Linker region of nebulin family members plays an important role in targeting these molecules to cellular structures

The nebulin family of actin-binding proteins plays an essential role in cytoskeletal dynamics and actin filament stability. All of the family members are modular proteins with their key defining structural feature being the presence of the 35-residue nebulin modules. The family members now include nebulin, nebulette, N-RAP, LASP-1, and LIM-nebulette. Nebulin and nebulette are associated with the thin filament/Z-line junction of striated muscle. LASP-1 and LIM-nebulette are found within focal adhesions, and N-RAP is associated with muscle cellular junctions. Although much investigation has focused on the role of the interactions between nebulin modules and actin, each of these proteins contains other domains that are essential for their cellular targeting and functions. The serine-rich linker region of nebulette has previously been shown to serve just such a purpose by targeting the association of the nebulin modules to the cardiac Z-line in cultured cardiomyocytes. In this report, we analyze the targeting functions of the homologous regions of LASP-1 and LIM-nebulette in their incorporation into focal adhesions. We have found that the linker region of LASP-1 is indeed important for its cellular localization and that the shortened linker region of LIM-nebulette drives the association of nebulin modules to focal adhesions. This work was supported by grants from the National Institutes of Health-HLB and the National Council of the American Heart Association to C.L.M.     

Chromosomal assignment of LASP1 and LASP2 genes and organization of the LASP2 gene in chicken

Lasp-1 and lasp-2 are actin-binding proteins that contain a LIM domain, two nebulin repeats and an SH3 domain with significant identity. We determined the chromosomal locations of the LASP1 and LASP2 genes in chicken by fluorescence in situ hybridization. The LASP1 gene was localized to a pair of microchromosomes and the LASP2 gene was localized to chromosome 2p3.1, indicating that the chromosomal locations of the LASP1 and LASP2 genes are highly conserved between chicken and human. The comparison of genomic and cDNA sequences of chicken lasp-2 and nebulette, a nebulin-related protein in muscle, suggested that both the corresponding mRNAs shared exons in the same manner as their human homologues. When compared with the domain structure of nebulette, another nebulin repeat was predicted for lasp-2, and all the nebulin repeats of lasp-2 were better conserved than those in nebulette. We also found the exon boundaries in nebulin repeats of lasp-2 were similar to those of other nebulin-related proteins.     

The nebulette repeat domain is necessary for proper maintenance of tropomyosin with the cardiac sarcomere

Nebulette is a cardiac-specific isoform of the giant actin-binding protein nebulin. Nebulette, having a mass of ∼ 100 kDa, is only predicted to extend 150 nm from the edge of the Z-lines. Overexpression of the nebulette C-terminal linker and/or SH3 domains in chicken cardiomyocytes results in a loss of endogenous nebulette with a concomitant loss of tropomyosin (TPM) and troponin, as well as a shortening of the thin filaments. These data suggest that nebulette's position in the sarcomere is important for the maintenance of TPM, troponin and thin filament length. To evaluate this hypothesis, N-terminal nested truncations tagged with GFP were expressed in chicken cardiomyocytes and the cells were analyzed for the distribution of myofilament proteins. Minimal effects on the myofilaments were observed with N-terminal deletions of up to 10 modules; however, deletion of 15 modules replicated the phenotype observed with expression of the C-terminal fragments. Expression of internal deletions of nebulette verifies that a site between module 10 and 15 is important for TPM maintenance within the sarcomeric lattice. We have additionally isolated TPM cDNAs from a yeast two hybrid (Y2H) analysis. These data indicate the importance of the nebulette-TPM interactions in the maintenance and stability of the thin filaments.     

Terminal regions of mouse nebulin: sequence analysis and complementary localization with N-RAP

The regions of mouse nebulin extending from the ends of the super repeats to the C-terminus and N-terminus were cloned and sequenced. Comparison of the mouse sequence with the previously published human sequence shows that the terminal regions of nebulin are highly conserved. The four phosphorylation motifs and SH3 domain found at the C-terminus of mouse nebulin are identical to those found in human nebulin, with the exception of four conservative substitutions. The modules linking this C-terminal region to the super repeats have deletions relative to both fetal and adult human nebulins that correspond to integral numbers of modules, making the mouse C-terminal simple repeat region among the shortest observed to date. The N-terminal region and the C-terminal modules were expressed in Escherichia coli and used for antibody production. Immunofluorescent labeling of these regions of nebulin in isolated myofibrils demonstrates that they are located near the center of the sarcomere and near the Z-line, respectively. Immunogold labeling with antibodies raised against the N-terminal nebulin sequence localizes this region in the A-band near the tips of the thin filaments. Nebulin localization is complementary to that of N-RAP, another muscle-specific protein containing nebulin-like super repeats; nebulin is exclusively found in the sarcomeres, while N-RAP is confined to the terminal bundles of actin filaments at the myotendinous junction. Cell Motil. Cytoskeleton 3:211-222, 2000 Published 2000 Wiley-Liss, Inc.     

Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein

Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.     

Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies

We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).     

Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1

Zyxin is a versatile component of focal adhesions in eukaryotic cells. Here we describe a novel binding partner of zyxin, which we have named LIM-nebulette. LIM-nebulette is an alternative splice variant of the sarcomeric protein nebulette, which, in contrast to nebulette, is expressed in non-muscle cells. It displays a modular structure with an N-terminal LIM domain, three nebulin-like repeats, and a C-terminal SH3 domain and shows high similarity to another cytoskeletal protein, Lasp-1 (LIM and SH3 protein-1). Co-precipitation studies and results obtained with the two-hybrid system demonstrate that LIM-nebulette and Lasp-1 interact specifically with zyxin. Moreover, the SH3 domain from LIM-nebulette is both necessary and sufficient for zyxin binding. The SH3 domains from Lasp-1 and nebulin can also interact with zyxin, but the SH3 domains from more distantly related proteins such as vinexin and sorting nexin 9 do not. On the other hand, the binding site in zyxin is situated at the extreme N terminus as shown by site-directed mutagenesis. LIM-nebulette and Lasp-1 use the same linear binding motif. This motif shows some similarity to a class II binding site but does not contain the classical PXXP sequence. LIM-nebulette reveals a subcellular distribution at focal adhesions similar to Lasp-1. Thus, LIM-nebulette, Lasp-1, and zyxin may play an important role in the organization of focal adhesions.     

Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling

The Xin actin-binding repeat–containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain–binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat–containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.     

Xin-repeats and nebulin-like repeats bind to F-actin in a similar manner

Xin and nebulette are striated muscle-specific actin-binding proteins that both contain multiple actin-binding repeats. The nature of these repeats is different: nebulette has nebulin-like repeats, while Xin contains its own unique repeats. However, the suggestion was made from biochemical data that the Xin-repeats may bind to multiple sites on the actin molecule as was found for nebulin. We have used electron microscopy and the iterative helical real space reconstruction to visualize complexes of F-actin with Xin fragments containing either three or six Xin-repeats, and with the CN5-nebulette fragment, containing five nebulin-like repeats. Our results indicate that Xin and nebulette fragments bind to F-actin in a similar manner and in two distinct modes: in one mode actin subdomain 1 is bound, while in the second mode the binding bridges between a different site on actin subdomains 1/2 of one protomer and subdomains 3/4 of an adjacent actin protomer. Taken together with published data about nebulin, tropomyosin and ADF/cofilin, our results suggest that the ability to bind in multiple modes to the actin protomer is a general property of many actin-binding proteins.     

Interactions between nebulin-like motifs and thin filament regulatory proteins

Nebulin (600-900 kDa) and nebulette (107-109 kDa) are two homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively. Both proteins are capped with a unique region at the amino terminus as well as a serine-rich linker domain and SH3 domains at the COOH terminus. Their significant size difference is attributed to the length of the central region wherein both proteins are primarily composed of approximately 35 amino acid repeats termed nebulin-like repeats or motifs. These motifs are marked by a conserved SXXXY sequence and high affinity binding to F-actin. To further characterize the effects that nebulin-like proteins may have on the striated muscle thin filament, we have cloned, expressed, and purified a five-motif chicken nebulette fragment and tested its interaction with the thin filament regulatory proteins. Both tropomyosin and troponin T individually bound the nebulette fragment, although the affinity of this interaction was significantly increased when tropomyosin-troponin T was tested as a binary complex. The addition of troponin I to the tropomyosin-troponin T complex decreased the binding to the nebulette fragment, indicating an involvement of the conserved T2 region of troponin T in this interaction. F-actin cosedimentation demonstrated that the nebulette fragment was able to significantly increase the affinity of the tropomyosin-troponin assembly for F-actin. The relationships provide a means for nebulin-like motifs to participate in the allosteric regulation of striated muscle contraction.     

Nebulin isoforms of extraocular muscle

The extraocular muscles (EOMs), which are responsible for reflexive and voluntary eye movements, have many unique biochemical, physiological, and ultrastructural features that set them apart from other skeletal muscles. For example, rodent EOMs lack M-lines and express EOM-specific myosin heavy chain (MYH13) and α-cardiac myosin heavy chain. Recent gene-expression profiling studies indicate the presence of other cardiac-specific proteins in adult EOMs. This interesting mixture of myofibrillar and cytoskeletal proteins poses the questions as to whether nebulette, as opposed to nebulin, might be expressed in EOM, and what isoforms of titin are expressed in the EOM. We have performed gel electrophoresis and immunological analyses to determine the titin and nebulin isoforms expressed in the EOM. We have found that the mass of the titin isoforms expressed in the EOM most closely resemble those found in the skeletal muscles tested, viz., the soleus and extensor digitorum longus (EDL). We also demonstrate that, although the EOM expresses cardiac isoforms of myosin, it does not express nebulette and contains a nebulin isoform with a mass consistent with that found in the prototypical fast hindlimb muscle EDL. This work was supported by grants from NIH-NHLB HL073089 to C.L.M. and NEI/NIH EY12998 to F.H.A.     

Targeting of nebulin fragments to the cardiac sarcomere

Nebulin, a vertebrate skeletal muscle actin binding protein, plays an important role in thin filament architecture. Recently, a number of reports have indicated evidence for nebulin expression in vertebrate hearts. To investigate the ability of nebulin to interact with cardiac myofilaments, we have expressed nebulin cDNA fragments tagged with green fluorescent protein (GFP) in chicken cardiomyocytes and PtK2 cells. Nebulin fragments from both the superrepeats and single repeats were expressed minus and plus the nebulin linker. Nebulin fragment incorporation was monitored by fluorescent microscopy and compared with the distribution of actin, alpha-actinin and titin. Expression of nebulin N-terminal superrepeats displayed a punctate cytoplasmic distribution in PtK2 cells and cardiomyocytes. Addition of the nebulin linker to the superrepeats resulted in association of the punctate staining with the myofibrils. Nebulin C-terminal superrepeats plus and minus the linker localized with stress fibers of PtK2 cells and associated with the cardiac myofilaments at the level of the Z-line. Expression of the single repeats plus and minus the nebulin linker region resulted in both a Z-line distribution and an A-band distribution. These data suggest that N-terminal superrepeat nebulin modules are incapable of supporting interactions with the cardiac myofilaments; whereas the C-terminal nebulin modules can. The expression of the N-terminal or C-terminal superrepeats did not alter the distribution of actin, alpha-actinin or titin in either atrial or ventricular cultures.     

The Nebulin family: an actin support group

Nebulin, a giant, actin-binding protein, is the largest member of a family of proteins (including N-RAP, nebulette, lasp-1 and lasp-2) that are assembled in a variety of cytoskeletal structures, and expressed in different tissues. For decades, nebulin has been thought to act as a molecular ruler, specifying the precise length of actin filaments in skeletal muscle. However, emerging evidence suggests that nebulin should not be viewed as a ruler but as an actin filament stabilizer required for length maintenance. Nebulin has also been implicated recently in an array of regulatory functions independent of its role in actin filament length regulation. In this review, we discuss the current evolutionary, biochemical, and functional data for the nebulin family of proteins - a family whose members, both large and small, function as cytoskeletal scaffolds and stabilizers.     

Targeted disruption of nebulette protein expression alters cardiac myofibril assembly and function

To evaluate nebulette's role in cardiac myofibrils, cardiomyocytes expressing green fluorescent protein (GFP)-nebulette constructs were monitored for their ability to contract and myofilament protein distribution was analyzed. Cells expressing full-length GFP-nebulette appear unaffected and exhibit normal beating frequencies. Expression of the GFP linker and SH3 results in loss of the endogenous nebulette and tropomyosin; however, Z-line and thick filaments are undisturbed. Cells expressing either of these domains have dramatically reduced beating frequencies, consistent with the loss of thin filament proteins. This loss was inhibited by the addition of protease inhibitors during culturing. The GFP repeat domain disrupts both myofibrillogenesis and contraction in spreading cardiomyocytes, whereas introduction of this protein into well-spread cardiomyocytes results in localization at the Z-line and a 50% reduction in beating frequency. Ultimately, these cells form bundles containing the GFP repeat and many myofilament proteins. Interestingly, butanedione monoxime inhibition of contraction inhibited the formation of these bundles. These results show that the GFP-nebulette domains have a dominant-negative effect on the distribution and function of the sarcomeric proteins. Taken together with the observation that nebulette colocalizes with alpha-actinin in the pre-, nascent, and mature myofibrils, our data demonstrate the importance of this cardiac-specific nebulin isoform in myofibril organization and function.     

Structural aspects of the functional modules in human protein kinase-Cα deduced from comparative analyses

Three-dimensional models of the five functional modules in human protein kinase Cα (PKCα) have been generated on the basis of known related structures. The catalytic region at the C-terminus of the sequence and the N-terminal auto-inhibitory pseudo-substrate have been modeled using the crystal structure complex of cAMP-dependent protein kinese (cAPK) and PKI peptide. While the N-terminal helix of the catalytic region of PKCα is predicted to be in a different location compared with cAPK, the C-terminal extension is modeled like that in the cAPK. The predicted permissive phosphorylation site of PKCα, Thr 497, is found to be entirely consistent with the mutagenesis studies. Basic Lys and Arg residues in the pseudo-substrate make several specific interactions with acidic residues in the catalytic region and may interact with the permissive phosphorylation site. Models of the two zinc-binding modules of PKCα are based on nuclear magnetic resonance and crystal structures of such modules in other PKC isoforms while the calcium phospholipid binding module (C2) is based on the crystal structure of a repeating unit in synaptotagmin I. Phorbol ester binding regions in zinc-binding modules and the calcium binding region in the C2 domain are similar to those in the basis structures. A hypothetical model of the relative positions of all five modules has the putative lipid binding ends of the C2 and the two zinc-binding domains pointing in the same direction and may serve as a basis for further experiments. © 1996 Wiley-Liss, Inc.     

Self-association of CIITA and its transactivation potential.

The major histocompatibility complex (MHC) class II transactivator (CIITA) regulates the expression of genes involved in the immune response, including MHC class II genes and the interleukin-4 gene. Interactions between CIITA and sequence-specific, DNA-binding proteins are required for CIITA to function as an activator of MHC class II genes. CIITA also interacts with the coactivators CBP (also called p300), and this interaction leads to synergistic activation of MHC class II promoters. Here, we report that CIITA forms complexes with itself and that a central region, including the GTP-binding domain is sufficient for self-association. Additionally, this central region interacts with the C-terminal leucine-rich repeat as well as the N-terminal acidic domain. LXXLL motifs residing in the GTP-binding domain are essential for self-association. Finally, distinct differences exist among various CIITA mutant proteins with regard to activation function, subcellular localization, and association with wild-type protein and dominant-negative potential.     

Expression of nebulette during early cardiac development

Nebulette, a cardiac homologue of nebulin, colocalizes with alpha-actinin in the pre-myofibrils of spreading cardiomyocytes and has been hypothesized to play a critical role in the formation of the thin-filament-Z-line complex early during myofibrillogenesis. Data from mesodermal explants or whole tissue mounts of developing hearts suggest that the pattern of myofibrillogenesis in situ may differ from observations of spreading cardiomyocytes. To evaluate the role of nebulette in myofibrillogenesis, we have analyzed the expression of nebulette in chicken heart rudiments by immunoblots and immunofluorescence. We detect the 110 kDa nebulette in heart rudiments derived from stage 9-10 using the anti-nebulin mAb, N114, or polyclonal anti-nebulette Abs by immunoblotting. Immunofluorescence analysis of explants stained with anti-nebulette and anti-alpha-actinin Abs demonstrates that both proteins localize along actin filaments in punctate to continuous manner at early stages of cardiac development and later give rise to striations. In both cases, the punctate staining had a periodicity of approximately 1.0 microm indicating a pre-myofibrils distribution at the earliest time points examined. We demonstrate that nebulette is indeed associated with premyofibrils in very early stages of myofibrillogenesis and suggest that nebulette may play an important role in the formation of these structures.     

The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments

Strict regulation of actin thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with actin filament capping proteins to regulate thin filament lengths. Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity. These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle.