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

Abstract

It is widely acknowledged that E2F1 and GSK3␤ are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3␤ 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), GSK3␤ was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3␤ and its kinase-defective mutant GSK3␤ KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3␤ and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3␤ colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3␤ 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 GSK3␤ 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.GSK3␤, a multifunctional serine/threonine (Ser/Thr) kinase that was originally identified as a regulator of glycogen metabolism, is now known to be a key kinase in several important signaling pathways. The kinase activity of GSK3␤ is positively regulated by phosphorylation of its Tyr 216 and negatively regulated by N-terminal phosphorylation of its Ser 9 (1). GSK3␤ is involved in several signaling pathways important for early central nervous system patterning and neuronal differentiation. For example, GSK3␤ down-regulates ␤-catenin via phosphorylation through the forming of a multicomponent complex with adenomatous polyposis coli and Axin, and thus inhibits the transcription of downstream genes in Wnt signaling (2). In addition, GSK3␤ regulates cell cycle by phosphorylating cyclin D1 and p21 (3-5). Also, it activates MRP2, an actin-binding protein of the kelch-related protein family, and promotes neurite outgrowth in NGF 3 -stimulated PC12 cells. Treatment of PC12 cells with GSK3␤-interfering RNAs results in the inhibition of neurite outgrowth (6). Moreover, GSK3␤ promotes dendrite formation through the ILK-GSK3␤ pathway in sympathetic neurons (7), and it regulates the phosphorylation of CRMP-2, adenomatous polyposis coli, and MAP1B, which are separately related to the polymerization, stabilization, and dynamics of microtubules, principal cytoskeletal components of axons (8).The E2F family has at least 10 members, classified as E2Fs (E2F1 to -8) and DPs (DP1 and -2). Physiological E2F exists as E2F/DP heterodimers in mammalian cells, binding and regulating E2F-targeted genes (9, 10). The E2F family plays a crucial role in regulating cell cycle progression at the G 1 -S transition mediated by pRB, a retinoblastoma tumor suppressor protein. pRB is phosphorylated in a cell cycle-dependent manner by cyclin-dependent kinases ...

APP and APLP1 are degraded through autophagy in response to proteasome inhibition in neuronal cells

“…As previously described (Zhou et al, 2008), PC12 cells grown on collagen coated glass coverslips were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 3% BSA in PBS for 60 min. The cells were then incubated with primary antibodies diluted in TBST (20 mmol/L Tris-HCl, pH 7.6, 137 mmol/L NaCl, 0.1% Tween 20) for 3 h and washed twice with p PBS and incubated with fluorescein isothiocyanate (FITC) conjugated anti-mouse or antirabbit antibodies for an additional 40 min.…”

“…As previously described (Zhang et al, 2004(Zhang et al, , 2006Zhou et al, 2008;Zhang et al, 2011), cells were lysed with 1 mL of lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 2 mmol/L EDTA, 25 mmol/L NaF, 1% Triton X-100) supplemented with protease inhibitors (Sigma) for 30 min at 4°C. After centrifugation at 12,000 g for 15 min, the lysates were immunoprecipitated with specific antibody and protein ASepharose (Zymed Laboratories Inc.) for 3 h at 4°C.…”

“…Sequences for the forward primer and reverse primer are as follows: 5′-CAGATTAATGAGGTGATGCGTGAA-3′ (APLP1 forward), 5′-GTCGGCTTTAGGCAGGTTCTT-3′ (APLP1 reverse); 5′-GCTGGCTGAACCCCAGATT-3′ (APP forward), 5′-CCCACTTCCCATTCTGGACAT-3′ (APP reverse); and 5′-AGATCATTGACCTCGTGTTGGA-3′ (Tubulin forward), 5′-ACCAGTTCCCCCACCAAAG-3′ (Tubulin reverse). The reaction system and procession was employed as previously described (Zhou et al, 2008).…”

Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1

“…E2F1 can control cell proliferation and differentiation. Overexpression of E2F1 significantly inhibits neuronal differentiation [26] . We have found that Omi is increased and E2F1 is decreased in differentiated N2a cells ( Figure 4A).…”

“…The expression of E2F1 is irreversibly downregulated during the differentiation of C2C12 myocytes [25] . Additionally, the association of GSK3β with E2F1 facilitates nerve growth factor-induced PC12 cell differentiation, and overexpression of E2F1 inhibits neurite outgrowth [26] .…”