Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death

Abstract

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.

Identification of synthetic lethality of PLK1 inhibition and microtubule-destabilizing drugs

“…Post-translational modifications of antiapoptotic BCL-2 family proteins have been reported as essential events for mitotic cell death. 13,15,21,22 In line with our data, MCL-1 has been described to be phosphorylated, ubiquitinated and degraded during prolonged mitotic arrest. [13][14][15] Also, phospho-defective BCL-2 and BCL-X L mutants were shown to block mitotic cell death compared with wild-type proteins, and mitotic phosphorylation of BCL-X L is known to reduce its ability to antagonize proapoptotic BAX.…”

“…[13][14][15] Also, phospho-defective BCL-2 and BCL-X L mutants were shown to block mitotic cell death compared with wild-type proteins, and mitotic phosphorylation of BCL-X L is known to reduce its ability to antagonize proapoptotic BAX. [21][22][23] Consistently, we found that BI 2536/VCR-stimulated BAX/BAK activation is essential for apoptosis, as BAK knockdown or BCL-2 overexpression significantly reduce BI 2536/VCR-induced apoptosis.…”

The Apoptotic Mechanism of Action of the Sphingosine Kinase 1 Selective Inhibitor SKI-178 in Human Acute Myeloid Leukemia Cell Lines

“…Several lines of evidence indicate that extensive phosphorylation of Bcl-2 at Ser70 and other sites by CDK1 inactivates its antiapoptotic function and is a key feature of apoptotic cell death in response to agents that induce prolonged mitotic arrest (Haldar et al, 1998;Barboule et al, 2005;Eichhorn et al, 2013). However, Dai et al (2013) recently demonstrated that phosphorylation of Bcl-2 at Ser70 enhances the antiapoptotic function of Bcl-2 by promoting Bim and Bak binding.…”

“…Bcl-2 is known to undergo transient cell cycle-dependent phosphorylation at multiple sites, including Ser70, during mitosis (Scatena et al, 1998;Barboule et al, 2005). In contrast, enhanced and prolonged levels of Bcl-2 phosphorylation in mitosis are commonly associated with apoptotic cell death (Haldar et al, 1998;Eichhorn et al, 2013). Therefore, we next examined the effects of SKI-178 on cell cycle progression to determine whether SKI-178-induced apoptosis is associated with mitotic progression.…”

“…Although our data suggest a strong correlation between Bcl-2 phosphorylation at Ser70 and induction of apoptotic cell death, the functional role of this phosphorylation is not yet clear. Although many studies have implicated this Bcl-2 phosphorylation in making cells more susceptible to apoptosis (Haldar et al, 1998;Eichhorn et al, 2013), there are recent studies that suggest it actually enhances its cytoprotective effects (Dai et al, 2013;Zhou et al, 2014). Nonetheless, Bcl-2 is not the only CDK1 substrate that displays extensive/prolonged phosphorylation during prolonged mitotic arrest.…”