Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/ mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP (ERK1 and ERK2) influences cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain (MLCK) activity leading to phosphorylation of chains (MLC). Inhibition causes decreased MLCK function, MLC phosphorylation, cell migration on proteins. In contrast, expression mutationally active...

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by Rho family of guanosine triphosphatases. Cytoskeletal dynamics primarily modulated interactions actin and myosin II that regulated light chain kinase (MLCK)–mediated phosphorylation regulatory (MLC). 1 (PAK1) phosphorylates MLCK, resulting decreased MLCK activity. activity MLC were decreased, cell spreading was inhibited baby hamster kidney–21 HeLa cells expressing constitutively active PAK1. These data...

The introduction of impermeant probes such as antibodies and other proteins into living cells without compromising physiological function is an important approach for studying cellular regulatory mechanisms. Many techniques including direct microinjection, liposome-mediated delivery, fusion red cell ghosts, osmotic lysis pinocytic vesicles have been used to introduce intact cells. We a modification the voltage-discharge technique physiologically responsive pheochromocytoma cultured In this...

A nuclear isoform of myosin I β that contains a unique 16–amino acid amino-terminal extension has been identified. An affinity-purified antibody to the peptide demonstrated staining. Confocal and electron microscopy revealed colocalized with RNA polymerase II in an α-amanitin– actinomycin D–sensitive manner. The coimmunoprecipitated blocked vitro synthesis. This appears be complex may affect transcription.

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by catalytic subunit cAMP-dependent protein kinase cGMP-dependent was investigated. When which has calmodulin bound is phosphorylated 1 mol phosphate incorporated per or platelet with no effect on activity. Phosphorylation when not results in incorporation 2 significantly decreases decrease activity due to a 5 7-fold increase amount required...

AbstractThe serine/threonine kinase PAK4 is an effector molecule for the Rho GTPase Cdc42. differs from other members of PAK family in both sequence and function. Previously we have shown that important function this to mediate induction filopodia response activated Since previous characterization was carried out only with wild-type kinase, generated a constitutively active mutant determine whether it has functions. Expression fibroblasts led transient filopodia, which consistent its role as...

The role of cyclic adenosine monophosphate-mediated phosphorylation myosin light chain kinase in relaxing smooth muscle was examined. immunoprecipitated from tissue extracts and the phosphate content determined. addition forskolin to resting or methacholine-contracted muscles resulted an increase a relaxation contracted muscles. These findings suggest that is one reactions process by which monophosphate causes muscle.

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in regulation epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either catalytic domain kinase (tMK) or beta-galactosidase gene (beta-gal). Autoradiograms sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis immunoprecipitated 32Pi-labeled...

The role of the intranuclear movement chromatin in gene expression is not well-understood. Herpes simplex virus forms replication compartments (RCs) infected cell nuclei as sites viral DNA and late transcription. These structures develop from small that grow size, move, coalesce. Quantitative analysis RC trajectories, derived 4D images, shows most RCs move by directed motion. Directed impaired presence actin myosin inhibitors well a transcription inhibitor. In addition, coalesce at...

The role of myosin phosphorylation in regulating smooth muscle contraction has been investigated by quantitating the phosphate content tracheal frozen during or relaxation. Myosin was purified from quick-frozen samples with aid antibodies prepared against myosin, and determined after separation nonphosphorylated phosphorylated subunits isoelectric focusing. increased an initial value 0.50 to 1.1 mol phosphate/mol within 3 min addition 100 microM methacholine resting muscle. coincided...

Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) is the major mediator of angiogenic effects VEGF. In addition to its well known role as a membrane receptor that activates multiple signaling pathways, VEGFR2 also has nuclear localization. However, what does in nucleus still unknown. present report we show that, endothelial cells, interacts with several proteins, including Sp1, transcription factor been implicated regulation genes needed for angiogenesis. By vivo chromatin...

Actin, a major component of the cytoplasm, is also abundant in nucleus. Nuclear actin involved variety nuclear processes including transcription, chromatin remodeling, and intranuclear transport. Nevertheless, regulation by posttranslational modifications has not been investigated. We now show that modified SUMO2 SUMO3 computational modeling site-directed mutagenesis identified K68 K284 as critical sites for SUMOylating actin. present model actin–SUMO complex SUMOylation required localization

The Swank Dacron blood filter has been clinically tested to determine if its use would improve hospital mortality following open heart surgery and the incidence of fat nonfat emboli. rate fell from 19.8% 6.5%. Cerebral emboli were reduced an 31% 4.1%. not affected by filter. It is concluded that effective means reducing microemboli during heart-lung bypass.

Actin is abundant in the nucleus and it clear that nuclear actin has important functions. However, mystery surrounds absence of classical filaments nucleus. To address this question, we investigated how polymerizing into persistent affected transcription by RNA polymerase II. Nuclear impaired dynamics sequestering actin. Polymerizing stable disrupted interaction with II correlated localization, dynamics, gene recruitment, reduced global cell proliferation. crosslinking vitro similarly...

Abstract Class I histone deacetylases (HDACs) are known to remove acetyl groups from tails. This liberates positive charges on the tail and allows for tighter winding of DNA, preventing transcription factor binding gene activation. Although functions HDAC proteins becoming apparent both biochemically clinically, how this class is regulated remains poorly understood. We identified a novel interaction between nuclear actin 1 2. Nuclear has been previously shown interact with growing list...

We produced and affinity-purified polyclonal antibodies to adrenal myosin I. These recognize I by Western blot analysis (116 kDa) inhibit the actin-activated ATPase activity of purified They also a 120-kDa protein in extracts prepared from many different cell lines. Fluorescence microscopy demonstrated presence immunoreactive material perinuclear region, leading edges, nuclei 3T3 cells. nuclear staining mouse oocytes at germinal vesicle stage pronuclei during fertilization. Confocal...

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization microinjection studies were performed using monospecific antibodies to IIA IIB isotypes. Each antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin demonstrate differential expression meiotic maturation following fertilization: only isoform detects metaphase spindles or accumulates mitotic cleavage furrow. In unfertilized oocyte, both are...

We have previously demonstrated that vasopressin increases the water permeability of inner medullary collecting duct (IMCD) by inducing trafficking aquaporin-2 to apical plasma membrane and this response is dependent on intracellular calcium mobilization calmodulin activation. Here, we address hypothesis mediated in part through activation calcium/calmodulin-dependent myosin light chain kinase (MLCK) regulation non-muscle II. Immunoblotting immunocytochemistry presence MLCK, regulatory...