faster moving band. MiTF S73A Inhibitors,Modulators,Libraries did not show any band shift nor degrada tion after UVC, while MiTF WT was phos phorylated and degraded.

To investigate whether poly ubiquitination is involved in MiTF regu lation after UVC radiation, NHMs were exposed to 3 mJ/cm2 of UVC and then collected 2 hours later Inhibitors,Modulators,Libraries for immunoprecipitation. As shown in Fig 3E, UVC dra matically enhanced poly ubiquitination of MiTF pro tein. Anti GFP antibody was used as a negative control for anti MiTF antibody. Taken together, these results Inhibitors,Modulators,Libraries suggest that Erk1/2 mediated MiTF phosphorylation on serine 73 is required for MiTF degradation after UVC. These results are consistent with previous observation that phosphorylation on serine 73 is essential for MiTF poly ubiquitination and degradation. Expression of MiTF WT led to a temporary G1 arrest and enhanced cell survival in A375 cells but expression of MiTF S73A did not Cells normally undergo cell cycle arrest after UVC expo sure to allow enough time for DNA damage repair.

To investigate the role of MiTF in UVC mediated DNA damage response and cell cycle control, A375 cells which carry a wild type p53 gene were transfected with QCXIP GFP, QCXIP MiTF WT or QCXIP MiTF S73A and then exposed to UVR. Cell cycle distribution was analyzed by fluores cence activated cell sorting at various time kinase inhibitor ARQ197 points after staining with Propidium Iodide. About 40% of cells were in G1 phase when un irradiated in all three groups.