Exploring the role of Cullin7/Ambra1 interaction in the regulation of cell proliferation

Recently, many evidences of a crosstalk between proliferation and autophagy have been reported, but the molecular mechanism behind this crosstalk has not been fully elucidated yet. AMBRA1 has been recently identified as an important regulator of proliferation by interacting and regulating the c-Myc activity. Cul7 is found to be mutated in a growth retardation syndrome (called 3M syndrome). Previously in our lab it was found, the interaction between AMBRA1 and CUL7 by proteomic analysis. Here we report a functional analysis of CUL7-AMBRA1 interaction in order to better understand the role of these proteins in cell proliferation. We found that AMBRA1 physically interacts with CUL7 at endogenous level and we mapped their sites of interaction. We also demonstrated that they are not involved in the regulation of their reciprocal expression and of their previously described pathways. Moreover, we showed the regulation of proliferation by CUL7 is associated to its ability that to positively regulate the stability of βTrCP, a substrate receptor of Cullin 1, suggesting a functional crosstalk between these two Cullins. Importantly, we found that the downregulation of AMBRA1 is able to prevent the degradation of βTrCP observed in CUL7-silenced cells. Associated to this observation, we found that AMBRA1 rescue of the proliferation arrest caused by CUL7 downregulated cells, indicating that these proteins contribute to the regulation of a common proliferative pathway. Unexpectedly, analysing the levels of known βTrCP substrates, we also found a decrease in the protein levels of some of them upon CUL7 downregulation, including the cell cycle regulator CDC25A. This result is consistent with a hyperactivation of the CUL1-βTrCP complex caused by an impaired deneddylation of this E3 ligase, which leads to excessive substrate degradation but also to autoubiquitination and degradation of βTrCP. Indeed, we found that a neddylation inhibitor is able to reduce the degradation of βTrCP complex upon CUL7 downregulation. Overall, our results demonstrated that AMBRA1 plays a role in the crosstalk between CUL7 and CUL1-βTrCP complex, which is possibly mediated by regulating the activity of the COP9 signalosome. Based on these evidences, we proposed a novel molecular mechanism by which these proteins regulate proliferation, which could contribute to the growth alteration observed in the 3M syndrome.