ping (7, 12, 27). These studies have also demonstrated central roles for myosin II in cell locomotion and in maintenance of cortical tension (17,27,38). Although the roles of myosin II in nonmuscle cells are generally becoming clear, the mechanisms regulating localization and activity of myosin II in these settings are not well understood. Myosin light chain (MLC) 1 phosphorylation has a clear role in regulating mammalian smooth and nonmuscle myosin II motor function, and is also thought to participate in regulation of filament assembly in nonmuscle cells (36). Although phosphorylation of the myosin II heavy chain (MHC) has also been observed in several types of mammalian nonmuscle cells in a variety of settings (4,18,23,26), the de-
Myosin II assembly and localization into the cytoskeleton is regulated by heavy chain phosphorylation in Dictyostelium. The enzyme myosin heavy chain kinase A (MHCK A) has been shown previously to drive myosin filament disassembly in vitro and in vivo. MHCK A is noteworthy in that its catalytic domain is unrelated to the conventional families of eukaryotic protein kinases. We report here the cloning and initial biochemical characterization of another kinase from Dictyostelium that is related to MHCK A. When the segment of this protein that is similar to the MHCK A catalytic domain was expressed in bacteria, the resultant protein displayed efficient autophosphorylation, phosphorylated Dictyostelium myosin II, and also phosphorylated a peptide substrate corresponding to a portion of the myosin II tail. We have therefore named this gene myosin heavy chain kinase B. These results provide the first confirmation that sequences in other proteins that are related to the MHCK A catalytic domain can also encode protein kinase activity. It is likely that the related segments of homology present in rat eukaryotic elongation factor-2 kinase and a putative nematode eukaryotic elongation factor-2 kinase also encode the catalytic domains of those enzymes.
Vascular smooth muscle cells (VSMCs) reversibly coordinate the expression of VSMC-specific genes and the genes required for cell cycle progression. Here we demonstrate that isoforms of the MEF2/RSRF transcription factor are expressed in VSMCs and in vascular tissue. The MEF2A DNA-binding activity was upregulated when quiescent VSMCs were stimulated to proliferate with serum mitogens. The serum-induction of MEF2A DNA-binding activity occurred approximately 4 h following serum activation, and this correlated with an increase in the level of MEF2A protein without changes in the level of MEF2A mRNA or protein stability. These results indicate that MEF2A induction by serum is regulated at the level of translation.
Myosin heavy chain kinase A (MHCK A) participates in the regulation of cytoskeletal myosin assembly in Dictyostelium, driving filament disassembly via phosphorylation of sites in the myosin tail. MHCK A contains an amino-terminal coiled-coil domain, a novel central catalytic domain, and a carboxyl-terminal domain containing a 7-fold WD repeat motif. We have overexpressed MHCK A truncation constructs to clarify the roles of each of these domains. Recombinant full-length MHCK A, MHCK A lacking the predicted coiled-coil domain, and MHCK A lacking the WD repeat domain were expressed at high levels in Dictyostelium cells lacking endogenous MHCK A. Biochemical analysis of the purified proteins demonstrates that the putative coiled-coil domain is responsible for the oligomerization of the MHCK A holoenzyme. Removal of the WD repeat domain had no effect on catalytic activity toward a synthetic peptide, but did result in a 95% loss of protein kinase activity when native myosin filaments were used as the substrate. Cellular analysis confirms that the same severe loss of activity against myosin occurs in vivo when the WD repeat domain is eliminated. These results suggest that the WD repeat domain of MHCK A serves to target this enzyme to its physiological substrate.Conventional myosin (myosin II) is involved in a wide range of contractile events in eukaryotic cells. In Dictyostelium, genetic and cellular analyses have demonstrated roles for myosin in the maintenance of cortical tension, cytokinesis, morphogenesis, capping of receptors, and cell locomotion (1-5). Although the roles of myosin II appear similar in many cell types, the in vivo mechanisms regulating myosin II assembly and activity in nonmuscle cells are not well understood.Assembly of Dictyostelium myosin II bipolar filaments can be regulated by phosphorylation on the myosin II heavy chain (MHC).1 Two distinct MHC kinases (MHCKs) have been purified and cloned from Dictyostelium. A 130-kDa MHCK (MHCK A), discussed below, is expressed during both growth and development. An 84-kDa MHCK that is expressed only during development appears to contain two distinct catalytic domains, one related to protein kinase C (6 -8) and one related to diacylglycerol kinases (9). Both MHCKs are capable of phosphorylating threonine residues on the myosin tail and can drive myosin bipolar filament disassembly in vitro. A third kinase, related to MHCK A, has recently been cloned from Dictyostelium (10), but is has not yet been established whether this protein regulates myosin assembly in vivo. The in vitro target sites of MHCK A have been mapped to threonines 1823, 1833, and 2029 in the tail region of the MHC (11, 12). The physiological significance of these sites was demonstrated by mutating the sites either to alanine (3X ALA myosin) to create a nonphosphorylatable MHC or to aspartic acid (3X ASP myosin) to mimic phosphorylated MHC (13). In vivo, cells expressing 3X ALA myosin display severe myosin II overassembly in the cytoskeleton, whereas cells expressing 3X ASP myosin display se...
In the promoters of many immediate early genes, including c-fos, CArG DNA regulatory elements mediate basal constituitive expression and rapid and transient serum induction. CArG boxes also occur in the promoters of muscle-specific genes, including skeletal alpha-actin, where it confers muscle-specific expression. These elements are regulated, at least in part, by the ubiquitous transcription factors serum response factor (SRF) and YY1. The homeobox transcription factor Phox1/MHox has also been implicated in regulation of the c-fos CArG element and is thought to function by facilitating SRF binding to DNA. Here, we provide in vitro and in vivo evidence that the mechanism of YY1 repression of CArG elements results from competition with SRF for overlapping binding sites. We describe in detail the binding sites of YY1 and SRF through serial point mutations of the skeletal alpha-actin proximal CArG element and identify a mutation that dramatically reduces YY1 binding but retains normal SRF binding. YY1 competes with SRF for binding to wild-type CArG elements, but not to this point mutant in vitro. This mutant is sufficient for muscle-specific expression in vivo but is much less sensitive to repression by YY1 overexpression. We utilized the YY1/SRF competition to address the role of Phox1 at these elements. Phox1 overexpression did not diminish YY1-mediated repression, suggesting that transcriptional activation by Phox1 does not result from enhanced SRF binding to these elements. These methods may prove to be useful for assessing interactions between other CArG element regulatory factors.
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