Barrier-to-Autointegration Factor Phosphorylation on Ser-4 Regulates Emerin Binding to Lamin A In Vitro and Emerin Localization In Vivo

Abstract

Barrier-to-autointegration factor (BAF) is a conserved 10-kDa chromatin protein essential in proliferating cells. BAF dimers bind double-stranded DNA, histone H3, histone H1.1, lamin A, and transcription regulators, plus emerin and other LEM-domain nuclear proteins. Two-dimensional gel analysis showed that endogenous human and Xenopus BAF are posttranslationally modified by phosphorylation and potentially other modifications and that they are hyperphosphorylated during mitosis. The invariant Ser-4 residue on BAF is a major site of phosphorylation during both interphase and mitosis. In HeLa cells that overexpressed the phosphomimetic BAF missense mutant S4E, but not S4A, emerin mislocalized from the nuclear envelope, suggesting Ser-4-nonphosphorylated BAF normally promotes emerin localization at the nuclear envelope. Supporting this model, wild-type BAF but not mutant S4E enhanced emerin binding to lamin A in vitro. Thus, Ser-4-unphosphorylated BAF has a positive role in localizing emerin; this role may be disease relevant because loss or mislocalization of emerin causes Emery-Dreifuss muscular dystrophy. Our findings further suggest Ser-4 phosphorylation inhibits BAF binding to emerin and lamin A, and thereby weakens emerin-lamin interactions during both mitosis and interphase.

Lamin A precursor induces barrier-to-autointegration factor nuclear localization

“…In particular, a very bright BAF nuclear staining (25% of cells) was detected Previously reported studies described BAF binding to mature lamin A and prelamin A in vitro. 19,22 In agreement with these results, we observed BAF interaction with WT-lamin A, nonfarnesylated (unprocessable) and farnesylated-carboxymethylated (uncleavable) lamin A precursor in living cells. A coimmunoprecipitation study was performed in HEK293 cells transfected with GFP-BAF in combination with LA-WT or each prelamin A mutant.…”

“…However, we cannot exclude that BAF translocation in HGPS cells could also be due to accumulation of wild-type prelamin A, as described for SUN1 translocation observed by other authors. 43 Moreover, we obtain BAF lamin A-∆50 coimmunoprecipitation in HEK293 transfected cells demonstrating that the 50 amino acid deletion of progerin (residues 607-657), extending within a previously in vitro determined prelamin A/BAF interaction sequence (residues 394-664), 19 does not affect BAF/progerin interaction in living cells. Since BAF is proposed as an RBBP4 binding protein, the loss of NURD-protein components observed in HGPS cells may appear in contrast with BAF increase in the nucleus.…”

“…[16][17][18] The regulation of BAF depends on its modification state and binding partners in the nucleus and nuclear membrane. 16,19,20 Recently, new BAF binding partners have been identified with chromatin-regulatory function. In particular, it has been reported that BAF and emerin dynamically interact with DNA damage response proteins of the CUL4-DDB-ROC1 complex.…”

“…In particular, it has been reported that BAF and emerin dynamically interact with DNA damage response proteins of the CUL4-DDB-ROC1 complex. 21 Because in vitro binding between prelamin A and BAF has been previously described, 19 we hypothesized that prelamin A could affect the localization and expression of this DNA binding protein, thus influencing chromatin organization. To test this hypothesis, we performed a study of BAF cellular distribution and protein expression in prelamin A accumulating cells.…”

“…19 BAF direct interaction with both prelamin A and emerin has been described. 19 Moreover, in vitro binding studies have demonstrated that the presence of BAF as a prelamin A-emerin complex component increases the molecular affinity between these proteins.…”