Characterization of vinblastine-induced Bcl-xL and Bcl-2 phosphorylation: evidence for a novel protein kinase and a coordinated phosphorylation/dephosphorylation cycle associated with apoptosis induction

Du, Lili; Lyle, Christopher S.; Chambers, Timothy C.

doi:10.1038/sj.onc.1208189

Cited by 75 publications

(101 citation statements)

References 29 publications

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“…Bcl-xL phosphorylation at Ser62 is well-documented and generally associated with microtubule poisoning [41][42][43][44][45][46][47][48][49]. Phospho-Bcl-xL(Ser62) functions have remained elusive until recently.…”

Section: Discussionmentioning

confidence: 99%

“…Bcl-xL also fulfills functions during the cell cycle [38][39][40]. Bcl-xL phosphorylation at Ser62, within the unstructured loop domain of the protein, has been detected most often in cells treated with microtubule poisons, including nocodazole, paclitaxel, vinblastine, vincristine, colchicine and pironetin [41][42][43][44][45][46][47][48][49], and coupled, more recently, with specific G2 and mitotic events (Wang et al, 2011; manuscripts submitted). Similarly, Bcl-xL phosphorylation at Thr47 and Thr115 has been documented in response to genotoxic stress induced by ionizing radiation [50].…”

Section: Introductionmentioning

confidence: 99%

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Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

Wang

Beauchemin

Bertrand

2011

Cellular Signalling

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Functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis more slowly after microtubule poisoning, than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be separable from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phosphoBcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL (Ser49) is found with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

Wang

Beauchemin

Bertrand

2011

Cellular Signalling

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“…[30][31][32] Here, we report that PINK1 is required to protect cells from CCCP-induced apoptosis, and that such protection strongly depends on its ability to interact with Bcl-xL and to phosphorylate its serine 62. In fact, we observed absence of protection by PINK1 in cells subjected to Bcl-xL silencing and, conversely, rescue from apoptosis by a Bcl-xL mutant that mimics the phosphorylation on the S62, confirming that Bcl-xL represents a downstream target of PINK1 kinase activity.…”

Section: Discussionmentioning

confidence: 99%

PINK1 protects against cell death induced by mitochondrial depolarization, by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage

et al. 2013

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Mutations in the PINK1 gene are a frequent cause of autosomal recessive Parkinson's disease (PD). PINK1 encodes a mitochondrial kinase with neuroprotective activity, implicated in maintaining mitochondrial homeostasis and function. In concurrence with Parkin, PINK1 regulates mitochondrial trafficking and degradation of damaged mitochondria through mitophagy. Moreover, PINK1 can activate autophagy by interacting with the pro-autophagic protein Beclin-1. Here, we report that, upon mitochondrial depolarization, PINK1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to Beclin-1. PINK1-Bcl-xL interaction does not interfere either with Beclin-1 release from Bcl-xL or the mitophagy pathway; rather it protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL. Our data provide a functional link between PINK1, Bcl-xL and apoptosis, suggesting a novel mechanism through which PINK1 regulates cell survival. This pathway could be relevant for the pathogenesis of PD as well as other diseases including cancer. Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease, with prevalence of 1% in the population older than 60 years. 1 Several biochemical abnormalities, including mitochondrial dysfunction, oxidative stress and misfolded protein damage, have been implicated in PD pathogenesis. 2 Although the majority of late-onset cases are sporadic, early-onset PD is frequently caused by mutations in genes with autosomal recessive inheritance, mainly Parkin (GeneID: 5071) and PINK1 (GeneID: 65018). 3 PINK1 encodes a 63 kDa mitochondrial protein kinase, which is processed by mitochondrial proteases to generate two smaller isoforms. [4][5][6][7] We and others have shown that PINK1 acts as a key neuroprotective protein, aimed at preventing mitochondrial dysfunction and apoptotic cell death in response to multiple stress conditions. [8][9][10] This pro-survival activity is exerted through several mechanisms, including phosphorylation of the mitochondrial proteins TRAP1 and Omi/HtrA2, and regulation of mitochondrial calcium buffering. [11][12][13][14] Increasing data now indicate that PINK1 acts upstream of Parkin in an evolutionary conserved pathway implicated in regulating mitochondrial biogenesis, trafficking and fusion/ fission events, to maintain mitochondrial network health. 15 In particular, upon mitochondrial depolarization, PINK1 processing is impaired, determining a marked accumulation of the full-length protein on the surface of dysfunctional mitochondria, where it recruits Parkin. This process results in the phosphorylation and/or ubiquitination of several mitochondrial substrates, leading to the selective quarantine of damaged mitochondria and their degradation through mitophagy. [16][17][18][19] In line with this, we reported that coexpression of mutant, but not wild-type (wt) PINK1, with mutant alpha-synuclein resulted in the formation of enlarged autophagosomes surrounding abnormal mitoch...

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“…However, studies implicating JNK in microtubule inhibitor-induced Bcl-xL/Bcl-2 phosphorylation (12,14,16) have been complicated by the fact that JNK also plays a key role in cell cycle progression. In a carefully designed study using synchronized cells examined over an extended time-course, we showed that when cells are treated with both a JNK inhibitor and a microtubule inhibitor, the JNK inhibitor slows the normal progression to mitotic arrest, and events such as Bcl-xL/Bcl-2 phosphorylation are delayed by many hours (17). Nonetheless, in the context of validated JNK inhibition, microtubule inhibitor-induced Bcl-xL/Bcl-2 phosphorylation still occurs to a normal extent (17).…”

mentioning

confidence: 99%

Identification of the Major Phosphorylation Site in Bcl-xL Induced by Microtubule Inhibitors and Analysis of Its Functional Significance

Upreti

Galitovskaya

Chu

et al. 2008

Journal of Biological Chemistry

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Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 3 Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 3 Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Coexpression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax.

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Characterization of vinblastine-induced Bcl-xL and Bcl-2 phosphorylation: evidence for a novel protein kinase and a coordinated phosphorylation/dephosphorylation cycle associated with apoptosis induction

Cited by 75 publications

References 29 publications

Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

PINK1 protects against cell death induced by mitochondrial depolarization, by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage

Identification of the Major Phosphorylation Site in Bcl-xL Induced by Microtubule Inhibitors and Analysis of Its Functional Significance

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