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  • br Discussion br A feedforward

    2019-10-03


    4. Discussion
    4.1. A feedforward loop involving Active Rac1, S70pBcl-2 and their interaction is critical for S70pBcl-2 maintenance and its anti-apoptotic function
    Our previous work has implicated the ‘pro-oxidant’ activity of active Rac1 or Bcl-2 in promoting cancer cell survival and/or chemo-re-sistance [5,8,9,16]. Interestingly, inhibiting Rac1 activity alleviated Bcl-2-induced increase in mitochondrial O2.- [8,9], thus suggesting a clear role for active Rac1 in the apoptosis inhibitory function of Bcl-2. In this ABT-888 (Veliparib) report we provide evidence that GTP-loaded Rac1 (active) ex-hibits higher affinity for Bcl-2 and that this physical proximity promotes 
    the sustained S70pBcl-2, which stabilizes its anti-apoptotic activity. Intriguingly, the observation that S70pBcl-2 further enhances the
    binding between Bcl-2 and active Rac1 argues in favor of a positive feedforward loop to sustain S70pBcl-2. The feedforward loop between Rac1 and S70pBcl-2 is corroborated by the inability of S70A to bind to Rac1, while S70E elicited avid interaction. A plausible explanation could be that the initial binding of active Rac1 to Bcl-2 is rapid and transient, which allows for the induction of ROS-dependent S70pBcl-2 to further stabilize the binding of Rac1 to Bcl-2. Alternatively, the binding of Rac1 to Bcl-2 could happen due to JNK-mediated en-dogenous S70pBcl-2, thereby first prompting the binding of active Rac1 to Bcl-2 and subsequently further enhancing S70pBcl-2 through active Rac1-induced ROS.
    The critical involvement of the BH3 domain and its juxtaposed non-structured loop region (contains the S70 residue) of Bcl-2 in the inter-action with active Rac1 are highlighted. The use of BH3-mimetic in-hibitors to disrupt Rac1/Bcl-2 interaction hints the possibility of a competitive binding between BH3-mimetic inhibitors and Rac1 at the BH3 domain of Bcl-2. Alternatively, the binding between BH3-mimetic inhibitors and Bcl-2 could change the conformation of Bcl-2, thereby disrupting the interaction between Rac1 and Bcl-2 at other domains; potentially the juxtaposed non-structured loop region. Nevertheless, our data provide support to the latter hypothesis, whereby a drop in S70pBcl-2 is accompanied by both decreases in Rac1 or NOXA inter-action with Bcl-2. Reciprocally, Bcl-2 concurrently binds to both Rac1 and NOXA upon active Rac1-induced S70pBcl-2 in Rac1V12 cells. Furthermore, Deng et al. demonstrated that maximal association of Bcl-2 to Bax requires S70pbcl-2 [26], which is correspondingly important
    (caption on next page)
    Fig. 5. S70 phosphorylation of Bcl-2 further secures interaction between Rac1 and Bcl-2.
    (a) Western blot analysis showing the immunoprecipitation of Bcl-2 and immunoblotting of Rac1 following 24-h treatment of 10 mM Tiron in M14 Melanoma cells stably expressing the Myc-tagged constitutively active Rac1 mutant, V12. n = 3. (b) Western blot analysis showing S70pBcl-2, Bcl-2 and β-actin following 6-h treatment of increasing doses of JNK inhibitor, SP600125, in M14 Melanoma cells transiently transfected with wild type Bcl-2. OE: Overexpression. n = 3. (c) Western blot analysis showing the immunoprecipitation of Bcl-2 and immunoblotting of Rac1 following 6-h treatment of increasing doses of JNK inhibitor, SP600125, in M14 Melanoma cells transiently transfected with wild type Bcl-2. n = 3. (d–e) Western blot analysis showing the immunoprecipitation of Bcl-2 and immunoblotting of Rac1 in M14 Melanoma cells transiently transfected with 2 μg empty vector, pcDNA3.1, wild type (WT) Bcl-2, phosphomimetic S70E mutant Bcl-2 and non-phosphorylatable S70A mutant Bcl-2 for 48 h and their respective immunoblotted inputs of S70pBcl-2, Bcl-2, Rac1 and β-actin. n = 3. (f) PLA showing red dot appearances (white arrow) upon Bcl-2 and Rac1 interaction of CEM cells transiently transfected with 2 μg pcDNA3.1, WT Bcl-2, S70A or S70E plasmids for 48 h. Red dots were calculated from 30 cells from randomly selected images. Blank images indicate only secondary antibodies used to check for non-specific signal. n = 3.