N52 monodeamidated Bcl-xL shows impaired oncogenic propertiesin vivoandin vitro

Abstract

Bcl-xL is a member of the Bcl-2 family, playing a critical role in the survival of tumor cells. Here, we show that Bcl-xL oncogenic function can be uncoupled from its anti-apoptotic activity when it is regulated by the post-translational deamidation of its Asn52.Bcl-xL activity can be regulated by post-translational modifications: deamidation of Asn52 and 66 into Asp residues was reported to occur exclusively in response to DNA damage, and to cripple its anti-apoptotic activity. Our work reports for the first time the spontaneous occurrence of monodeamidated Asp52Bcl-xL in control conditions, in vivo and in vitro. In the normal and cancer cell lines tested, no less than 30% and up to 56% of Bcl-xL was singly deamidated on Asn52. Functional analyses revealed that singly deamidated Bcl-xL retains anti-apoptotic functions, and exhibits enhanced autophagic activity while harboring impaired clonogenic and tumorigenic properties compared to native Bcl-xL. Additionally, Asp52Bcl-xL remains phosphorylatable, and thus is still an eligible target of anti-neoplasic agents. Altogether our results complement the existing data on Bcl-xL deamidation: they challenge the common acceptance that Asn52 and Asn66 are equally eligible for deamidation, and provide a valuable improvement of our knowledge on the regulation of Bcl-xLoncogenic functions by deamidation.

Improved Electrophoretic Separation to Assist the Monitoring of Bcl-xL Post-Translational Modifications

“…In addition, one single Asn→Ala substitution generated a shift in the migration distance (compare lanes “AD” and “N66D”, and compare lanes “N52D” and “DA” on gels c, d, and e in Figure 1). The fine separation of proteins granted by these new gel compositions can indeed be used to accurately pinpoint that the modified form (labeled * in WT lanes of Figure 1) above unmodified Bcl-x L co-migrates exactly with the single mutant Bcl-x L N52D, thus confirming what we previously reported with long tris–glycine PAGE [7]. However, these gels still show a difference in the migration distances of Bcl-x L AD and Bcl-x L DA (as 25 cm-long tris–glycine SDS-PAGE already did) even though both proteins have the exact same molecular weight.…”

“…Finally, in a previous study asking whether Bcl-x L could accumulate both deamidation and phosphorylation, we had provided compelling evidence that monodeamidated Bcl-x L could be phosphorylated in response to microtubule inhibition. However, we and others had failed to determine whether doubly-deamidated Bcl-x L could be phosphorylated because of insufficient electrophoretic resolution provided by tris–glycine PAGE [7,8]. We show here that taurine–glycine gels proved efficient to overcome this limitation and found that doubly-deamidated Bcl-x L is eligible for regulation by phosphorylation.…”

“…Bcl-x L undergoes deamidation on Asn52 and Asn66, and mutagenesis on these two positions can be used either to recapitulate deamidation by Asn→Asp conversion or to prevent it by Asn→Ala conversion. The migration distances of these mutants, listed in Figure 1, have previously been compared by tris–glycine PAGE to determine why Bcl-x L migrates as a doublet in all the cell extracts we analyzed [7], and to identify the PTM responsible for the slower migration of a fraction of cellular Bcl-x L (labeled * in WT lanes of gels in Figure 1) [7]. Long 25 cm tris–glycine SDS-PAGE was needed (Figure 1a) to obtain a resolution that 6 cm mini-gels did not allow (Figure 1b).…”

“…As a result, for more than ten years, Bcl-x L was essentially described as a doubly deamidated protein on N52 and N66. Only recently did the use of 25 cm-long SDS-PAGE allow us to emphasize the existence of N52 monodeamidated Bcl-x L , thus pinpointing that N52 and N66 were not equally susceptible to deamidation and that a sequential mechanism was actually at play, always involving N52 first [7].…”

Renal Mesangial Cells Isolated from Sphingosine Kinase 2 Transgenic Mice Show Reduced Proliferation and are More Sensitive to Stress-Induced Apoptosis

“…Bcl-XL appears as a double band because of deamidation of two amino acids Asn 52 and Asn 65 into Asp. Thus, the most upper band represents the double deamidated form, which is the only form that is functionally characterized [25]. In mesangial from wildtype mice (Wt), hSK2-ctrl mice (expressing no Cre recombinase, -Cre), and hSK2-tg mice (hSK2tg, expressing Cre recombinase, +Cre), were separated by SDS-PAGE and subjected to Western blot analysis using antibodies against human SK-2 (hSK2, upper panels), mSK-2 (middle panels) and GAPDH (lower panels).…”