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.
Highlights
The Bcl-2 family of proteins, including Bcl-xL [1], stands out among key regulators of apoptosis, executing crucial functions and controlling whether cells will live or die during development and cellular stress [2]
Studies were conducted in BJ cells expressing human influenza hemagglutinin (HA)-tagged Bcl-xL(wt), (S49A), (S49D), (S62A), (S62D) or dual (S49/62A) and (S49/62D) phosphorylation mutants
The cells were infected with lentiviruses expressing various cDNAs at early cell population doubling ranging from 25.42 to 27, as indicated on graph (x axis, time-point 0), and the kinetics of cell population doubling were monitored over a period of 4 months post-infection, until cell populations stop proliferating
Summary
The Bcl-2 family of proteins, including Bcl-xL [1], stands out among key regulators of apoptosis, executing crucial functions and controlling whether cells will live or die during development and cellular stress [2]. Studies have revealed that members of the Bcl-2 family, in addition to their central role in apoptosis, are involved in membrane dynamics and remodelling [3, 4], cell cycle regulation [5,6,7,8,9,10,11,12], DNA damage responses, repair and recombination [13,14,15,16,17], effects that are generally distinct from their function in apoptosis. A subset of the Bcl-xL protein pool undergoes dynamic phosphorylation at Ser during the S and G2 phases of the cell cycle, followed by a high phosphorylation peak during the early step of mitosis [11, 12]. Polo kinase 1 (PLK1) and mitogen-activated protein kinase 9 / c-jun N-terminal kinase 2 (MAPK9/JNK2) are major protein kinases associated with progressive phosphorylation of Bcl-xL(S62) during G2, where it accumulates in nuclear structures, including nucleoli and Cajal bodies [11]
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