scholarly journals Keratins Turn over by Ubiquitination in a Phosphorylation-Modulated Fashion

2000 ◽  
Vol 149 (3) ◽  
pp. 547-552 ◽  
Author(s):  
Nam-On Ku ◽  
M. Bishr Omary
Keyword(s):  
Intermediate Filament ◽  
Liver Diseases ◽  
Direct Evidence ◽  
Proteasome Inhibition ◽  
Type I ◽  
Ubiquitinated Proteins ◽  
Antibody Staining ◽  
Keratin Polypeptides ◽  
Proteasome Pathway ◽  
Turn Over

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.

Vimentin's tail interacts with actin-containing structures in vivo

1994 ◽  
Vol 107 (6) ◽  
pp. 1609-1622 ◽  
Author(s):  
R.B. Cary ◽  
M.W. Klymkowsky ◽  
R.M. Evans ◽  
A. Domingo ◽  
J.A. Dent ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Direct Evidence ◽  
Type I ◽  
Tail Domain ◽  
Primary Sequence ◽  
Net Charge ◽  
Physiological Conditions

The tail domain of the intermediate filament (IF) protein vimentin is unnecessary for IF assembly in vitro. To study the role of vimentin's tail in vivo, we constructed a plasmid that directs the synthesis of a ‘myc-tagged’ version of the Xenopus vimentin-1 tail domain in bacteria. This polypeptide, mycVimTail, was purified to near homogeneity and injected into cultured Xenopus A6 cells. In these cells the tail polypeptide co-localized with actin even in the presence of cytochalasin. Two myc-tagged control polypeptides argue for the specificity of this interaction. First, a similarly myc-tagged lamin tail domain localizes to the nucleus, indicating that the presence of the myc tag did not itself confer the ability to co-localize with actin (Hennekes and Nigg (1994) J. Cell Sci. 107, 1019–1029). Second, a myc-tagged polypeptide with a molecular mass and net charge at physiological pH (i.e. -4) similar to that of the mycVimTail polypeptide, failed to show any tendency to associate with actin-containing structures, indicating that the interaction between mycVimTail and actin-containing structures was not due to a simple ionic association. Franke (1987; Cell Biol. Int. Rep. 11, 831) noted a similarity in the primary sequence between the tail of the type I keratin DG81A and vimentin. To test whether the DG81A tail interacted with actin-containing structures, we constructed and purified myc-tagged DG81A tail polypeptides. Unexpectedly, these keratin tail polypeptides were largely insoluble under physiological conditions and formed aggregates at the site of injection. While this insolubility made it difficult to determine if they associated with actin-containing structures, it does provide direct evidence that the tails of vimentin and DG81A differ dramatically in their physical properties. Our data suggest that vimentin's tail domain has a highly extended structure, binds to actin-containing structures and may mediate the interaction between vimentin filaments and microfilaments involved in the control of vimentin filament organization (Hollenbeck et al. (1989) J. Cell Sci. 92, 621; Tint et al. (1991) J. Cell Sci. 98, 375).

scholarly journals Anomalous placement of introns in a member of the intermediate filament multigene family: an evolutionary conundrum.

1986 ◽  
Vol 6 (5) ◽  
pp. 1529-1534 ◽  
Author(s):  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Intermediate Filament ◽  
Multigene Family ◽  
Protein Gene ◽  
Sequence Data ◽  
Complete Sequence ◽  
Acidic Protein ◽  
Type I ◽  
Neurofilament Protein ◽  
Gene Encoding

The origin of introns and their role (if any) in gene expression, in the evolution of the genome, and in the generation of new expressed sequences are issues that are understood poorly, if at all. Multigene families provide a favorable opportunity for examining the evolutionary history of introns because it is possible to identify changes in intron placement and content since the divergence of family members from a common ancestral sequence. Here we report the complete sequence of the gene encoding the 68-kilodalton (kDa) neurofilament protein; the gene is a member of the intermediate filament multigene family that diverged over 600 million years ago. Five other members of this family (desmin, vimentin, glial fibrillary acidic protein, and type I and type II keratins) are encoded by genes with six or more introns at homologous positions. To our surprise, the number and placement of introns in the 68-kDa neurofilament protein gene were completely anomalous, with only three introns, none of which corresponded in position to introns in any characterized intermediate filament gene. This finding was all the more unexpected because comparative amino acid sequence data suggest a closer relationship of the 68-kDa neurofilament protein to desmin, vimentin, and glial fibrillary acidic protein than between any of these three proteins and the keratins. It appears likely that an mRNA-mediated transposition event was involved in the evolution of the 68-kDa neurofilament protein gene and that subsequent events led to the acquisition of at least two of the three introns present in the contemporary sequence.

Diurnal variation in concentrations of various markers of bone metabolism in growing female goats and sheep

2003 ◽  
Vol 77 (2) ◽  
pp. 197-203 ◽  
Author(s):  
A. Liesegang ◽  
M.-L. Sassi ◽  
J. Risteli
Keyword(s):  
Type I Collagen ◽  
Bone Markers ◽  
Degradation Products ◽  
Bone Marker ◽  
Type I ◽  
Blood Samples ◽  
Bone Specific Alkaline Phosphatase ◽  
Growing Goats ◽  
Turn Over ◽  
Carboxyterminal Telopeptide

AbstractTwelve 6-month-old growing female goats and sheep were used in this study. Blood samples were obtained in the morning before goats and sheep were given food and then at 2-h intervals for 24 h (part I). This procedure was repeated 2 weeks later (part II). Concentrations of osteocalcin (OC), activities of total (tAP) and bone-specific alkaline phosphatase (bAP), degradation products of C-terminal telopeptide of type-I collagen (CrossLaps™ CL), and carboxyterminal telopeptide of type-I collagen (ICTP) were measured in serum.In both parts of the study, all bone marker concentrations were significantly higher in goats than in sheep. The OC concentrations in goats increased in the late afternoon/evening and decreased thereafter to reach values similar to those obtained at the beginning. The ICTP concentrations in goats slowly decreased until 14:00 h, increased, and decreased again. The concentrations in sheep decreased continuously but not significantly, towards the morning sampling. The CL concentrations increased in both sheep and goats during the night but at 06:00 h started to decrease to levels found at the beginning of testing. The bAP activities decreased in goats from 20:00 to 22:00 h. Changes in the concentrations of bone markers were mainly observed in goats of this study. As documented for bone resorption and formation in other species, circadian rhythms were evident for concentrations of ICTP, CL, bAP and OC. The present study indicates that growing goats may have a physiologically higher bone turn-over than growing sheep, because the bone marker concentrations were always higher.

scholarly journals Multiple Aspects of PIP2 Involvement in C. elegans Gametogenesis

2018 ◽  
Vol 19 (9) ◽  
pp. 2679 ◽  
Author(s):  
Livia Ulicna ◽  
Jana Rohozkova ◽  
Pavel Hozak
Keyword(s):  
Mammalian Cells ◽  
Chromosome Structure ◽  
Type I ◽  
Nuclear Speckles ◽  
C Elegans ◽  
Meiotic Progression ◽  
Prophase I ◽  
Binding Partners ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

One of the most studied phosphoinositides is phosphatidylinositol 4,5-bisphosphate (PIP2), which localizes to the plasma membrane, nuclear speckles, small foci in the nucleoplasm, and to the nucleolus in mammalian cells. Here, we show that PIP2 also localizes to the nucleus in prophase I, during the gametogenesis of C. elegans hermaphrodite. The depletion of PIP2 by type I PIP kinase (PPK-1) kinase RNA interference results in an altered chromosome structure and leads to various defects during meiotic progression. We observed a decreased brood size and aneuploidy in progeny, defects in synapsis, and crossover formation. The altered chromosome structure is reflected in the increased transcription activity of a tightly regulated process in prophase I. To elucidate the involvement of PIP2 in the processes during the C. elegans development, we identified the PIP2-binding partners, leucine-rich repeat (LRR-1) protein and proteasome subunit beta 4 (PBS-4), pointing to its involvement in the ubiquitin–proteasome pathway.

Mutations in the non-helical linker segment L1-2 of keratin 5 in patients with Weber-Cockayne epidermolysis bullosa simplex

1994 ◽  
Vol 107 (4) ◽  
pp. 765-774
Author(s):  
Y.M. Chan ◽  
Q.C. Yu ◽  
J. LeBlanc-Straceski ◽  
A. Christiano ◽  
L. Pulkkinen ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Intermediate Filament ◽  
Point Mutations ◽  
Epidermolysis Bullosa Simplex ◽  
Keratin Gene ◽  
Filament Assembly ◽  
Keratin Polypeptides ◽  
Epidermolytic Hyperkeratosis ◽  
Hands And Feet ◽  
Linker Segment

Keratins are the major structural proteins of the epidermis. Analyzing keratin gene sequences, appreciating the switch in keratin gene expression that takes place as epidermal cells commit to terminally differentiate, and elucidating how keratins assemble into 10 nm filaments, have provided the foundation that has led to the discoveries of the genetic bases of two major classes of human skin diseases, epidermolysis bullosa simplex (EBS) and epidermolytic hyperkeratosis (EH). These diseases involve point mutations in either the basal epidermal keratin pair, K5 and K14 (EBS), or the suprabasal pair, K1 and K10 (EH). In severe cases of EBS and EH, mutations are found in the highly conserved ends of the alpha-helical rod domain, regions that, by random mutagenesis, had already been found to be important for 10 nm filament assembly. In order to identify regions of the keratin polypeptides that might be more subtly involved in 10 nm filament assembly and to explore the diversity in mutations within milder cases of these diseases, we have focused on Weber-Cockayne EBS, where mild blistering occurs primarily on the hands and feet in response to mechanical stress. In this report, we show that affected members of two different W-C EBS families have point mutations within 1 residue of each other in the non-helical linker segment of the K5 polypeptide. Genetic linkage analyses, the absence of this mutation in > 150 wild-type alleles and filament assembly studies suggest that these mutations are responsible for the W-C EBS phenotype. These findings provide the best evidence to date that the non-helical linker region in the middle of the keratin polypeptides plays a subtle but significant role in intermediate filament structure and/or intermediate filament cytoskeletal architecture.

Genes for intermediate filament proteins and the draft sequence of the human genome

2001 ◽  
Vol 114 (14) ◽  
pp. 2569-2575 ◽  
Author(s):  
Michael Hesse ◽  
Thomas M. Magin ◽  
Klaus Weber
Keyword(s):  
Human Genome ◽  
Dna Sequences ◽  
Intermediate Filament ◽  
Muscle Protein ◽  
Gene Families ◽  
Type I ◽  
Type Ii ◽  
Draft Sequence ◽  
Intermediate Filament Proteins ◽  
Processed Pseudogenes

We screened the draft sequence of the human genome for genes that encode intermediate filament (IF) proteins in general, and keratins in particular. The draft covers nearly all previously established IF genes including the recent cDNA and gene additions, such as pancreatic keratin 23, synemin and the novel muscle protein syncoilin. In the draft, seven novel type II keratins were identified, presumably expressed in the hair follicle/epidermal appendages. In summary, 65 IF genes were detected, placing IF among the 100 largest gene families in humans. All functional keratin genes map to the two known keratin clusters on chromosomes 12 (type II plus keratin 18) and 17 (type I), whereas other IF genes are not clustered. Of the 208 keratin-related DNA sequences, only 49 reflect true keratin genes, whereas the majority describe inactive gene fragments and processed pseudogenes. Surprisingly, nearly 90% of these inactive genes relate specifically to the genes of keratins 8 and 18. Other keratin genes, as well as those that encode non-keratin IF proteins, lack either gene fragments/pseudogenes or have only a few derivatives. As parasitic derivatives of mature mRNAs, the processed pseudogenes of keratins 8 and 18 have invaded most chromosomes, often at several positions. We describe the limits of our analysis and discuss the striking unevenness of pseudogene derivation in the IF multigene family. Finally, we propose to extend the nomenclature of Moll and colleagues to any novel keratin.

scholarly journals Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading

2019 ◽  
Vol 47 (20) ◽  
pp. 10754-10770 ◽  
Author(s):  
Anming Huang ◽  
Leopold Kremser ◽  
Fabian Schuler ◽  
Doris Wilflingseder ◽  
Herbert Lindner ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Posttranslational Modifications ◽  
Centromeric Chromatin ◽  
Phosphorylated Form ◽  
Specific Loading ◽  
Histone H3 Variant ◽  
Proteasome Pathway ◽  
Turn Over ◽  
Fine Tune

Abstract Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.

Aldosterone binding sites along nephron of Xenopus and rabbit

1989 ◽  
Vol 257 (1) ◽  
pp. R87-R95 ◽  
Author(s):  
A. Gnionsahe ◽  
M. Claire ◽  
N. Koechlin ◽  
J. P. Bonvalet ◽  
N. Farman
Keyword(s):  
Binding Sites ◽  
Direct Evidence ◽  
Distal Tubule ◽  
Distal Segment ◽  
The Other ◽  
Type I ◽  
Thick Ascending Limb ◽  
Type Ii ◽  
Straight Segment ◽  
Dry Film

Distal segment of several amphibians exhibits aldosterone-modulated ion transport properties. On the other hand, A6 cells, derived from Xenopus laevis (XL) kidney, are aldosterone sensitive. We examined the distribution of aldosterone binding sites in isolated tubules of XL compared with rabbit. After incubation with 2 nM [3H]aldosterone, microdissected tubular segments from proximal (PT), distal straight segment (DST), and flask cell collecting (CT) tubules from XL and from rabbit cortical thick ascending limb (CTAL), connecting (CNT), and collecting (CCD) tubules were processed for dry film autoradiography. In XL, specific nuclear labeling of type I (mineralocorticoid) sites was restricted to DST. Labeling of type II (glucocorticoid) sites was present all along the tubule. No specific cytoplasmic labeling was observed, except for type II sites in PT. In the rabbit, aldosterone binds to both type I and type II sites in the three tubular segments studied. In these segments, the binding was about fourfold higher than in DST of XL. These results bring direct evidence in designating the distal tubule of amphibians as a target epithelium for aldosterone. In addition, they suggest that A6 cell line may derive from DST of the Xenopus nephron.

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