Purification and Characterization of an α-Actinin-binding PDZ-LIM Protein That Is Up-regulated during Muscle Differentiation

α-Actinin is required for the organization and function of the contractile machinery of muscle. In order to understand more precisely the molecular mechanisms by which α-actinin might contribute to the formation and maintenance of the contractile apparatus within muscle cells, we performed a screen to identify novel α-actinin binding partners present in chicken smooth muscle cells. In this paper, we report the identification, purification, and characterization of a 36-kDa smooth muscle protein (p36) that interacts with α-actinin. Using a variety of in vitro binding assays, we demonstrate that the association between α-actinin and p36 is direct, specific, and saturable and exhibits a moderate affinity. Furthermore, native co-immunoprecipitation reveals that the two proteins are complexed in vivo. p36 is expressed in cardiac muscle and tissues enriched in smooth muscle. Interestingly, in skeletal muscle, a closely related protein of 40 kDa (p40) is detected. The expression of p36 and p40 is dramatically up-regulated during smooth and skeletal muscle differentiation, respectively, and p40 colocalizes with α-actinin at the Z-lines of differentiated myotubes. We have established the relationship between p36 and p40 by molecular cloning of cDNAs that encode both proteins and have determined that they are the products of a single gene. Both proteins display an identical N-terminal PDZ domain and an identical C-terminal LIM domain; an internal 63-amino acid sequence present in p36 is replaced by a unique 111-amino acid sequence in p40. Analysis of the sequences of p36 and p40 suggest that they are the avian forms of the actinin-associated LIM proteins (ALPs) recently described in rat (Xia, H., Winokur, S. T., Kuo, W.-L., Altherr, M. R., and Bredt, D. S. (1997) J. Cell Biol. 139, 507–515). The expression of the human ALP gene has been postulated to be affected by mutations that cause facioscapulohumeral muscular dystrophy; thus, the characterization of ALP function may ultimately provide insight into the mechanism of this disease. α-Actinin is required for the organization and function of the contractile machinery of muscle. In order to understand more precisely the molecular mechanisms by which α-actinin might contribute to the formation and maintenance of the contractile apparatus within muscle cells, we performed a screen to identify novel α-actinin binding partners present in chicken smooth muscle cells. In this paper, we report the identification, purification, and characterization of a 36-kDa smooth muscle protein (p36) that interacts with α-actinin. Using a variety of in vitro binding assays, we demonstrate that the association between α-actinin and p36 is direct, specific, and saturable and exhibits a moderate affinity. Furthermore, native co-immunoprecipitation reveals that the two proteins are complexed in vivo. p36 is expressed in cardiac muscle and tissues enriched in smooth muscle. Interestingly, in skeletal muscle, a closely related protein of 40 kDa (p40) is detected. The expression of p36 and p40 is dramatically up-regulated during smooth and skeletal muscle differentiation, respectively, and p40 colocalizes with α-actinin at the Z-lines of differentiated myotubes. We have established the relationship between p36 and p40 by molecular cloning of cDNAs that encode both proteins and have determined that they are the products of a single gene. Both proteins display an identical N-terminal PDZ domain and an identical C-terminal LIM domain; an internal 63-amino acid sequence present in p36 is replaced by a unique 111-amino acid sequence in p40. Analysis of the sequences of p36 and p40 suggest that they are the avian forms of the actinin-associated LIM proteins (ALPs) recently described in rat (Xia, H., Winokur, S. T., Kuo, W.-L., Altherr, M. R., and Bredt, D. S. (1997) J. Cell Biol. 139, 507–515). The expression of the human ALP gene has been postulated to be affected by mutations that cause facioscapulohumeral muscular dystrophy; thus, the characterization of ALP function may ultimately provide insight into the mechanism of this disease. actinin-associated LIM protein amino acids chicken embryo fibroblasts polyacrylamide gel electrophoresis bovine serum albumin base pair(s) In vertebrates, there are three types of muscle: skeletal, cardiac, and smooth, that contract by an actin- and myosin-dependent mechanism. Locomotion depends on the ability of skeletal muscle to contract rapidly, blood circulation depends on cardiac muscle contraction, and involuntary movements such as peristalsis of the gastrointestinal tract depend on smooth muscle function. In skeletal and cardiac muscle cells, the contractile apparatus is organized into functional units called sarcomeres, each of which is bordered by a structure known as the Z-disc. The Z-discs serve to anchor the actin filaments at the ends of the sarcomere. In smooth muscle cells, the actin- and myosin-rich contractile machinery is organized quite differently; rather than appearing in semicrystalline sarcomeric arrays, the contractile elements are obliquely organized. The smooth muscle contractile apparatus exhibits no Z-discs but instead has two other structures, dense bodies and dense plaques, that are thought to anchor and integrate the actin filaments within the muscle cells (2Small J.V. BioEssays. 1995; 17: 785-792Crossref PubMed Scopus (84) Google Scholar). Z-discs, dense bodies, and dense plaques thus appear to play parallel, central roles in muscle cytoarchitecture and function. Perhaps not surprisingly, given their similar roles in different muscle types, Z-discs, dense bodies and dense plaques are all enriched in α-actinin, a major structural protein present in all muscle cells (3Masaki T. Endo M. Ebashi S. J. Biochem. ( Tokyo ). 1967; 62: 630-632Crossref PubMed Scopus (183) Google Scholar, 4Geiger B. Dutton A.J. Tokuyasu K.T. Singer S.J. J. Cell Biol. 1981; 91: 614-628Crossref PubMed Scopus (256) Google Scholar). α-Actinin is an actin filament cross-linking protein that exists as an antiparallel homodimer in muscle and nonmuscle cells (5Burridge K. Feramisco J.R. Nature. 1981; 294: 565-567Crossref PubMed Scopus (177) Google Scholar, 6Endo T. Masaki T. J. Biochem. ( Tokyo ). 1982; 92: 1457-1468Crossref PubMed Scopus (53) Google Scholar). In nonmuscle cells, α-actinin is found periodically along the actin stress fibers, where it is thought to be involved in bundling actin thin filaments into stress fibers (7Lazarides E. Burridge K. Cell. 1975; 6: 289-298Abstract Full Text PDF PubMed Scopus (409) Google Scholar). Nonmuscle α-actinin is also present at the ends of the stress fibers, in focal adhesions, where it binds the cytoplasmic domain of the integrin β1 subunit, an observation that suggests a molecular mechanism by which α-actinin might link microfilaments to the cell membrane (8Otey C.A. Pavalko F.M. Burridge K. J. Cell Biol. 1990; 111: 721-729Crossref PubMed Scopus (650) Google Scholar, 9Pavalko F.M. Burridge K. J. Cell Biol. 1991; 114: 481-491Crossref PubMed Scopus (125) Google Scholar). α-Actinin also appears to play a key role in organizing the actin machinery in muscle; for example, high resolution electron microscopic analyses have illustrated that, in the Z-discs of striated muscle, α-actinin forms cross-links that anchor actin filaments (10Cheng N. Deatherage J.F. J. Cell Biol. 1989; 108: 1761-1774Crossref PubMed Scopus (29) Google Scholar, 11Deatherage J.F. Cheng N. Bullard B. J. Cell Biol. 1989; 108: 1775-1782Crossref PubMed Scopus (25) Google Scholar). Genetic studies have clarified substantially the central role of α-actinin in muscle structure and function. In Drosophila, α-actinin loss-of-function mutations perturb Z-disc integrity and disrupt myofibrillar attachments to tendon cells (12Fyrberg E. Kelly M. Ball E. Fyrberg C. Reedy M.C. J. Cell Biol. 1990; 110: 1999-2011Crossref PubMed Scopus (111) Google Scholar, 13Roulier E.M. Fyrberg C. Fyrberg E. J. Cell Biol. 1992; 116: 911-922Crossref PubMed Scopus (53) Google Scholar). These structural abnormalities are associated with reduced muscle function and lead to progressive paralysis and larval lethality (12Fyrberg E. Kelly M. Ball E. Fyrberg C. Reedy M.C. J. Cell Biol. 1990; 110: 1999-2011Crossref PubMed Scopus (111) Google Scholar). Certain weaker α-actinin alleles affect the morphology and function of thoracic muscles, leading to a flightless phenotype (12Fyrberg E. Kelly M. Ball E. Fyrberg C. Reedy M.C. J. Cell Biol. 1990; 110: 1999-2011Crossref PubMed Scopus (111) Google Scholar). It appears that, in Drosophila, α-actinin is not absolutely required for the assembly of the contractile machinery during development, since embryogenesis proceeds normally. Rather, α-actinin appears to play a critical role in anchoring and stabilizing the contractile filaments against the forces of muscle contraction. α-Actinin-rich structures also perform critical functions in muscle of the nematode Caenorhabditis elegans. In C. elegans, actin filaments of the body wall muscle cells are attached to the plasma membrane through α-actinin-rich structures called dense bodies (14Francis G.R. Waterston R.H. J. Cell Biol. 1985; 101: 1532-1549Crossref PubMed Scopus (198) Google Scholar). The function of the dense bodies resembles that of the Z-lines and the dense plaques of the vertebrate striated and smooth muscles, respectively. Although mutations in the C. elegans gene encoding α-actinin have not been described, mutations that affect other dense body constituents have been characterized. For example, nematode dense bodies contain vinculin and worms that lack vinculin function display disorganized muscle and are paralyzed (15Barstead R.J. Waterston R.H. J. Cell Biol. 1991; 114: 715-724Crossref PubMed Scopus (135) Google Scholar). Thus, a defect in the organization of the α-actinin-rich dense bodies compromises muscle cytoarchitecture and function. Despite the well established and apparently universal importance of α-actinin-rich structures for the subcellular organization and function of diverse muscle types, little is known about other proteins that cooperate with α-actinin in the establishment and maintenance of the contractile machinery. In order to better understand the molecular mechanism by which α-actinin participates in the stabilization of the contractile elements during muscle contraction, we sought to identify novel α-actinin-binding partners. Here we report the identification, purification, and characterization of a 36-kDa α-actinin-binding partner (p36) that is expressed in cardiac and smooth muscle. By a variety of binding studies, we demonstrate that the association of p36 with α-actinin is direct, specific, and saturable. We have also identified a higher molecular weight isoform, called p40, that is expressed exclusively in skeletal muscle and is colocalized with α-actinin at the Z-lines. Furthermore, the expression of both p36 and p40 is induced upon muscle differentiation, the that proteins play a critical role in the organization of actin filament within muscle cells. of the domain structures of p36 and p40 has the of an N-terminal PDZ domain C. BioEssays. PubMed Scopus Google and a C-terminal LIM domain M.C. Cell. Full Text PDF PubMed Scopus Google in each that the proteins described are to be the avian of the actinin-associated LIM protein a for the protein affected in facioscapulohumeral muscular J. Cell Biol. PubMed Scopus Google Scholar). chicken to avian smooth muscle proteins as described M.C. J. Biol. 1991; Full Text PDF PubMed Google Scholar). present in the with of and α-Actinin the M.C. J. Cell Biol. 1992; 116: PubMed Scopus (198) Google Scholar). The 36-kDa α-actinin-binding called the the different performed at The in of and against The of proteins on an with The proteins that to the to a in The proteins with a of in p36 on polyacrylamide and to p36 and to to the described by J. M. D. Biochem. 1992; PubMed Scopus Google Scholar). The by high a of p36 performed on a protein α-actinin as described M.C. J. Cell Biol. 1992; 116: PubMed Scopus (198) Google Scholar, M.C. J. Cell Biol. PubMed Scopus Google Scholar). The of the protein by by performed the described M.C. J. Cell Biol. 1992; 116: PubMed Scopus (198) Google Scholar). proteins by and to and the for in the of of In the α-actinin, a of protein to the to at with an by to the of Nature. PubMed Scopus Google with a of polyacrylamide in this For proteins by and to against chicken p36 chicken α-actinin by K. by to protein binding performed in as described M.C. J. Cell Biol. PubMed Scopus Google that the with chicken p36 at The in to a of of for in with of α-actinin, the determined a The of the chicken p36 by gel The protein the gel to a in The gel to the albumin and The of the chicken p36 determined by as described M.C. J. Biol. 1991; Full Text PDF PubMed Google Scholar). The in The proteins in albumin and The native molecular and the of p36 as described by and Scopus Google a of for the the of p36 on the amino acid sequence described embryo fibroblasts in with bovine The cell in with bovine serum and serum induced by the cells into with serum The against chicken of cells performed as described M.C. J. Cell Biol. PubMed Scopus Google Scholar). cells on for on for For we the by an and an by a cells on for in and performed as described by S. Cell. 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Full Text Full Text PDF PubMed Scopus Google Scholar). chicken in the and chicken in the of to of each in and the for and of each a cells the cDNAs the the on two and by of the chicken These to a the on the sequence of this two and that to internal sequence and to a the Using the we a chicken embryo and of the plaques and on both reveals that the two cDNAs encode proteins that are in a central of the we that the two cDNAs encode the p36 and p40 that we have In order to understand more precisely the mechanisms involved in the assembly of the during we have an to identify α-actinin-binding partners in muscle cells a avian smooth muscle by with of and present in each of by similar gel to and with α-actinin The α-actinin interacts with two The protein present in the is a LIM protein that we have as a binding partner for α-actinin M.C. J. Cell Biol. PubMed Scopus Google Scholar, M.C. J. Cell Biol. PubMed Scopus Google Scholar). identified in the screen is present in the and at a molecular of kDa called The of the α-actinin in this is in C. In an to the of the we performed a The smooth proteins in the by to and the with α-actinin in the of protein in the of an of α-actinin in the α-actinin interacts with Furthermore, the binding of to p36 is in the of α-actinin but not in of an of a and between the α-actinin, and a 36-kDa protein expressed in smooth muscle cells. 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In we two at and a at in the encoding p36 are replaced by a and a respectively, in the encoding that are to at the Analysis of the amino acid sequences of avian p36 and p40 a high of to recently described rat and human muscle proteins called the J. Cell Biol. PubMed Scopus Google Scholar). Thus, we that the proteins we have described the and avian of We suggest the muscle on in smooth muscle, and the muscle on in skeletal muscle. In this paper, we the identification, purification, and characterization of avian a cardiac and smooth muscle protein that interacts with the α-actinin. We have a for avian smooth muscle and have variety of binding to demonstrate a direct, specific, and saturable between and α-actinin. and α-actinin display a moderate in vitro with an of we have native to demonstrate that and α-actinin are present in the molecular vivo. for the in of the their within cells. In the of studies, we also identified a protein that is closely related to and that is expressed exclusively in skeletal muscle. Thus, all three vertebrate muscle types expression of an ALP than which a protein of smooth muscle, muscle cells appear to be the of ALP expression in the The that appear to be expressed substantially in nonmuscle suggests that their role may be related to differentiated function of muscle. In of this we that ALP expression during smooth muscle and skeletal muscle is to the of differentiated a role for ALP in muscle organization function. The rat skeletal muscle of ALP described recently by J. Cell Biol. PubMed Scopus Google identified the protein in the of a for PDZ domain proteins in skeletal muscle and described an between ALP and α-actinin a and performed studies to that the gene encoding ALP to human J. Cell Biol. PubMed Scopus Google to a of that is in with facioscapulohumeral muscular the of muscle C. PubMed Scopus Google Scholar). It has been postulated that the the expression of gene that is for of muscle function 1995; PubMed Scopus (29) Google Scholar). Thus, the ALP gene has as a for the gene affected in facioscapulohumeral muscular might be for a protein that an role in muscle J. Cell Biol. PubMed Scopus Google that rat dramatically up-regulated expression during skeletal muscle on the avian and the of by a for of native ALP muscle, the characterization of and association with α-actinin, and the of a smooth muscle of The of ALP of role in muscle. cloning and of the cDNAs encoding chicken and has their relationship to each The N-terminal and C-terminal of the proteins are identical in the two in an internal present in are replaced by a unique sequence of in on the of the sequences this central it appears that p36 and p40 are by by We that the unique central in the ALP functional the of the different muscle in which they are exclusively be required to the of novel In to the in the central of and we also two that are to affect the amino acid sequence of the the at in to and the at in to In both we an in the skeletal muscle and a in the It is that during the of the and have no that they be the of to which in an to in the of a and thus protein structure and function Biochem. Full Text PDF PubMed Scopus Google Scholar). are present at in skeletal muscle J. 17: PubMed Scopus Google with the observation that the that is a skeletal muscle exhibits an the of the are an as for since is required for the Biochem. Full Text PDF PubMed Scopus Google Scholar). The sequence by of that the amino acid at in of is a thus, it not be be be required to the at the of the cDNAs has The domain structures of both and suggest their ability to protein partners. The two proteins each display an identical N-terminal PDZ domain and C-terminal LIM PDZ are acid that C. BioEssays. PubMed Scopus Google Scholar). LIM are amino acids in Nature. 1990; PubMed Scopus Google that two M.C. S. PubMed Scopus Google and also serve as protein binding M.C. Cell. Full Text PDF PubMed Scopus Google Scholar). The of two protein binding in and suggests that the proteins as a within the contractile machinery of muscle cells. into the of the α-actinin binding of ALP has recently domain that it is the PDZ domain of that interacts with α-actinin J. Cell Biol. PubMed Scopus Google Scholar). LIM also protein binding it be of importance in the to protein partner partners with the LIM domain present in The that ALP are expressed in muscle cells, are up-regulated during muscle differentiation, and are associated with α-actinin at key for muscle cytoarchitecture the that proteins may cooperate with α-actinin to the contractile machinery of muscle cells. this and the human that suggest the ALP gene as a for the gene that is affected in facioscapulohumeral muscular J. Cell Biol. PubMed Scopus Google it be of to the of ALP in facioscapulohumeral muscular and to role in muscle structure and function. We the of the for and in S. for with protein and J. for in We are to for K. 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