Dataset Information

Characterization of the nonmuscle myosin heavy chain IIB promoter: regulation by E2F.

ABSTRACT: To identify DNA sequences important for the transcriptional regulation of the nonmuscle myosin heavy chain IIB (NMMHC-IIB) gene we isolated and sequenced genomic clones that contain the promoter of the gene for both human and mouse. In addition to considerable homology in the first (untranslated) exon (91%) we found 80% sequence identity in the 700 base pairs immediately upstream of the major start of transcription (+1) as well as significant homologies as far as 1500 base pairs upstream. The promoter region was characterized using luciferase reporter constructs transiently transfected into NIH3T3 cells. Consensus binding sites for several known transcription factors are present that are completely conserved between the mouse and human genes, including CRE/ATF, Sp1, CAAT, and the cell-cycle transcription factor E2F. Gel shift assays indicated that E2F can bind to its putative binding site in vitro. To test whether this site is functional we cotransfected NMMHC-IIB promoter constructs driving luciferase with a vector expressing E2F-1. The E2F-1 vector stimulated luciferase activity from an intact promoter whereas mutation of the site eliminates binding and diminishes transactivation. These data provide strong evidence that E2F or an E2F-related transcription factor is involved in the regulation of nonmuscle myosin expression.

SUBMITTER: Weir L

PROVIDER: S-EPMC6148259 | biostudies-literature | 1996

REPOSITORIES: biostudies-literature

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Characterization of the nonmuscle myosin heavy chain IIB promoter: regulation by E2F.

Weir L L Chen D D

Gene expression 19960101 1

To identify DNA sequences important for the transcriptional regulation of the nonmuscle myosin heavy chain IIB (NMMHC-IIB) gene we isolated and sequenced genomic clones that contain the promoter of the gene for both human and mouse. In addition to considerable homology in the first (untranslated) exon (91%) we found 80% sequence identity in the 700 base pairs immediately upstream of the major start of transcription (+1) as well as significant homologies as far as 1500 base pairs upstream. The pr ...[more]

PMID: 8931991

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Project description:Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.Significance of the studyPhosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

Dataset Information

Characterization of the nonmuscle myosin heavy chain IIB promoter: regulation by E2F.

Publications

Characterization of the nonmuscle myosin heavy chain IIB promoter: regulation by E2F.

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