FIP200 YN together with YC and YN did not gener ate any significant signals above the background level, but we detected the GFP signal in the cells co transfected with both wild type COP1 YC and FIP200 YN after UV exposure. The restored signal was predom inantly in the cytoplasm with some in the nucleus too. We also detected interaction between http://www.selleckchem.com/products/epz-5676.html COP1 YN and COP1 YC as a control. In this case, the sig nal was both in the nucleus and in the cytoplasm. Quantification Inhibitors,Modulators,Libraries by flow cytometric analysis showed that the COP1 COP1 interaction was constitutive, whereas the COP1 FIP200 interaction was inducible in re sponse to UV treatment. Importantly, inter action was diminished when we used a COP1 mutant, which contains a serine to alanine substitution at the conserved ATM ATR phosphorylation site at the 389th codon, suggesting that UV mediated phosphorylation of COP1 is required for the efficient for mation of a complex between COP1 and FIP200.

Taken together, while COP1 stably forms a multimeric complex in the cell, its binding to FIP200 Inhibitors,Modulators,Libraries in the cytoplasm is enhanced by UV stimulation. Ectopic expression of COP1 reduced the expression of a certain form of FIP200 protein and exhibited tumorigenisity in response to UV To examine the effect of COP1 on FIP200, Inhibitors,Modulators,Libraries we ectopi cally expressed GFP tagged COP1 in NIH3T3 cells. Figure 4A shows that the level of ectopic expression was approximately the same as that of the endogenous protein. Interestingly, the faster migrating form of FIP200 was downregulated in NIH GFP COP1 cells compared to that of the control cells transfected with the control GFP vector, whereas the slower migrating form remained the same.

Because it is known that FIP200 forms a complex with ULK1, Atg13, and Atg101 to function downstream of mTOR to induce autophagy, we investigated their expression. Figure 4A shows that ectopic expres sion of COP1 affected differently, ULK1 was almost un affected, Atg13 was upregulated and Atg101 was slightly downregulated. We did not detect any direct binding of COP1 with Inhibitors,Modulators,Libraries ULK1, Atg13, and Atg101, suggesting that COP1 affects these components through interaction with FIP200. Interestingly, treatment of cells Cilengitide with an inhibitor to proteasome, MG132, reversed the effect of COP1 overexpression. When we investigated autophagy in these cells, however, autophagy was fully induced in response to amino acid starvation.

COP1 may affect other activities asso ciated with FIP200. Because interaction between COP1 and FIP200 was enhanced by UV stimulation and SA mutation dimin ished the interaction, we ectopically expressed wild type and SA mutant form of COP1 in NIH3T3 cells, and examined the effect of UV on FIP200. Figure 4B, left panel shows that both wild type and SA mutant COP1 selleck compound were successfully overexpressed. Immunoprecipitation of the HA tagged exogenous COP1 protein with the anti body to the HA epitope brought down the endogenous COP1 protein, indicating that COP1 formed a dimer or a larger multimeric complex, which was expect