Myocyte enhancer factor 2 (MEF2) has been implicated in the complex hierarchical regulation of muscle-specific gene expression and differentiation. While the MyoD family members are able to initiate the skeletal muscle differentiation program, whether MEF2 is sufficient in directing skeletal muscle differentiation is still controversial. Furthermore, how MEF2 transactivates its target genes is not fully understood. It has been suggested that the interactions of MEF2 with other factors modify its transcriptional activity. Therefore, the identification of MEF2-interacting factors may be important in understanding the mechanism by which MEF2 activates its target genes. In this study, a mitogen-activated protein kinase (MAP kinase), ERK5/BMK1 was found to interact with MEF2 in a yeast two hybrid screen. The interaction was confirmed by a glutathione S -transferase-pull down assay and a co-immunoprecipitation study indicating that endogenous ERK5 and MEF2 interact with each other in vivo . The interacting domain of MEF2 was mapped to the N-terminus which contains the highly conserved MADS and MEF2 domains. Functionally, ERK5/BMK1 was able to phosphorylate MEF2 in vitro . Furthermore, when cotransfected with ERK5/BMK1, the transactivation capacity of MEF2 was enhanced. These results suggest that the functions of MEF2 could be regulated through ERK5/BMK1.

Our previous studies showed immunological and functional similarities, as well as partial sequence homology, between the enzymatically inactive alternatively spliced variant of human ␤-galactosidase (S-gal) and the 67-kDa elastin/laminin-binding protein (EBP) from sheep. To define the genetic origin of the EBP further, a full-length human S-gal cDNA clone was constructed and subjected to in vitro transcription/translation. The cDNA was also transfected into COS-1 cells and into the EBP-deficient smooth muscle cells (SMC) from sheep ductus arteriosus (DA). In vitro translation yielded an unglycosylated form of the S-gal protein, which immunoreacted with anti-␤-galactosidase antibodies and bound to elastin and laminin affinity columns. S-gal cDNA transfections into COS-1 and DA SMC increased expression of a 67-kDa protein that immunolocalized intracellularly and to the cell surface and, when extracted from the cells, bound to elastin. The S-gal-transfected cells displayed increased adherence to elastin-covered dishes, consistent with the cell surface distribution of the newly produced S-gal-encoded protein. Transfection of DA SMC additionally corrected their impaired elastic fiber assembly. These results conclusively identify the 67-kDa splice variant of ␤-galactosidase as EBP.Virtually all cell types, including tumor cells, interact with the extracellular matrix (ECM) 1 during certain stages of their development. Such contacts may be strictly adhesive or can transduce signals from the ECM to the intracellular machinery (1). These significant cell matrix interactions are mediated through specialized cell surface receptors (2, 3). Interactions between cells and elastin are mediated by a non-integrin cell surface receptor complex consisting of three protein subunits (4 -6). Two of these subunits (61-and 55-kDa subunits) are cell membrane-associated proteins that immobilize the third, a 67-kDa peripheral subunit called the elastin-binding protein (EBP). The EBP binds predominantly to the repeating VGVAPG hydrophobic domains on elastin, but it may also bind to other similar hydrophobic domains on elastin (7), and to the LGTIPG sequence on laminin (5, 8). Moreover, the EBP also interacts with moieties containing ␤-galactosugars through a separate "lectin-like" binding domain. However, binding of ␤-galactosugar-bearing moieties to the lectin domain of the EBP causes such conformational changes in the 67-kDa protein that it loses its affinity for elastin and separates from the other subunits of the elastin receptor. Thus, the EBP can be shed from the cell surface by interactions with galactosugars (galactose, lactose) or with N-acetylgalactosamine-containing glycosaminoglycans (chondroitin sulfate, dermatan sulfate), which bind to its lectin site (9-11). The EBP appears to be directly involved in the generation of intracellular signal transmission after contact with its matrix ligands (6). Binding of elastin-derived peptides to the EBP, when present on the cell surface, resulted in a rapid and transient increase in free ...

To study the primary structure of human acetylcholinesterase (AcChoEase; EC 3.1.1.7) and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with greater than or equal to 50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase (BtChoEase; EC 3.1.1.8). However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy (-117 kcal/mol) downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the BtChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification.

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase (BtChoEase; EC 3.1

We introduce a method by which periplasmic proteins can be released rapidly, simply, and quantitatively by treating cells with chloroform. All the amino acid-binding proteins tested maintained their activity during chloroform treatment. This method makes practical the analysis of the periplasmic protein complement of a large number of strains.