Identification of a novel muscle A-type lamin-interacting protein (MLIP)

Abstract

Mutations in the A-type lamin (LMNA) gene are associated with age-associated degenerative disorders of mesenchymal tissues, such as dilated cardiomyopathy, Emery-Dreifuss muscular dystrophy, and limb-girdle muscular dystrophy. The molecular mechanisms that connect mutations in LMNA with different human diseases are poorly understood. Here, we report the identification of a Muscle-enriched A-type Lamin-interacting Protein, MLIP (C6orf142 and 2310046A06rik), a unique single copy gene that is an innovation of amniotes (reptiles, birds, and mammals). MLIP encodes alternatively spliced variants (23-57 kDa) and possesses several novel structural motifs not found in other proteins. MLIP is expressed ubiquitously and most abundantly in heart, skeletal, and smooth muscle. MLIP interacts directly and co-localizes with lamin A and C in the nuclear envelope. MLIP also co-localizes with promyelocytic leukemia (PML) bodies within the nucleus. PML, like MLIP, is only found in amniotes, suggesting that a functional link between the nuclear envelope and PML bodies may exist through MLIP. Down-regulation of lamin A/C expression by shRNA results in the up-regulation and mislocalization of MLIP. Given that MLIP is expressed most highly in striated and smooth muscle, it is likely to contribute to the mesenchymal phenotypes of laminopathies.

FIGURE 1.

Interaction of LMNA with MLIP. A, yeast two-hybrid interaction assay that identified six independent MLIP clones from a human heart cDNA library (Clontech). B, yeast-two hybrid assay of the LMNA_1–230, LMNA_1–130 and LMNA_120–230, and MLIP clone 1 and MLIP clone 3 interactions. Twenty microliters of the culture was plated on His⁺ (left) and His⁻ (right) plates at a cell density of 1 × 10⁶/ml.

FIGURE 2.

MLIP is a single copy, evolutionarily conserved amniota gene. Phylogenetic and gene synteny analysis centered on (A) human MLIP (C6orf142) gene and (B) human LMNA gene was performed by Genomicus v58.01 genome browser (50).

FIGURE 3.

Alignment and predicted secondary structure for human MLIP. A, the deduced amino acid sequence of human MLIP and other selected species (mouse and chicken) were aligned by ClustalW algorithm. MLIP-specific polyclonal antibodies were raised against the two highlighted (yellow) mouse MLIP peptides. The highlighted red sequence is primarily β-strand. The consensus sequence is denoted by asterisks (*) as identical, periods as conserved substitutions, and colons as semiconservative substitutions. Numbers indicate the position of the last amino acid of each line. B, the predicted secondary structure for human MLIP is represented by red, α-helical; green, β-strand; black, coil and turn. Hydrophobicity (by Kyte-Doolittle plot) of human MLIP is represented below the secondary amino acid structure of human MLIP.

FIGURE 4.

MLIP specifically binds lamin A/C. A, purified recombinant MLIP binds directly within the first 230 amino acids of recombinant lamins A and C in an in vitro immunoprecipitation assay. Purified recombinant His₆-MLIP and GST-lamin A were mixed together (as indicated) with complexes precipitated through the His tag. Western blot analysis was performed using anti-GST (Cell Signaling) and anti-MLIP polyclonal antibodies. A 1:10 dilution of the total starting material was run on the same gel (left panels). The assay was repeated two additional times with similar results. B, MLIP peptide neutralization of the anti-MLIP serum demonstrates the specificity of the MLIP antibodies in Western blot analysis. C, MLIP co-immunoprecipitates with lamin A/C from adult mouse hearts. Total mouse heart lysates were divided 45%:45%:10% with 45% incubated with either anti-MLIP serum or MLIP peptide neutralized anti-MLIP serum (Control). The input control for the immunoprecipitation represents ∼2% of total heart lysate.

FIGURE 5.

MLIP co-localizes with lamin A/C and PML bodies in mouse C2C12 myoblasts. A–D, mouse C2C12 myoblasts were analyzed by indirect immunofluorescence microscopy (Carl Zeiss AxioImager Z1 Microscope) with antibodies against MLIP and lamin A/C. DNA was stained with DAPI (A), lamin A/C (B), and MLIP (C). D, merged images of DAPI (blue), lamin A/C (red), and MLIP (green) staining from A–C. Arrow indicates nuclear envelope and co-localization of MLIP with lamin A/C. E–H, C2C12 myoblasts were analyzed by indirect immunofluorescence and sequential scanning confocal microscopy with antibodies against MLIP and PML. DNA was stained with DAPI (E), PML (F), and MLIP (G). The arrows indicate co-localization of MLIP with PML. H, merged images of DAPI (blue), PML (red), and MLIP (green) staining from E–G. Scale bar, 10 μm.

FIGURE 6.

MLIP tissue expression profile. Expression of MLIP mRNA in mouse (A) and human (B) tissue by Northern analysis revealed two transcripts. 10 μg of poly(A)-enriched RNA was loaded per lane. A human β-actin cDNA fragment was used to probe the blots as an internal loading control. The β-actin probe cross-hybridizes with cardiac α-actin and skeletal α-actin, which show up in the heart and skeletal muscle lanes. C, endogenous tissue distribution of MLIP protein by Western analysis. 5 μg of total soluble protein lysates from each tissue was loaded per lane as represented by Coomassie Blue staining of total protein, lower panel. D, normalized tissue distribution of MLIP expression in adult mouse tissue (n = 3, mean ± S.D.) as determined by quantitative PCR.

FIGURE 7.

MLIP mouse tissue expression by indirect immunohistochemistry. A–C, brain; D–E, heart ventricles; G–I, atria; J–L, skeletal muscle; M–O, liver; and P–R, lung. F and I, magnified image of heart (E and H, respectively) as indicated by box. Nucleus is identified by arrow. MLIP is expressed primarily in fast oxidative muscle fibers of soleus muscle. MLIP (S) and peroxiredoxin-3 (PRDX3) (T) expression profiles in serial soleus sections. The arrows mark similar fibers in S and T. Magnification is indicated for each image.

FIGURE 8.

MLIP is an alternatively spiced gene. A, RT-PCR was performed on mRNA isolated from mouse hearts using primers targeted to the 5′- and 3′-UTR of MLIP. The RT-PCR product was TA cloned into pCR-II plasmid (Invitrogen) and transformed into bacteria. Direct PCR was performed with primers targeting flanking regions of the MLIP insertion site amplified with each PCR product sequenced. Cloned MLIP cDNA length and predicted protein mass are identified for each of the bands (left and center columns). Calculated protein mass based on migration rates for each of the MLIP bands is presented in Fig. 6C (right column). B, based on these data, an alternative splice map of MLIP was constructed. The numbers below each splice map reflect the transcript size and predicted translated protein size.

FIGURE 9.

Lamin A is required for normal MLIP expression and cellular localization. A, Western blot of whole cell lysates showing reduced lamin A protein. C2C12 myoblasts down-regulated for lamin A by shRNA treatment or control cells infected with nonspecific shRNA were analyzed by Western blotting 48 h after infection using antibodies against lamin A/C, MLIP, or GAPDH. B and C, quantification of lamin A/C down-regulation (B) and MLIP isoform expression by LMNA shRNAi (C) was compared with nonspecific shRNAi. Each lamin A/C or MLIP isoform was normalized to the GAPDH internal loading control, and the data presented in B and C are the means ± S.D. of three independent experiments. D–G, control C2C12 myoblasts infected with nonspecific shRNAi were analyzed by indirect immunofluorescence microscopy with antibodies against MLIP and LMNA. DNA was stained with DAPI (D), MLIP (E), and lamin A/C (F). G, merged images of DAPI (blue), MLIP (green), and lamin A/C (red) staining from D–F are shown. H–K, C2C12 myoblasts infected with LMNA-specific shRNAi were analyzed by indirect immunofluorescence microscopy (Carl Zeiss AxioImager Z1 Microscope) with antibodies against MLIP and LMNA. DNA was stained with DAPI (H), MLIP (I), and lamin A/C (J). K, Merged images of DAPI (blue), MLIP (green) and lamin A/C (red) staining from H–J are shown. Scale bar, 10 μm.