Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation during Mitosis Prevents Chromosome Instability and Aneuploidy in Normal Human Diploid Fibroblasts

Abstract

Bcl-xL proteins undergo dynamic phosphorylation/dephosphorylation on Ser49 and Ser62 residues during mitosis. The expression of Bcl-xL(S49A), (S62A) and dual (S49/62A) phosphorylation mutants in tumor cells lead to severe mitotic defects associated with multipolar spindle, chromosome lagging and bridging, and micro-, bi- and multi-nucleated cells. Because the above observations were made in tumor cells which already display genomic instability, we now address the question: will similar effects occur in normal human diploid cells? We studied normal human diploid BJ foreskin fibroblast cells expressing Bcl-xL (wild type), (S49A), (S49D), (S62A), (S62D) and the dual-site (S49/62A) and (S49/62D) mutants. Cells expressing S49 and/or S62 phosphorylation mutants showed reduced kinetics of cell population doubling. These effects on cell population doubling kinetics correlated with early outbreak of senescence with no impact on the cell death rate. Senescent cells displayed typical senescence-associated phenotypes including high-level of senescence-associated β-galactosidase activity, interleukin-6 (IL-6) secretion, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 activation as well as γH2A.X-associated nuclear chromatin foci. Fluorescence in situ hybridization analysis and Giemsa-banded karyotypes revealed that the expression of Bcl-xL phosphorylation mutants in normal diploid BJ cells provoked chromosome instability and aneuploidy. These findings suggest that dynamic Bcl-xL(S49) and (S62) phosphorylation/dephosphorylation cycles are important in the maintenance of chromosome integrity during mitosis in normal cells. They could impact future strategies aiming to develop and identify compounds that could target not only the anti-apoptotic domain of Bcl-xL protein, but also its mitotic domain for cancer therapy.

The long unstructured region of Bcl‐xl modulates its structural dynamics

“…Certainly phosphorylation is one of such possible perturbation relevant in the context of biology. Indeed, two residues S49 and S62 present on the loop and have already been reported as phosphorylation site but its mechanism of influence on the function of Bcl‐xl is yet to be revealed . Grossly, the Phosphorylation turns a neutral residue into a negatively charged one and it is also involved in the regulation of the function of protein through electrostatic perturbation including the allosteric routes as well .…”

“…Indeed, two residues S49 and S62 present on the loop and have already been reported as phosphorylation site but its mechanism of influence on the function of Bcl-xl is yet to be revealed. 15,25,26 Grossly, the Phosphorylation turns a neutral residue into a negatively charged one and it is also involved in the regulation of the function of protein through electrostatic perturbation including the allosteric routes as well. 46,47 As the effect of the UNSTR was found to be transmitted from H1, H2 Effect of phosphorylation on the structured region of protein Phosphorylation of S49 and S62 has altered the structure and dynamics of the binding pocket of protein.…”

“…However, in 2011 Raghav et al have tried to explore the structural changes on Bcl‐2 with modelled long unstructured region when it binds to the pro‐apoptotic proteins but the impact of the loop on the structure and dynamics of the helical region of protein remained unclear. Two phosphorylation sites S49 and S62 were reported in the large unstructured region to have some regulatory evidences but their structural impact is not known. Although the structure of Bcl‐xl with and without this large unstructured region is available in PDB, but those static structures are not sufficient to reveal the difference in their flexibility and intrinsic nature of the dynamics which directly impact the function, for example, binding of other proteins/ligands.…”

Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner

“…5A, Lower lists the corresponding ratios that are elevated only on a hard, plastic surface. Each of the proteins whose phosphorylation is up-regulated in NMIIA-deleted GBM cells on a soft surface is connected to the Ras-Raf-MEK-ERK pathway (31)(32)(33)(34)(35)(36). Western blots of these lysates demonstrate that when grown on a soft (0.5 kPa) substrate, NMIIA-deleted cells enhance ERK1/2 phosphorylation approximately fourfold compared with NMIIA-intact cells.…”

BCL-xL, a Mitochondrial Protein Involved in Successful Aging: From C. elegans to Human Centenarians

“…Phosphorylation of BCL-xL at Ser-62 and Ser-49, have been well characterized and can alter BCL-xL intracellular localization and its loop conformation [32], which regulates the molecular association with other proteins such as BH3-only proteins and p53 [23]. Interestingly, BCL-xL proteins undergo dynamic phosphorylation/dephosphorylation on Ser-49 and Ser-62 residues during mitosis are important in the maintenance of chromosome integrity in normal cells [33]. The function of BCL-xL is regulated by several kinases, including PLK3 [34], JNK [35], CDK2 [36], and MST1 [37].…”