scholarly journals Spatial and temporal control of nonmuscle myosin localization: identification of a domain that is necessary for myosin filament disassembly in vivo.

1991 ◽  
Vol 112 (4) ◽  
pp. 677-688 ◽  
Author(s):  
T T Egelhoff ◽  
S S Brown ◽  
J A Spudich
Keyword(s):  
Critical Role ◽  
Surface Proteins ◽  
Myosin Filament ◽  
Chain Gene ◽  
Multicellular Development ◽  
Filament Assembly ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

Myosin null mutants of Dictyostelium are defective for cytokinesis, multicellular development, and capping of surface proteins. We have used these cells as transformation recipients for an altered myosin heavy chain gene that encodes a protein bearing a carboxy-terminal 34-kD truncation. This truncation eliminates threonine phosphorylation sites previously shown to control filament assembly in vitro. Despite restoration of growth in suspension, development, and ability to cap cell surface proteins, these delta C34-truncated myosin transformants display severe cytoskeletal abnormalities, including excessive localization of the truncated myosin to the cortical cytoskeleton, impaired cell shaped dynamics, and a temporal defect in myosin dissociation from beneath capped surface proteins. These data demonstrate that the carboxy-terminal domain of myosin plays a critical role in regulating the disassembly of the protein from contractile structures in vivo.

scholarly journals FCP1, a Phosphatase Specific for the Heptapeptide Repeat of the Largest Subunit of RNA Polymerase II, Stimulates Transcription Elongation

2002 ◽  
Vol 22 (21) ◽  
pp. 7543-7552 ◽  
Author(s):  
Subhrangsu S. Mandal ◽  
Helen Cho ◽  
Sungjoon Kim ◽  
Kettly Cabane ◽  
Danny Reinberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Carboxy Terminal Domain ◽  
Transcription Factor Iif ◽  
Transcript Elongation ◽  
Rnap Ii ◽  
Carboxy Terminal

ABSTRACT FCP1, a phosphatase specific for the carboxy-terminal domain of RNA polymerase II (RNAP II), was found to stimulate transcript elongation by RNAP II in vitro and in vivo. This activity is independent of and distinct from the elongation-stimulatory activity associated with transcription factor IIF (TFIIF), and the elongation effects of TFIIF and FCP1 were found to be additive. Genetic experiments resulted in the isolation of several distinct fcp1 alleles. One of these alleles was found to suppress the slow-growth phenotype associated with either the reduction of intracellular nucleotide concentrations or the inhibition of other transcription elongation factors. Importantly, this allele of fcp1 was found to be lethal when combined individually with two mutations in the second-largest subunit of RNAP II, which had been shown previously to affect transcription elongation.

scholarly journals Tubulin phosphorylation by casein kinase II is similar to that found in vivo.

1987 ◽  
Vol 105 (4) ◽  
pp. 1731-1739 ◽  
Author(s):  
L Serrano ◽  
J Díaz-Nido ◽  
F Wandosell ◽  
J Avila
Keyword(s):  
Neuroblastoma Cells ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Carboxy Terminal Domain ◽  
Phosphatase Treatment ◽  
Microtubule Protein ◽  
Carboxy Terminal ◽  
Brain Tubulin

Purified brain tubulin subjected to an exhaustive phosphatase treatment can be rephosphorylated by casein kinase II. This phosphorylation takes place mainly on a serine residue, which has been located at the carboxy-terminal domain of the beta-subunit. Interestingly, tubulin phosphorylated by casein kinase II retains its ability to polymerize in accordance with descriptions by other authors of in vivo phosphorylated tubulin. Moreover, the V8 phosphopeptide patterns of both tubulin phosphorylated in vitro by casein kinase II and tubulin phosphorylated in vivo in N2A cells are quite similar, and different from that of tubulin phosphorylated in vitro by Ca/calmodulin-dependent kinase II. On the other hand, we have found an endogenous casein kinase II-like activity in purified brain microtubule protein that uses GTP and ATP as phosphate donors, is inhibited by heparin, and phosphorylates phosphatase-treated tubulin. Thus it appears that a casein kinase II-like activity should be considered a candidate for the observed phosphorylation of beta-tubulin in vivo in brain or neuroblastoma cells.

scholarly journals Myosin storage myopathy mutations yield defective myosin filament assembly in vitro and disrupted myofibrillar structure and function in vivo

2017 ◽  
Vol 26 (24) ◽  
pp. 4799-4813 ◽  
Author(s):  
Meera C Viswanathan ◽  
Rick C Tham ◽  
William A Kronert ◽  
Floyd Sarsoza ◽  
Adriana S Trujillo ◽  
...  
Keyword(s):  
Structure And Function ◽  
Myosin Filament ◽  
Filament Assembly ◽  
Myosin Storage Myopathy ◽  
And Function

scholarly journals Sequence and transmembrane topology of MEC-4, an ion channel subunit required for mechanotransduction in Caenorhabditis elegans.

1996 ◽  
Vol 133 (5) ◽  
pp. 1071-1081 ◽  
Author(s):  
C C Lai ◽  
K Hong ◽  
M Kinnell ◽  
M Chalfie ◽  
M Driscoll
Keyword(s):  
Caenorhabditis Elegans ◽  
Ion Channel ◽  
Critical Role ◽  
Comparative Sequence Analysis ◽  
Mechanical Stimuli ◽  
Transmembrane Topology ◽  
C Elegans ◽  
Carboxy Terminal

The process by which mechanical stimuli are converted into cellular responses is poorly understood, in part because key molecules in this mode of signal transduction, the mechanically gated ion channels, have eluded cloning efforts. The Caenorhabditis elegans mec-4 gene encodes a subunit of a candidate mechanosensitive ion channel that plays a critical role in touch reception. Comparative sequence analysis of C. elegans and Caenorhabditis briggsae mec-4 genes was used to initiate molecular studies that establish MEC-4 as a 768-amino acid protein that includes two hydrophobic domains theoretically capable of spanning a lipid bilayer. Immunoprecipitation of in vitro translated mec-4 protein with domain-specific anti-MEC-4 antibodies and in vivo characterization of a series of mec-4lacZ fusion proteins both support the hypothesis that MEC-4 crosses the membrane twice. The MEC-4 amino- and carboxy-terminal domains are situated in the cytoplasm and a large domain, which includes three Cys-rich regions, is extracellular. Definition of transmembrane topology defines regions that might interact with the extracellular matrix or cytoskeleton to mediate mechanical signaling.

scholarly journals Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II

2000 ◽  
Vol 20 (1) ◽  
pp. 104-112 ◽  
Author(s):  
Christine R. Rodriguez ◽  
Eun-Jung Cho ◽  
Michael-C. Keogh ◽  
Claire L. Moore ◽  
Arno L. Greenleaf ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Capping Enzyme ◽  
Carboxy Terminal ◽  
Polyadenylation Factor ◽  
Enzyme Targeting

ABSTRACT The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase–CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, whilesrb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

scholarly journals IRES-mediated translation of the carboxy-terminal domain of the horizontal cell specific connexin Cx55.5 in vivo and in vitro

2008 ◽  
Vol 9 (1) ◽  
pp. 52 ◽  
Author(s):  
Mahboob Ul-Hussain ◽  
Georg Zoidl ◽  
Jan Klooster ◽  
Maarten Kamermans ◽  
Rolf Dermietzel
Keyword(s):  
Horizontal Cell ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

Calpain is a signal transducer and activator of transcription (STAT) 3 and STAT5 protease

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1850-1852 ◽  
Author(s):  
Atsushi Oda ◽  
Hiroshi Wakao ◽  
Hiroyoshi Fujita
Keyword(s):  
Genetic Engineering ◽  
Cysteine Protease ◽  
Posttranslational Modification ◽  
Dominant Negative ◽  
Signal Transducer ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

Truncation of signal transducer and activator of transcription (STAT) 5 at the carboxy-terminal domain, either by genetic engineering or by proteolytic cleavage, results in generation of dominant-negative forms. A nuclear serine protease expressed in the myeloid precursor cells is known to mediate this cleavage, but other proteases responsible for this reaction were unknown. We found that calpain, a ubiquitously expressed cysteine protease, also trims STAT5 in vivo and in vitro, within the carboxy-terminal domain. Nuclear element is not necessary for calpain-mediated STAT5 cleavage, since this process occurs in platelets. We also found that STAT3 is a substrate for calpain in vivo and in vitro, indicating that calpain-mediated cleavage is a common feature of STAT3 and STAT5. Thus, our study reveals a novel pathway for posttranslational modification of STAT3 and STAT5.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

Molecular genetic truncation analysis of filament assembly and phosphorylation domains of Dictyostelium myosin heavy chain

1994 ◽  
Vol 107 (10) ◽  
pp. 2875-2886 ◽  
Author(s):  
R.J. Lee ◽  
T.T. Egelhoff ◽  
J.A. Spudich
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Molecular Motor ◽  
Molecular Genetic ◽  
Surface Receptors ◽  
Cell Functions ◽  
Filament Assembly ◽  
Carboxy Terminal

Conventional myosin (‘myosin II’) is a major component of the cytoskeleton in a wide variety of eukaryotic cells, ranging from lower amoebae to mammalian fibroblasts and neutrophils. Gene targeting technologies available in the Dictyostelium discoideum system have provided the first genetic proof that this molecular motor protein is essential for normal cytokinesis, capping of cell surface receptors, normal chemotactic cell locomotion and morphogenetic shape changes during development. Although the roles of myosin in a variety of cell functions are becoming clear, the mechanisms that regulate myosin assembly into functional bipolar filaments within cells are poorly understood. Dictyostelium is currently the only system where mutant forms of myosin can be engineered in vitro, then expressed in their native context in cells that are devoid of the wild-type isoform. We have utilized this technology in combination with nested truncation and deletion analysis to map domains of the myosin tail necessary for in vivo and in vitro filament assembly, and for normal myosin heavy chain (MHC) phosphorylation. This analysis defines a region of 35 amino acids within the tail that is critical for filament formation both for purified myosin molecules and for myosin within the in vivo setting. Phosphorylation analysis of these mutants in intact cytoskeletons demonstrates that the carboxy-terminal tip of the myosin heavy chain is required for complete phosphorylation of the myosin tail.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

Sign in / Sign up

Export Citation Format

Share Document