VRK2A is an A-type lamin-dependent nuclear envelope kinase that phosphorylates BAF

Abstract

The nuclear envelope (NE) is critical for numerous fundamental cellular functions, and mutations in several NE constituents can lead to a heterogeneous spectrum of diseases. We used proximity biotinylation to uncover new constituents of the inner nuclear membrane (INM) by comparative BioID analysis of lamin A, Sun2 and a minimal INM-targeting motif. These studies identify vaccinia-related kinase-2 (VRK2) as a candidate constituent of the INM. The transmembrane VRK2A isoform is retained at the NE by association with A-type lamins. Furthermore, VRK2A physically interacts with A-type, but not B-type, lamins. Finally, we show that VRK2 phosphorylates barrier to autointegration factor (BAF), a small and highly dynamic chromatin-binding protein, which has roles including NE reassembly, cell cycle, and chromatin organization in cells, and subtly alters its nuclear mobility. Together these findings support the value of using BioID to identify unrecognized constituents of distinct subcellular compartments refractory to biochemical isolation and reveal VRK2A as a transmembrane kinase in the NE that regulates BAF.

FIGURE 1:

Harnessing a NE-targeting fragment of Nup153 to generate INM-BioID for comparison with BioID2-LaA and BioID2-Sun2. (A) The NETC domain (aa 1–144) of Nup153 was fused to BioID2 to generate N144-BioID2 as a means to detect NE constituents. (B) N144-GFP3 (green) was transiently expressed in BJ cells and its localization at the NE observed by fluorescence colocalization with LaA/C. (C) IF analysis of BJ cells stably expressing BioID2-only, N144-BioID2, BioID2-LaA, and BioID2-Sun2. Fusion proteins were detected by antibodies against BioID2 (red). Streptavidin (green) was used to label biotinylated proteins. For all IF images, DNA was labeled with Hoechst dye 33342 (blue in DNA merge). Scale bar, 10 µm. (D) By IB analysis, BioID2 fusion proteins were detected with anti-BioID2, and proteins biotinylated by BioID2-only, N144-BioID2, BioID2-LaA, and BioID2-Sun2 were detected with streptavidin-HRP. (E) Venn diagram showing the number of proteins differentially detected by the three NE baits (see Materials and Methods for inclusion criteria). (F) Functional annotation clustering using DAVID analysis of unique or enriched (see Materials and Methods for inclusion criteria) proteins differentially identified by the three BioID2 baits.

FIGURE 2:

The relative abundance and number of TM or NE candidate proteins identified by BioID2 baits. Percentage of total relative peak intensity (abundance; A) and relative number of TM or NE candidate proteins (B) identified by N144-BioID2, BioID2-LaA, or BioID2-Sun2. Percentage of identified TM or NE candidate proteins by abundance (C, E, G) and percentage of TM or NE candidate proteins among identified candidates (D, F, H) for comparative analysis of N144-BioID2 and BioID2-LaA (C, D), N144-BioID2 and BioID2-Sun2 (E, F), and BioID2-LaA and BioID2-Sun2 (G, H).

FIGURE 3:

VRK2A is a novel constituent of the NE. (A) Schematic showing two predominant isoforms of VRK2—VRK2A and VRK2B—with TM and kinase domains labeled. (B) GFP-tagged VRK2A and VRK2B were transiently expressed in BJ cells and their localization monitored by IF. Anti-Nup153 (red) was used as the NE marker. DNA was labeled with Hoechst dye 33342 (blue). The experiment was repeated at least three times. Scale bar, 10 µm.

FIGURE 4:

A-type lamins retain and physically interact with VRK2A at the NE. (A) siRNA-mediated knockdown of Lmna was performed on C2C12 cells stably expressing GFP-VRK2A. Lmna knockdown was confirmed by anti-lamin A (red). Anti-Nup153 (not in merged) was used as an additional NE marker. The experiment was repeated twice. (B) Transient expression of GFP-VRK2A in WT Lmna and Lmna-gene deleted (KO) MEF cells. (C) Coexpression of GFP-VRK2A with A-type lamins (HA–lamin A; top) and B-type lamins (HA–lamin B1; bottom) in Lmna KO MEF cells. DNA was labeled with Hoechst dye 33342 (blue). The experiment was repeated three times. Scale bar, 10 µm. (D) Anti-GFP coIP from total lysates of transiently expressed GFP empty vector and GFP-VRK2A and -VRK2B in HeLa cells, followed by immunoblotting with anti–lamin A/C. (E) Anti-GFP coIP in parental Saos-2 and Saos-2 cells stably expressing GFP-VRK2A followed by immunoblotting with anti-LaA.

FIGURE 5:

VRK2 phosphorylates BAF in cells and subtly alters its mobility at the NE. (A) tdTomato-tagged empty vector and -VRK1 and -VRK2A were expressed transiently in HeLa cells and labeled with anti-pBAF (red). Red arrowheads depict cells overexpressing cytoplasmic VRK1 or NE/ER/cytoplasmic VRK2A; green arrowheads show cells with enriched pBAF labeling. (B) siRNA-mediated knockdown of VRK1 and VRK2 was performed in MDA-MB-231 cells and labeled for pBAF and tBAF (red) for IF analysis (B) or immunoblotted with VRK1, VRK2, pBAF, tBAF, and tubulin as a loading control (C). Band intensities for pBAF were quantified using ImageJ, normalized relative to tubulin, and expressed as ratios relative to control. For the IF images in A and B, DNA was labeled with Hoechst dye 33342 (blue). The experiments were repeated twice. Scale bar, 10 µm. (D) FLIP analysis was performed on GFP-BAF HeLa cells 48 h after VRK2 siRNA–mediated depletion; 44 control and VRK2-depleted cells from two independent FLIP experiments were analyzed. IF analysis (inset) shows a HeLa GFP-BAF cell before and after photobleaching. The red circle represents the bleached ROI, and the yellow rectangle denotes the ROI analyzed for the loss of GFP signal. VRK2 depletion is observed by IB analysis (inset). (E, F) FLIP analysis was performed on HeLa GFP-BAF cells stably expressing tdTomato-empty and -VRK2A WT (E) or -VRK2A K169E (F). For E and F, FLIP analysis was performed on 30 cells from three independent FLIP experiments. Data are presented as the normalized average intensity ± SEM. None of these FLIP studies reached clear statistical significance, but they did exhibit consistent trends for independent experiments.