1 is involved in many biological responses such as tumorigenesis, DNA damage response, lipid metabolism, and gluconeogenesis by targeting different substrates for degradation, which include p53, c Jun, Ets1 2, TRB3, and TORC2. Particularly, in a DNA damage responsive pathway, COP1 functions downstream of ATM ATR kinases by direct phosphorylation, but the precise mechanism remains to be determined. Considering a wide range of COP1 action in various biological responses, components and pathways downstream of COP1 are not fully under stood yet. To better understand the COP1 signaling pathway, we searched for novel COP1 interacting proteins by yeast two hybrid screening and identified FIP200 as one such candidate.
FIP200 was first reported as a regulator of the retinoblastoma protein, identified as a tumor suppressor in human breast cancer, and recently rediscovered as a mammalian counterpart of Atg17 in the yeast Atg1 Atg13 Atg17 complex. The mamma lian ULK1 Atg13 FIP200 complex func tions downstream of mTOR, and, together with the Beclin 1 Vps34 kinase pathway and the Atg5 Atg12 and LC3 conjugation systems, plays a key role in the induc tion of autophagy, an intracellular lysosomal degradation system for cytoplasmic proteins and organelles. In this study, we investigated the interaction between COP1 and FIP200 by the yeast two hybrid assay, the GST pulldown assay, and the Split GFP assay. Proliferat ing mammalian cells expressed several different forms of FIP200 protein, and one of them was downregulated Brefeldin_A by the ectopic overexpression of COP1 protein, suggesting that COP1 modulates FIP200 associated biological activities in a certain occasion, which may contribute to the complexity of the COP1 associated function.
Results Identification of FIP200 as an interactor with COP1 To explore the novel signaling pathway mediated by COP1, we sought a candidate for interactors with COP1 by yeast two hybrid screening of the human K562 erythroleu kemia cDNA library. Out of 1. 6 �� 106 transformants, we chose 13 potential clones that repeatedly exhibited positive signals. These clones contained part of two independent cDNAs, one for Jun D and one for FIP200 RB1 indu cible Coiled Coil 1. The presence of the former cDNA was anticipated given that c Jun is a sub strate of COP1 and that JunD is highly homologous to c Jun, both of which belong to the same family of AP1 transcription factors.
The latter component, FIP200, also termed RB1CC1, was originally shown to control retino blastoma protein and functions as a tumor suppressor in human breast cancer. FIP200 was recently rediscov ered as a component of the mammalian ULK1 Atg13 FIP200 complex and plays an important role in the induction of autophagy. Therefore, we decided to investigate the COP1 FIP200 interaction and the role of COP1 in terms of UV response and induction of autophagy. A yeast two hybrid analysis using deletion mutants of COP1 indicated that the RING domain at the N terminus of COP1, but not the WD40