AMBRA1 and BECLIN 1 interplay in the crosstalk between autophagy and cell proliferation

Abstract

Autophagy-promoting proteins and stimuli are often associated with inhibition of cell proliferation; in this context, we recently described a key role for the pro-autophagic protein AMBRA1. Indeed, AMBRA1, through its direct interaction with the protein phosphatase PP2A, tightly regulates the stability of the oncoprotein and pro-mitotic factor c-Myc. Moreover, the AMBRA1-mediated regulation of c-Myc affects both cell proliferation rate and tumorigenesis. Interestingly, AMBRA1/PP2A activity is under the control of the master regulator of autophagy and cell growth, the protein kinase mTOR. Besides the mechanistic details of this regulation pathway which we dissected previously, any possible interplay(s) between AMBRA1 and its interactor BECLIN 1 was not investigated in this scenario. Here we show that both AMBRA1 and BECLIN 1 affect c-Myc regulation, but through two different pathways. Nevertheless, these two pro-autophagic proteins are, together with PP2A, in the same macromolecular complex, whose functional significance of which will be addressed in future studies.

Keywords: EGFR degradation; PP2A; c-Myc; cancer; cell cycle.

Figure 1.

SEC analysis of AMBRA1, BECLIN 1 and PP2A-C complex. (A) Protein extracts from HEK293 cells were separated into different fractions and the presence of AMBRA1, BECLIN 1 and PP2A-C was assessed by Western Blot, using anti-AMBRA1, anti-BECLIN 1 and anti-PP2A-C antibodies. (B) Protein extracts, collected from HEK293 cells upon autophagy induction (EBSS treatment; starvation), were treated as in (A). Of note, variations of AMBRA1 and PP2A-C profiles can be observed upon autophagy induction [(B), fractions 23-27], with respect to the control condition (A). Although the meaning of these observations needs further investigation, upon starvation changes in the composition of the macromolecular complex can be hypothesized, most likely due ambra1, BECLIN 1 and PP2A-C interactors joining or leaving the complex.

Figure 2.

BECLIN 1 and AMBRA1 affect c-Myc phosphorylation in ERK1/2 dependent and independent manner, respectively. (A) H1975 lung cancer cells were treated with oligo-interference against BECLIN 1 (siBECLIN 1) or with aspecific oligonucleotides (siCTRL). Protein extracts were analyzed by SDS-PAGE and Western Blot using antibodies against BECLIN 1, P c-Myc^S62, c-Myc, P ERK1/2, ERK1/2 and ACTIN. (B) Densitometric analysis of the Western Blot shown in (A), performed using the ImageJ software; the average of the values from different experiments related to the control ratio was arbitrarily defined as 1.00. The band density ratio of phosphorylated proteins (P c-Myc^S62 and P ERK1/2) relative to total proteins (c-Myc and ERK1/2, respectively), are analyzed, with the control ratio arbitrarily defined as 1.00. n = 3 extracts prepared from independent experiments; data are presented as means ± s.d. and significance is *P ≤ 0.05 and **P ≤ 0.005. (C) Cells were treated as in (A) and, where indicated, were incubated with U0126 (5 μM) for 6 hours. (D) Densitometric analysis of the Western Blots shown in (C) and (E) was performed as described in (B). The band density ratio of P c-Myc^S62 relative to ACTIN is analyzed, with the control ratio arbitrarily defined as 1.00. n = 3 extracts prepared from independent experiments; data are presented as means ± s.d. and significance is *P ≤ 0.05.