Effects Of GSK3β And E2F1 Association On Neural Cell Differentiation

It is widely acknowledged that E2F1 and GSK3βare both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3βin cell differentiation has yet to be discovered.Here, we provide evidences that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3βwas increased at both the mRNA and protein levels, while E2F1 at these two levels was decreased. Distinguished from previous deduction that GSK3βkinase activity dependently inhibits E2F1 via cyclin D/Cdks complex and Rb protein, we found that both wild-type GSK3βand itskinase-activity-defective mutant GSK3βKM could inhibit the transcriptional activity of E2F1. And one of the special kinase inhibitor of GSK3βhad no effect on the inhibition caused by GSK3β. As an autoregulated factor, E2F1 has binding site in its own promoter and the changed protein level will affect its transcriptional activity and mRNA level. Furthermore, we discovered that GSK3βinhibited E2F1 by promoting its ubiquitination through physical interaction in a kinase-activity-independent manner.Moreover, we investigated the effect of the association of GSK3βand E2F1 on the neural cell differentiation. Firstly, the colocalization of GSK3βand E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. GSK3βand E2F1 mainly localized in the cytoplasm before NGF stimulation. However, most of GSK3βand E2F1 translocated into the nuclear and led to the stronger interaction in nuclei after the PC12 cells were stimulated by NGF. Secondly, at tissue level, GSK3βcolocalized and interacted with E2F1 in mouse hippocampus. And in mature neuron of mouse hippocampus region, GSK3βand E2F1 seemed absolutely localized in the cytoplasm discovered by immunofluorescence, which imply that it is necessary for GSK3βand E2F1 to stay in the cytoplasm to keep the terminally differentiated neurons in a quiet state without proliferation. Furthermore, GSK3βfacilitated neurite outgrowth by rescuing the promoter activities of Cdks inhibitors p21 and p15 from the inhibition caused by E2F1. However, a randomly selected GSK3βdeletion mutant GSK3βC1 that can not interact with and inhibit E2F1 had no effect on neurite outgrowth, which shows that the interaction between GSK3βand E2F1 is critical to GSK3β-induced neurite outgrowth.To summerize, our findings suggest that GSK3βcan promote the ubiquitation of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.