The Association of GSK3β with E2F1 Facilitates Nerve Growth Factor-induced Neural Cell Differentiation

Fangfang Zhou,Long Zhang,Aijun Wang,Bo Song,Kai Gong,Lihai Zhang,Min Hu,Xiufang Zhang,Nanming Zhao,Yandao Gong

doi:10.1074/jbc.m706136200

Fangfang Zhou, Long Zhang + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m706136200

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Abstract

It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination 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.

Highlights

Scription of downstream genes in Wnt signaling [2]
GSK3␤ Inhibits E2F1 antibody (E2F1) in the Process of Neural Cell Differentiation—Given that E2F1 and GSK3␤ have both been shown to relate with neural cell differentiation, their expressions were evaluated in the differentiation of PC12 cells induced by nerve growth factor (NGF)
We found that the association between endogenous E2F1 and GSK3␤ in PC12 cells was enhanced by NGF treatment, which implies that the interaction between E2F1 and GSK3␤ is involved in cell differentiation (Fig. 3E)

Summary

NGF was purchased from Xiamen

Mouse neuroblastoma N2a cells were activity of the Cdk-cyclin D1 complex This would decrease the cultured in 50% Dulbecco’s modified Eagle’s medium and 50%. Its targeted genes, including itself [3, 9, 17] These together PC12 cells were grown in RPMI 1640 medium supplemented with offered indirect evidence for the regulation of E2F1 by GSK3␤. We discovered that GSK3␤, fied Eagle’s medium supplemented with 1% fetal bovine serum and independent of its kinase activity, greatly suppressed E2F-lucif- NGF at a final concentration of 50 ng/ml. The nuclei were counterstained with 4Ј,6-diamidino-2-phenylindole (Sigma)

Frozen sections of mouse brain

RESULTS

DISCUSSION