The Mr 46,000 nuclear scaffold ATP-binding protein: identification of the putative nucleoside triphosphatase by proteolysis and monoclonal antibodies directed against lamins A/C

Previous work suggested that the major Mr 46,000 ATP-binding protein [a putative nucleoside triphosphatase (NTPase)] found in rat liver nuclear scaffold (NS) may be proteolytically derived from lamins A/C. To definitively establish this identification, we undertook a series of photolabeling, proteolysis, and immunoprecipitation experiments. Mice were immunized with human lamin C expressed in bacteria, and monoclonal antibody-producing hybridomas were obtained. The purified monoclonal antibodies all recognized lamins A and C on immunoblots of NS, as well as Mr 46,000 or 34,000 proteolytic fragments as minor components. The Mr 46,000 photolabeled band was the only major NS component photolabeled with low concentrations of azido-ATP, and it was immunoprecipitated with anti-lamin monoclonal antibodies. To preclude the possibility that the photolabeled Mr 46,000 protein represented a minor component which comigrated with the Mr 46,000 lamin fragment and which specifically associated with lamins A/C during immunoprecipitation, a series of proteolytic digestions were undertaken. Digestion of the photolabeled Mr 46,000 peptide with chymotrypsin and staphylococcal protease V8 produced a limited number of photolabeled fragments, all of which comigrated with major stainable fragments produced from the Mr 46,000 lamin fragment. Cyanogen bromide cleavage of the photolabeled Mr 46,000 polypeptide, followed by polyacrylamide gel electrophoresis or high performance liquid chromatography/amino acid analyses, defined the COOH-terminal cleavage site as the Y residue at amino acid 376 and localized the photolabeled site to the COOH-terminal region (amino acids 372-376). In support of this proposed proteolytic cleavage site, specific assays with tyrosine-containing thiobenzyl ester substrate documented the presence of NS protease activity which cleaves at tyrosine residues; this activity shows a Km of 0.2 mM and a Kcat of approximately 250/s. Parallel experiments with mildly proteolyzed cloned lamin C preparations showed selective photolabeling of an Mr 34,000 fragment, which corresponds to a proteolytic breakdown product of the Mr 46,000 NS polypeptide; this Mr 34,000 photolabeled fragment was also immunoprecipitated with anti-lamin monoclonal antibodies and contained the same photolabeled site as the Mr 46,000 peptide. Cloned lamin C preparations were inactive in NTPase assays but did exhibit substantial ATP binding with an apparent KD = 4 x 10(-5) M ATP. These results indicate that the major Mr 46,000 photoaffinity-labeled protein in NS, which represents the putative NTPase thought to participate in nucleocytoplasmic transport, is derived from lamin A or lamin C by NS proteolytic activity which exposes a cryptic ATP-binding site near the highly conserved end of coil-2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of cyclin D3 as a new interaction partner of lamin A/C

Lamin A/C is a major component of the nuclear lamina. An intact nuclear lamina has been proposed to be necessary for muscle differentiation. Cyclin D3 is known to be upregulated in differentiated muscle cells and to form insoluble complexes with cell-cycle regulatory factors in these cells. We have examined the possibility of direct binding interactions between lamin A/C and cyclin D3 by in vitro binding assays and co-immunoprecipitation studies with muscle cells. Our results indicate that cyclin D3 binds specifically to amino acid residues 383-474 of lamin A/C and associates with lamin A/C in muscle cells. The identification of cyclin D3 as a novel binding partner of lamin A/C has important implications for a role for lamin A/C in muscle differentiation.     

Interaction between emerin and nuclear lamins

Emerin is an inner nuclear membrane protein that is involved in X-linked recessive Emery-Dreifuss muscular dystrophy (X-EDMD). Although the function of this protein is still unknown, we revealed that C-terminus transmembrane domain-truncated emerin (amino acid 1-225) binds to lamin A with higher affinity than lamin C. Screening for the emerin binding protein and immunoprecipitation analysis showed that lamin A binds to emerin specifically. We also used the yeast two-hybrid system to clarify that this interaction requires the top half of the tail domain (amino acid 384-566) of lamin A. Lamin A and lamin C are alternative splicing products of the lamin A/C gene that is responsible for autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD). These results indicate that the emerin-lamin interaction requires the tail domains of lamin A and lamin C. The data also suggest that the lamin A-specific region (amino acids 567-664) plays some indirect role in the difference in emerin-binding capacity between lamin A and lamin C. This is the first report that refers the difference between lamin A and lamin C in the interaction with emerin. These data also suggest that lamin A is important for nuclear membrane integrity.     

A lamin A/C beta-strand containing the site of lipodystrophy mutations is a major surface epitope for a new panel of monoclonal antibodies

Using a phage-displayed peptide library, we have identified the epitope recognized by a new panel of five monoclonal antibodies (mAbs) raised against full-length recombinant human lamin A. The mAbs were found to recognize both lamin A and C by Western blotting and immunolocalization at the nuclear rim. A nine-amino acid consensus sequence PLLTYRFPP in the common immunoglobulin-like (Ig-like) domain of lamin A/C contains the binding site for all five mAbs. Three-dimensional structure of the Ig-like domain of lamin A/C shows this sequence is a complete beta-strand. This sequence includes arginine-482 (R482) which is mutated in most cases of Dunnigan-type familial partial lipodystrophy (FPLD). R482 may be part of an interaction site on the surface of lamin A/C for lamin-binding proteins associated with lipodystrophy.     

Disease-associated mutations in the coil 2B domain of human lamin A/C affect structural properties that mediate dimerization and intermediate filament formation

The lamin proteins are essential components of the nuclear lamina of eukaryotic cells, that are involved in a complex association mechanism to attain a functional supermolecular structure. Mutations of the lamin A/C gene are associated with several different neuromuscular diseases, and the detailed effect of disease-associated amino acid substitutions on the structure and stability of human lamin dimers is yet unknown. Here we present a structural and thermodynamic characterization by means of molecular dynamics simulations of the effect of pathological mutations (S326T, R331P, R331Q, E347K, E358K, M371K, and R377H) on the association of the coil 2B domains that mediate lamin A/C oligomerization. The structures attained during the simulations, along with the quantification of the contribution of each residue to the dimerization energies, support a lamin association mechanism mediated by homophilic intermolecular interactions promoted by dissociative conformational changes at distinct positions in the coiled coil. The pathogenic mutations can both increase or decrease the stability of lamin A/C dimers, and a possible correlation between the effect of the amino acid substitutions and disease onset and severity is presented.     

A new lamin in Xenopus somatic tissues displays strong homology to human lamin A

The nuclear lamina of vertebrates is composed of several major polypeptides that range in mol. wt from 60 to 80 kd. In mammals, the three major lamin proteins are designated A, B and C. Two major lamins have been described in Xenopus somatic tissues; two other lamins are expressed primarily in germ cells. We have analysed a cDNA clone encoding a Xenopus lamin that is highly homologous to human lamins A and C. The predicted protein has the carboxy-terminal domain characteristic of human lamin A and is thus a lamin A homologue. Surprisingly, the lamin encoded by the cDNA clone is not one of the known Xenopus lamins. The encoded protein is distinct in size from the oocyte lamin LIII and the two somatic lamins LI and LII. Monoclonal antibodies specific for LII, LIII and LIV (the lamin of male germ cells) do not recognize the protein encoded by the cDNA clone; conversely, a polyclonal antibody against the encoded protein does not recognize any of the known Xenopus lamins. This lamin is expressed late in embryonic development, and is present in all adult somatic cells examined, except erythrocytes. Thus frogs and mammals are similar in having three major somatic lamins that fall into distinct structural classes.     

Amino acid sequence and molecular characterization of murine lamin B as deduced from cDNA clones

The nuclear lamina is the karyoskeletal structure, intimately associated with the nuclear envelope, that is widespread among the diverse types of eukaryotic cells. A family of proteins, termed lamins, has been shown to be a prominent component of this lamina, and various members of this family are differentially expressed in different cell types. In mammals, three major lamins (A, B, C) have been identified, and in all cells so far examined lamin B is constitutively expressed while lamins A and C are not, suggesting that lamin B is sufficient to form a functional lamina. Because of this key importance of lamin B, cDNA clones encoding mammalian lamin B were isolated by screening murine cDNA libraries, representing F9 teratocarcinoma cells and fetal liver, with the corresponding cDNA probe of lamin LI of Xenopus laevis. The nucleotide sequence of the murine lamin B mRNA (approximately 2.9 kb) was determined. The deduced amino acid sequence of the encoded polypeptide (587 amino acids; mol. wt. 66760) is highly homologous to X. laevis lamin LI (72.9% identical residues) but displays lower similarity to A-type lamins (53.8% identical amino acid residues with human lamin A). Lamin B also conforms to the general molecular organization principle of the members of the intermediate filament (IF) protein family, i.e., an extended alpha-helical rod domain that is interrupted by two non alpha-helical linkers and flanked by non-alpha-helical head (amino-terminal) and tail (carboxy-terminal) domains. The tail domain, which does not reveal a hydrophobic region of considerable length, contains a typical karyophilic signal sequence and an uninterrupted stretch of eight negatively charged amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear lamin LI of Xenopus laevis: cDNA cloning, amino acid sequence and binding specificity of a member of the lamin B subfamily.

Lamins are karyoskeletal proteins associated with the nuclear envelope which can be divided into two groups, i.e. the type A lamins of near neutral pI and the more acidic lamins, including mammalian lamin B. We have isolated cDNA clones encoding a representative of the type B subfamily from Xenopus laevis, and have deduced its amino acid sequence from the coding portion of the approximately 2.9 kb mRNA. The polypeptide (mol. wt 66,433) is identified as a typical lamin by its homology to Xenopus human type A lamins, but detailed sequence comparison shows that LI is less related to Xenopus lamin A than the latter is to human lamin A. The conformation predicted for LI conforms to the general model of lamins and intermediate filament proteins and is characterized by an extended central alpha-helical coiled coil domain, flanked by non-alpha-helical domains, i.e. a relatively short N-terminal head and a long C-terminal tail. As in lamins A and C, the head of lamin LI is positively charged and the tail presents a similar C-terminal pentapeptide, a putative nuclear accumulation signal, a very negatively charged region and a number of short regions that are highly homologous in all lamins. However, LI differs from the type A lamins by the absence of the oligo-histidine stretch and a di-proline motif in the tail region and by a significantly lower number of identical amino acid positions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a novel, highly variable amino-terminal amino acid sequence element in the nuclear intermediate filament protein lamin B(2) from higher vertebrates

By comparing newly available cDNA sequences of the human intermediate filament protein lamin B(2) with published sequences, we have identified an additional translation initiation codon 60 nucleotides upstream of the previously assumed translation start. In addition, corresponding sequences were identified in the chimpanzee, mouse, rat and bovine genes and cDNAs, respectively. Therefore, we generated antibodies against these potential 20 new amino acids of the human sequence. By immunoblot analysis and immunofluorescence microscopy we show that human lamin B(2) is indeed synthesized as a longer version than previously reported, because it contains these additional 20 amino acids. Notably, the sequence homology to mouse, rat and bovine lamin B(2) is significantly lower in this segment than in that between the second methionine codon and the start of the alpha-helical rod indicating that the tip of the "head" is engaged in more species-specific functions. Forced expression of the GFP-tagged authentic "long" and the 20 amino acid shorter version of lamin B(2) in human cultured SW-13 cells demonstrated that both the longer and the shorter version are properly integrated into the nuclear lamina, although the shorter version exhibited a tendency to disturb envelope architecture at higher expression levels.     

Perturbation of wild-type lamin A metabolism results in a progeroid phenotype

Mutations in the lamin A/C gene cause the rare genetic disorder Hutchinson-Gilford progeria syndrome (HGPS). The prevalent mutation results in the production of a mutant lamin A protein with an internal 50 amino acid deletion which causes a cellular aging phenotype characterized by growth defects, limited replicative lifespan, and nuclear membrane abnormalities. However, the relevance of these findings to normal human aging is unclear. In this study, we demonstrate that increased levels of wild-type lamin A in normal human cells result in decreased replicative lifespan and nuclear membrane abnormalities that lead to apoptotic cell death and senescence in a manner that is strongly reminiscent of the phenotype shown by HGPS cells. In contrast to the accelerated aging defects observed in HGPS cells, the progeroid phenotype resulting from increased expression of wild-type lamin A can be rescued by overexpression of ZMPSTE24, the metalloproteinase responsible for the removal of the farnesylated carboxyl terminal region of lamin A. Furthermore, farnesyltransferase inhibitors also serve to reverse the progeroid phenotype resulting from increased lamin A expression. Significantly, cells expressing elevated levels of lamin A display abnormal lamin A localization and similar alterations in the nuclear distribution of lamin A are also observed in cells from old-age individuals. These data demonstrate that the metabolism of wild-type lamin A is delicately poised and even in the absence of disease-linked mutations small perturbations in this system are sufficient to cause prominent nuclear defects and result in a progeroid phenotype.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone resonance assignment of the lamin C-terminal region specific to prelamin A

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.     

Molecular characterization of protein kinase C-alpha binding to lamin A

Previous results from our laboratory have identified lamin A as a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are crucial for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of rat PKC-alpha, we have established that binding occurs through both the V5 region and a portion of the C2 region (i.e., the calcium-dependent lipid binding (CaLB) domain) of the kinase. In particular, we have found that amino acid 200-217 of the CaLB domain are essential for binding lamin A, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain and lamin A. We also show that the presence of four lysine residues of the CaLB domain (K205, K209, K211, and K213) was essential for the binding. We have determined that binding of elements of PKC-alpha to lamin A does not require the presence of cofactors such as phosphatidylserine (PS) and Ca(2+). We have also found that the binding site of lamin A for the CaLB domain of PKC-alpha is localized in the carboxyl-terminus of the lamin, downstream of amino acid 499. Our findings may prove to be important to clarify the mechanisms regulating PKC function within the nucleus and may also lead to the synthesis of isozyme-specific drugs to attenuate or reverse PKC-dependent nuclear signaling pathways important for the pathogenesis of cancer.     

The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains

The lamin B receptor is a previously identified integral membrane protein in the nuclear envelope of turkey erythrocytes that associates with the nuclear intermediate filament protein lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). In the present report, we use cell fractionation and antibodies against the lamin B receptor to localize it to an 8-M urea-extracted membrane fraction of chicken liver nuclei, supporting an inner nuclear membrane localization. We deduced the amino acid sequence of the chicken lamin B receptor from overlapping clones obtained by screening cDNA libraries with a probe generated by the polymerase chain reaction with primers based on the partial protein sequence of the isolated protein. The mature lamin B receptor has a calculated molecular mass of 73,375 D and eight segments of hydrophobic amino acids that could function as transmembrane domains as determined by hydropathy analysis. Preceding the first putative transmembrane segment is a highly charged 204-residue-long amino terminal region that contains two consensus sites for phosphorylation by protein kinase A. Since the lamin B receptor has been shown to be phosphorylated by protein kinase A in vitro and in vivo and this phosphorylation affects lamin B binding (Applebaum, J., G. Blobel, and S. D. Georgatos. 1990. J. Biol. Chem. 265:4181-4185), it is likely that this amino terminal region faces the nucleoplasm. The amino terminal region also contains three DNA-binding motifs that are found in gene regulatory proteins and histones, suggesting that the lamin B receptor may additionally play a role in gene regulation and/or chromatin organization.     

The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins.

The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.     

In vitro posttranslational modification of lamin B cloned from a human T-cell line.

Autoimmune diseases are characterized by spontaneously occurring autoantibodies which have proven to be useful reagents for the characterization of specific nuclear proteins. Using a monoclonal autoantibody (72B9) derived from a murine lupus strain, we have cloned a cDNA from the human T-cell line MOLT-4, which encodes nuclear lamin B. The identity of the encoded protein as lamin B was established by both biochemical and immunological criteria. Inspection of the deduced amino acid sequence of lamin B revealed the presence in coil 1B of the alpha-helical domain of a leucine heptad repeat region. Analysis of mRNA in HL60 and MOLT-4 cells, which express only lamin B, or HeLa cells, which express all three major lamins (A, B, and C), together with the comigration of in vitro-translated product with isolated HeLa cell lamin B by two-dimensional gel electrophoresis, suggests that a single lamin B is expressed in mammalian somatic cells. In vitro translation with the cDNA clone revealed an EDTA-sensitive posttranslational modification which resulted in an increase in the apparent molecular weight to that equivalent to the native in vivo-synthesized lamin B protein. This in vitro modification included incorporation of a product of mevalonolactone and required an intact carboxy terminus.     

Human lamin B contains a farnesylated cysteine residue

We recently showed that HeLa cell lamin B is modified by a mevalonic acid derivative. Here we identified the modified amino acid, determined its mode of linkage to the mevalonic acid derivative, and established the derivative's structure. A cysteine residue is modified because experiments with lamin B that had been biosynthetically labeled with [3H]mevalonic acid or [35S]cysteine and then extensively digested with proteases yielded 3H- or 35S-labeled products that co-chromatographed in five successive systems. A thioether linkage rather than a thioester linkage is involved because the mevalonic acid derivative could be released from the 3H-labeled products in a pentane-extractable form by treatment with Raney nickel but not with methanolic KOH. The derivative is a farnesyl moiety because the Raney nickel-released material was identified as 2,6,10-trimethyl-2,6,10-dodecatriene by a combination of gas chromatography and mass spectrometry. The thioether-modified cysteine residue appears to be located near the carboxyl end of lamin B because treatment of 3H-labeled lamin B with cyanogen bromide yielded a single labeled polypeptide that mapped toward this end of the cDNA-inferred sequence of human lamin B.     

Phorbol esters induce transient changes in the accessibility of the carboxy-terminal domain of nuclear lamin A.

Treatment of human epithelial cells in culture with phorbol esters (TPA) gives rise to a transient and reversible loss of accessibility to antibodies of the nonhelical carboxy-terminal domain of nuclear lamin A that distinguishes it from lamin C. No change in the accessibility of epitopes present in the common domain of lamins A and C was observed. Loss of accessibility of lamin A was not due to proteolytic degradation nor to modification of the isoelectric point of lamin A and did not depend upon protein kinase C activation nor protein synthesis. Perturbation of desmosome organization by growth in low calcium blocked the effect of TPA on lamin A. Prolonged exposure to nocodazole, one of the effects of which is a perinuclear collapse of intermediate filaments, also blocked the effect of TPA on lamin A. These results suggest that the initial target of TPA may be at the level of cell-cell contacts and that the perturbation induced by TPA may be propagated via the structural link formed by intermediate filaments between the cell surface and the nucleus, giving rise to a change in conformation of the carboxy-terminal domain of lamin A or to an interaction of this domain with another nuclear component. These results form the basis for the hypothesis that the interphase nuclear lamina may play an active role in the process of mechanochemical signal transduction.     

Role of nuclear lamins in nuclear segmentation of human neutrophils

Nuclear breakdown leading to the formation of apoptotic bodies has been postulated to involve degradation of nuclear structural proteins, such as lamins A/C and B. Although nuclear segmentation occurs during the maturation of polymorphonuclear leukocytes (neutrophils), its mechanism is not known. We found that human neutrophils have lamin B but lack lamins A/C while mononuclear cells possess all three types of lamin as assessed by immunoblotting. Differentiation of human promyelocytic HL-60 cells into neutrophil-like cells was also accompanied by the down-regulation of lamins A/C but not of lamin B. Moreover, when compared with normal cells, neutrophils with the Pelger-Hu?t anomaly of nuclear hyposegmentation exhibited significantly lower activity of caspase-6, a lamin A/C-cleaving enzyme. Differentiated HL-60 cells showed higher activity of caspase-6 than that of untreated cells. These observations allow us to speculate that remodeling of nuclear lamins might underlie the mechanism for nuclear segmentation of neutrophils.     

In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.     

Novel progerin-interactive partner proteins hnRNP E1, EGF, Mel 18, and UBC9 interact with lamin A/C

The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is an apparent accelerated aging disorder of childhood. Recently, HGPS has been characterized as one of a growing group of disorders known as laminopathies, which result from genetic defects of the lamin A/C (LMNA) gene. The majority of HGPS mutant alleles involve a silent mutation, c.2063C>T resulting in G608G, that generates a cryptic splicing site in exon 11 of LMNA and consequently truncates 50 amino acids near the C-terminus of pre-lamin A/C. To explore possible mechanisms underlying the development of HGPS, we began a search for proteins that would uniquely interact with progerin (the truncated lamin A in HGPS) using a yeast two-hybrid system. Four new progerin interactive partner proteins were identified that had not been previously found to interact with lamin A/C: hnRNP E1, UBC9 (ubiquitin conjugating enzyme E2I), Mel-18, and EGF1. However, using control and progeria fibroblasts, co-immunoprecipitation studies of endogenous proteins did not show differential binding affinity compared to normal lamin A/C. Thus, we did not find evidence for uniquely interacting partner proteins using this approach, but did identify four new lamin A/C interactive partners.