Identification, tissue expression and chromosomal localization of human Obscurin-MLCK, a member of the titin and Dbl families of myosin light chain kinases

Members of the Dbl family of guanine nucleotide exchange factors (GEFs) have important roles in the organization of actin-based cytoskeletal structures of a wide variety of cell types. Through the activation of members of the Rho family of GTP signaling molecules, these exchange factors elicit cytoskeletal alterations that allow cellular remodeling. As important regulators of RhoGTPase activity, members of this family are candidates for mediating the RhoGTPase activation and cytoskeletal changes that occur during cardiac development and during the myocardial response to hypertrophic stimuli. In this study, we characterize a novel human gene that is expressed in skeletal and cardiac muscle and has putative functional domains similar to those found in members of both the Dbl family of GEFs and the titin family of myosin light chain kinases (MLCK). The cDNA sequence of this gene, which has been designated Obscurin-myosin light chain kinase (Obscurin-MLCK), would be predicted to encode for at least 68 immunoglobulin domains, two fibronectin domains, one calcium/calmodulin binding domain, a RhoGTP exchange factor domain, and two serine-threonine kinase domains. The combination of the putative Rho GEF and two kinase domains has not been noted in any other members of the titin or Dbl families. Alternative splicing allows the generation of a number of unique Obscurin-MLCK isoforms that contain various combinations of the functional domains. One group of isoforms is comparable to Unc-89, a Caenorhabditis elegans sarcomere-associated protein, in that they contain a putative RhoGEF domain and multiple immunoglobulin repeats. Other isoforms more closely resemble MLCK, containing one or both of the putative carboxy-terminal serine-threonine kinase domains. The modular nature of the Obscurin-MLCK isoforms indicates that it may have an array of functions important to cardiac and skeletal muscle physiology.     

Orthologous relationship of obscurin and Unc-89: phylogeny of a novel family of tandem myosin light chain kinases

Myosin light chain kinases (MLCK) are a family of signaling proteins that are required for cytoskeletal remodeling in myocytes. Recently, two novel MLCK proteins, SPEG and obscurin-MLCK, were identified with the unique feature of two tandemly-arranged MLCK domains. In this study, the evolutionary origins of this MLCK subfamily were traced to a probable orthologue of obscurin-MLCK in Drosophila melanogaster, Drosophila Unc-89, and the MLCK kinase domains of zebrafish SPEG, zebrafish obscurin-MLCK, and human SPEG were characterized. Phylogenetic analysis of the MLCK domains indicates that the carboxy terminal kinase domains of obscurin-MLCK, SPEG and Unc-89 are more closely related to each other than to the amino terminal kinase domains or to other MLCKs, supporting the assertion that obscurin-MLCK is the vertebrate orthologue of Caenorhabditis elegans Unc-89, a giant multidomain protein that is required for normal myofibril assembly. The apparent lack of an invertebrate orthologue of SPEG and the conserved exon structure of the kinase domains between SPEG and obscurin-MLCK suggests that SPEG arose from obscurin-MLCK by a gene duplication event. The length of the primary amino acid sequence between the immunoglobulin (Ig) domains associated with the MLCK motifs is conserved in obscurin-MLCK, SPEG and C. elegans Unc-89, suggesting that these putative protein interaction domains may target the kinases to highly conserved intracellular sites. The conserved arrangement of the tandem MLCK domains and their relatively restricted expression in striated muscle indicates that further characterization of this novel MLCK subfamily may yield important insights into cardiac and skeletal muscle physiology.Edited by D. Tautz     

Obscurin-like 1, OBSL1, is a novel cytoskeletal protein related to obscurin

Cytoskeletal adaptor proteins serve vital functions in linking the internal cytoskeleton of cells to the cell membrane, particularly at sites of cell-cell and cell-matrix interactions. The importance of these adaptors to the structural integrity of the cell is evident from the number of clinical disease states attributable to defects in these networks. In the heart, defects in the cytoskeletal support system that surrounds and supports the myofibril result in dilated cardiomyopathy and congestive heart failure. In this study, we report the cloning and characterization of a novel cytoskeletal adaptor, obscurin-like 1 (OBSL1), which is closely related to obscurin, a giant structural protein required for sarcomere assembly. Multiple isoforms arise from alternative splicing, ranging in predicted molecular mass from 130 to 230 kDa. OBSL1 is located on human chromosome 2q35 within 100 kb of SPEG, another gene related to obscurin. It is expressed in a broad range of tissues and localizes to the intercalated discs, to the perinuclear region, and overlying the Z lines and M bands of adult rat cardiac myocytes. Further characterization of this novel cytoskeletal linker will have important implications for understanding the physical interactions that stabilize and support cell-matrix, cell-cell, and intracellular cytoskeletal connections.     

The effect of hyperbaric oxygenation on thein vitro growth ofEscherichia coli in environments with and without blood cells

The aim of this study was to investigate whether thein vitro presence of blood cells influences the anti-microbial activity of hyperbaric oxygen (HBO) againstEscherichia coli. FiftyE. coli isolates from clinical samples were used in the study. A small number of colonies belonging to each isolate from the nutrient media were transferred into two K₃EDTA tubes (the blood group) and two Mueller-Hinton broth tubes (the broth group). Then, both groups were divided into subgroups according to whether HBO was administered (HBO subgroup) or not (non-HBO subgroup). HBO treatment was applied for one hour at 2.5 absolute atmospheres. The tubes in the non-HBO subgroup were left at room temperature during this period. Subsequently, all the tubes were cultured on Mueller-Hinton and Eosin Methylene Blue agar using the quantitative counting technique. After 18 to 24 h incubation at 37 °C, the colonies formed in the plates were counted. In the blood group, compared with non-HBO subgroup samples, the number of colonies decreased in 56% of samples, increased in 32% of samples and did not change in 12% of samples in the HBO subgroup. Whereas, in the broth group the number of colonies decreased in only 32% of samples increased in 38% of samples and did not change in 30% of samples in the HBO subgroup compared with the non-HBO subgroup. The difference between the blood and the broth groups revealed a statistical significance using Pearson’s Chi-square test (P=0.025). We concluded that the antibacterial effect of HBO onE. coli increases in the cellular environment belonging to the host organism.     

Developmental expression and differential cellular localization of obscurin and obscurin‐associated kinase in cardiac muscle cells

The article to which this erratum refers, J Cell Biochem 2007: 10.1002/jcb.21551 , was originally published online in Wiley InterScience 27 Nov 2007. © 2008 Wiley‐Liss, Inc.     

Targeted deletion of the zebrafish obscurin A RhoGEF domain affects heart, skeletal muscle and brain development

Obscurin is a giant structural and signaling protein that participates in the assembly and structural integrity of striated myofibrils. Previous work has examined the physical interactions between obscurin and other cytoskeletal elements but its in vivo role in cell signaling, including the functions of its RhoGTPase Exchange Factor (RhoGEF) domain have not been characterized. In this study, morpholino antisense oligonucleotides were used to create an in-frame deletion of the active site of the obscurin A RhoGEF domain in order to examine its functions in zebrafish development. Cardiac myocytes in the morphant embryos lacked the intercalated disks that were present in controls by 72 and, in the more severely affected embryos, the contractile filaments were not organized into mature sarcomeres. Neural abnormalities included delay or loss of retinal lamination. Rescue of the phenotype with co-injection of mini-obscurin A expression constructs demonstrated that the observed effects were due to the loss of small GTPase activation by obscurin A. The immature phenotype of the cardiac myocytes and the retinal neuroblasts observed in the morphant embryos suggests that obscurin A-mediated small GTPase signaling promotes tissue-specific cellular differentiation. This is the first demonstration of the importance of the obscurin A-mediated RhoGEF signaling in vertebrate organogenesis and highlights the central role of obscurin A in striated muscle and neural development.