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  • br Results br Active Rac induced Rac Bcl interaction is

    2019-10-03


    3. Results
    3.1. Active Rac1-induced Rac1/Bcl-2 interaction is implicated in S70pBcl-2 induction
    A previous report suggested the involvement of active Rac1 in the phosphorylation of Bcl-2 albeit in monkey kidney fibroblast-like Resiniferatoxin (COS-7) [25], however, the underlying mechanism(s) particularly from the standpoint of redox-modifying activity of Rac1 remains obscure. Similarly, although Rac1 has been shown to interact with Bcl-2 [9],
    (caption on next page)
    (a) Western Blot analysis showing S70pBcl-2, Bcl-2, Rac1 and β-actin following 48-h Rac1 knockdown with either a Rac1 specific siRNA sequence or a Rac1 specific siRNA smartpool (150 nM) in CEM leukemia cells stably overexpressing Bcl-2 (CEM/Bcl-2). n = 4. Bar chart showing fold change in densitometry of S70pBcl-2 normalized to Bcl-2. (b–c) Western Blot analysis showing S70pBcl-2, Bcl-2, Rac1 and β-actin following 24-h treatment of increasing dose of Rac1 inhibitor, NSC23766, in CEM/Bcl-2 and Jurkat cells. n = 3, n = 5 respectively. Bar charts showing fold change in densitometry of S70pBcl-2 normalized to Bcl-2. (d) Measurement of Rac1 activity following 24-h treatment of 50 μM NSC23766 in CEM/Bcl-2 cells. n = 3. (e) Western Blot analysis showing S70pBcl-2, Bcl-2, Myc-tagged Rac1 (N17) and β-actin in CEM/Bcl-2 cells transiently transfected with Myc-tagged dominant negative Rac1 mutant, N17, or empty vector, pIRES. n = 3. Bar chart showing fold change in densitometry of S70pBcl-2 normalized to Bcl-2. All bar charts displaying mean and SD. Paired T-test was used for experiments with 2 samples. One way-ANOVA and Tukey's multiple comparisons tests were used for experiments with more than 2 samples. * and ** indicate P-value < 0.05 and < 0.02 respectively.
    there is no evidence to demonstrate if an active GTP-bound Rac1 is crucial for its interaction with Bcl-2 as well as a prerequisite for the induction of S70pBcl-2. Intrigued by these mechanistic gaps, we pro-ceeded to investigate the possibility of a physical crosstalk between active Rac1 and Bcl-2 in S70pBcl-2 induction. Firstly, we confirmed the involvement of active Rac1 in promoting S70pBcl-2 in CEM and Jurkat leukemia as well as M14 melanoma cancer cells. We genetically knocked down RAC1 with a single sequence-specific or smart-pool siRNA in Bcl-2 overexpressing CEM cells (CEM/Bcl-2), or inhibited Rac1 activation with NSC23766 or transiently expressed the dominant negative mutant Rac1T17N (Rac1N17) in CEM/Bcl-2 and Jurkat cells. Conversely, constitutively active Rac1G12V (Rac1V12) was stably ex-pressed in M14 melanoma cells. Indeed, we observed that gene knock down of RAC1 or Rac1N17 expression as well as pharmacological in-hibition of Rac1 activity reduced S70pBcl-2 in CEM/Bcl-2 and Jurkat cells (Fig. 1a–c, 1e). Rac1 inhibition with NSC23766 was also accom-panied by reduced Rac1 activity (Fig. 1d, S1; positive control). On the other hand, stable overexpression of Rac1V12 showed enhanced S70pBcl-2 in M14 cells (Fig. S2). These data collectively implicate ac-tive Rac1 in sustaining S70pBcl-2 in cancer cells.
    Given that Rac1-induced S70pBcl-2 is implicated in cancer cells, we proceeded to investigate if active Rac1 is required for the physical in-teraction between Rac1 and Bcl-2. Indeed, co-IP analysis showed that inhibition of Rac1 activation by NSC23766 or transient transfection of Rac1N17 prevented the interaction between Rac1 and Bcl-2 in CEM/Bcl-2 cells. This is accompanied by a reduction in S70pBcl-2 (Fig. 2a, S3a). As S70pBcl-2 stabilizes the anti-apoptotic activity of Bcl-2, we observed that S70pBcl-2 reduction also resulted in a decrease in Bcl-2 seques-tration of the BH3-only pro-apoptotic protein NOXA (Figs. S3a–b). Reciprocally, co-IP analysis and PLA revealed interaction between Rac1 and Bcl-2 (Fig. 2b–d) as well as higher S70pBcl-2 (Fig. 2b) in con-stitutively active Rac1V12 M14 cells, compared to pIRES vector-trans-fected cells. Similarly, sustained S70pBcl-2 facilitates binding to NOXA (Fig. S3c). In addition, blank samples of PLA with only secondary an-tibodies or IgG controls for co-IP showed absence of non-specific signals (Fig. 2c–d, S3d). To ascertain that active Rac1-induced interaction is important for sustained S70pBcl-2, we proceeded to use two different Bcl-2 inhibitors, HA14-1 and BH3-mimetic ABT199 (venetoclax), to disrupt the interaction between Rac1 and Bcl-2 (Fig. 2e–f). Indeed, as