Lamina-associated Polypeptide 2-α Forms Homo-trimers via Its C Terminus, and Oligomerization Is Unaffected by a Disease-causing Mutation

Abstract

The nucleoplasmic protein, Lamina-associated polypeptide (LAP) 2␣, is one of six alternatively spliced products of the LAP2gene, which share a common N-terminal region. In contrast to the other isoforms, which also share most of their C termini, LAP2␣ has a large unique C-terminal region that contains binding sites for chromatin, A-type lamins, and retinoblastoma protein. By immunoprecipitation analyses of LAP2␣ complexes from cells expressing differently tagged LAP2␣ proteins and fragments, we demonstrate that LAP2␣ forms higher order structures containing multiple LAP2␣ molecules in vivo and that complex formation is mediated by the C terminus. Solid phase binding assays using recombinant and in vitro translated LAP2␣ fragments showed direct interactions of LAP2␣ C termini. Cross-linking of LAP2␣ complexes and multiangle light scattering of purified LAP2␣ revealed the existence of stable homo-trimers in vivo and in vitro. Finally, we show that, in contrast to the LAP2␣-lamin A interaction, its self-association is not affected by a disease-linked single point mutation in the LAP2␣ C terminus.The nuclear envelope comprises the inner and outer nuclear membranes, the nuclear pore complexes, and the nuclear lamina, which underlies the inner nuclear membrane (1, 2). The nuclear lamina is the major structural framework in the nucleus of multicellular eukaryotes and is composed of a filamentous meshwork of type V intermediate filament proteins, the lamins. B-type lamins, encoded by two human genes (LMNB1 and LMNB2), are essential for cell viability. In contrast, the four A-type lamins (A, C, C2, and A⌬10), representing splicing isoforms of the LMNA gene, are dispensable for viability of individual cells but have crucial functions in tissue organization after birth (3, 4).In addition to the lamins, the nuclear lamina contains a number of integral membrane proteins of the inner nuclear membrane, the best characterized of which are the Lamin B receptor, Lamina-associated polypeptide (LAP), 4 and the three LEM domain-containing proteins LAP2␤, emerin and MAN1 (5, 6). All these proteins interact with lamin A/C and/or B and contribute to anchorage of the nuclear membrane to the lamina. The LEM domain, a conserved 40-amino-acid motif located near the N terminus of the LEM family proteins, interacts with the DNA-binding protein barrier-to-autointegration factor (BAF) and mediates the binding of these proteins to chromatin (7). In LAP2 proteins, a LEM-like segment at the very N terminus has been shown to interact with DNA directly (8).The family of LAP2 proteins includes six alternatively spliced isoforms derived from the same gene (9). Most LAP2 isoforms are closely related structurally and functionally and are localized to the inner nuclear membrane, such as LAP2␤. In contrast, LAP2␣ shares only the N-terminal 187 amino acids with the other isoforms, including the LEM and LEM-like domains, but otherwise possesses a unique 506-amino-acid C-terminal region without a transmembrane domain (see Fig. 1A), encoded by one large exon...

Nuclear lamina genetic variants, including a truncated LAP2, in twins and siblings with nonalcoholic fatty liver disease

“…S9A). Notably, the LAP2α R690C variant migrated as high-molecular-weight species similar to wild-type LAP2α, consistent with a prior report (35). In addition, >50% of high-molecular-weight LAP2α species were Triton-soluble, which did not vary between wild-type LAP2α and the R690C variant.…”

“…To address this, we expressed myc-tagged LAP2α in Huh7 cells, then performed SDS-PAGE under semi-native, denaturing/non-reducing, and denaturing/reducing conditions. Similar to prior findings (35), >50% of LAP2α migrated as high-molecular weight species (>250 kDa) under semi-native conditions (not shown), with identical results under denaturing non-reducing conditions (Fig. S9A).…”

“…Previous work demonstrated that the carboxy-terminal portion of LAP2α can dimerize or trimerize in vitro and that multimeric LAP2α migrates as a high molecular species on semi-native SDS-PAGE (34,35). Given that lamin A forms intra- and intermolecular disulfide bonds (36), we hypothesized that multimeric species of LAP2α might also be disulfide-linked.…”

Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype

“…The reduced intensity could be due to epitope masking of the LAP2α antibodies, likely due to a posttranslational modification or to conformational change in LAP2α. According to the crystal structure of the C-terminal coiled-coil domain, LAP2α can form dimers [ 50 ] or even trimers [ 51 ]. Therefore, in cells expressing Nup98 chimeras, LAP2α might form other oligomeric structures due to presence or absence of a yet to identify binding partner.…”

Lamina-Associated Polypeptide (LAP)2α and Other LEM Proteins in Cancer Biology

“…While most LAP2 isoforms differ in their up to ~300 aa long C-terminus only due to the inclusion/exclusion of small, alternatively spliced domains, the ~500 aa long LAP2α C-terminus is encoded by a single exon unique for LAP2α [30]. Unlike the other major LAP2 isoforms, LAP2α’s C-terminus lacks a C-terminal transmembrane domain and folds as an extensive four-stranded antiparallel coiled coil dimer [31] that can also form higher oligomers [32]. In addition, while the membrane bound LAP2 isoforms localize at the INM and interact primarily with B-type lamins of the peripheral lamina [33], LAP2α specifically binds A-type lamins via its unique C-terminus [34] in the nucleoplasm [35].…”