Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jan 10;289(2):1119-27.
doi: 10.1074/jbc.M113.492777. Epub 2013 Nov 21.

Dephosphorylation of Barrier-To-Autointegration Factor by Protein Phosphatase 4 and Its Role in Cell Mitosis

Affiliations
Free PMC article

Dephosphorylation of Barrier-To-Autointegration Factor by Protein Phosphatase 4 and Its Role in Cell Mitosis

Xiaolei Zhuang et al. J Biol Chem. .
Free PMC article

Abstract

Barrier-to-autointegration factor (BAF or BANF1) is highly conserved in multicellular eukaryotes and was first identified for its role in retroviral DNA integration. Homozygous BAF mutants are lethal and depletion of BAF results in defects in chromatin segregation during mitosis and subsequent nuclear envelope assembly. BAF exists both in phosphorylated and unphosphorylated forms with phosphorylation sites Thr-2, Thr-3, and Ser-4, near the N terminus. Vaccinia-related kinase 1 is the major kinase responsible for phosphorylation of BAF. We have identified the major phosphatase responsible for dephosphorylation of Ser-4 to be protein phosphatase 4 catalytic subunit. By examining the cellular distribution of phosphorylated BAF (pBAF) and total BAF (tBAF) through the cell cycle, we found that pBAF is associated with the core region of telophase chromosomes. Depletion of BAF or perturbing its phosphorylation state results not only in nuclear envelope defects, including mislocalization of LEM domain proteins and extensive invaginations into the nuclear interior, but also impaired cell cycle progression. This phenotype is strikingly similar to that seen in cells from patients with progeroid syndrome resulting from a point mutation in BAF.

Keywords: BAF; BAF BANF1; BAF BANF1 PP4C; Cell Cycle; Chromosomes; Mitosis; Nuclear Membrane; Phosphorylation.

Figures

FIGURE 1.
FIGURE 1.
pBAF is dephosphorylated by the PP4C complex. A, specificity of the pBAF antibody. Left panel, HEK293 cells were transfected with BAF siRNA or VRK1 siRNA, and the resulting lysates were separated on a 4–12% SDS-PAGE gel. The gel was transferred to PDVF membrane and blotted with pBAF antibody, tBAF antibody, or VRK1 antibody. Right panel, empty vector, BAF WT, BAF-S4D, or BAF-S4A was overexpressed in HEK293 cells. The resulting lysates were immunoprecipitated by GFP antibody and immunoblotted with both of pBAF or tBAF antibody. B, whole HEK293 cell lysates were incubated with increasing amounts of the indicated phosphatase inhibitor at 30 °C for 0.5 h. The lysates were then immunoblotted with pBAF antibody to check the effect of the phosphatase inhibitor on pBAF dephosphorylation by endogenous phosphatases present in the extract, with tBAF antibody as the control. Lane 1 shows the lysate control without incubation. C, HEK293 cells were transfected with PP2A siRNA pool or PP4C siRNA pool. The resulting cell lysates were blotted with the indicated antibodies. D, individual PP4C siRNA 6, 7, and 8 were transfected into HEK293 cells, and the effect on BAF phosphorylation was evaluated by immunoblotting. E, whole HEK293 cell lysate was immunodepleted with PP4C antibody and IgG (used as control). The level of remaining PP4C was evaluated by Western blot. F, immunodepleted cell lysate was incubated at 30 °C for 0.5 h, and cell lysate depleted with IgG was used as control. pBAF and tBAF were detected by immunoblotting. G, PP4C wild type and its inactive mutants PP4C-H56Q and PP4C-R85A were transfected into HEK293 cells separately, immunopurified by FLAG beads, and eluted with 3× FLAG peptides. Eluted PP4C or mutants was incubated with pBAF for 30 min and immunoblotted with PP4C or pBAF antibody. H and I, HEK 293 cells were transfected with individual R1 siRNA 6, 7, and 8 (H) or R2 siRNA 6, 7, and 8 (I). The cell lysates were immunoblotted with the indicated antibody.
FIGURE 2.
FIGURE 2.
Localization of pBAF and tBAF through cell mitosis. A, HEK293 cells at interphase were immunostained with pBAF and tBAF antibodies. DAPI was used to stain the nucleus. B, schematic diagram of the core region and non-core region of early telophase chromosomes. The core region is indicated in red, and the non-core region is colored green. C, HEK293 cells at telophase were fixed and stained with the pBAF antibody and DAPI (DNA). Arrows indicate the signal at the core region. D, HEK293 cells during mitosis were stained with tBAF and DAPI. Arrows indicate the signal at the non-core region. Images were taken with a confocal microscope.
FIGURE 3.
FIGURE 3.
Effects of phosphorylation on pBAF and tBAF localization during telophase. A and B, HEK293 cells transfected with VRK1, PP4C, or R2 siRNA were immunostained with pBAF (A) or tBAF (B) antibody and DAPI at early telophase. C, fluorescence intensity of pBAF at the core region (B) was measured and graphed. Fluorescence intensity was normalized to the signals of the control (>20 separate cells were measured) and analyzed by t test (*, p < 0.05 versus control). D, GFP signals in phospho-mimic mutants overexpressing cells at early telophase were observed by confocal microscopy.
FIGURE 4.
FIGURE 4.
BAF phosphorylation affects the core localization of emerin and NE formation. A and B, HEK293 cells transfected with VRK1 siRNA or PP4C siRNA were fixed and co-stained with pBAF antibody, emerin antibody, and DAPI. Cells at early telophase were photographed (A). Fluorescence intensities of pBAF and emerin at the core region were measured and normalized to % control (B) (>20 separate cells were measured; *, p < 0.05 versus control). C and D, HEK293 cells transfected with the indicated siRNAs were fixed and stained with lamin B1 antibody and DAPI (C). NE invaginations are indicated by arrows. The percentage of cells with NE invaginations was graphed (D). E and F, HEK293 cells transfected with BAF-S4D, BAF-S4A, or BAF WT GFP fusion proteins were fixed and stained with lamin B1 antibody and DAPI (E). Arrows indicate transfected cells, and arrowheads indicate non-transfected cells. The percentage of cells with NE invaginations was graphed (F).
FIGURE 5.
FIGURE 5.
BAF phosphorylation perturbs normal cell cycle progression. A, synchronized HeLa cells at different cell cycle stages (M, G1, S, and G2 stage) were lysed and analyzed by immunoblotting with the indicated antibodies. Cyclin B1 and cyclin E, which peak at M phase and S phase, respectively, were immunoblotted to confirm cell cycle stages. B and C, HEK293 cells transfected with the indicated siRNA were labeled with 5-ethynyl-2′-deoxyuridine and stained with PI. Percentages of cells in G0/G1, S, and G2/M phase were analyzed by flow cytometry (B). S phase cells were further resolved into early, middle, and late stages using additional thresholds of PI staining intensity (C). Values are presented as the percentage of total population of triplicate measurements. Two separate experiments were repeated. D, HEK293 cells transfected with BAF WT or indicated BAF mutant constructs were stained with PI and analyzed cell cycle by flow cytometry. Values are presented as percentage of total cell population of triplicate measurements from two separate experiments. E, HeLa cells overexpressing BAF WT or indicated BAF mutants were synchronized by double thymidine blocks and released for 8 h. Cells were stained by PI and analyzed by flow cytometry. Results represent the percentage of the total population from triplicate samples.

Similar articles

See all similar articles

Cited by 17 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback