Caspase Cleavage of Keratin 18 and Reorganization of Intermediate Filaments during Epithelial Cell Apoptosis

Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light–induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH2-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.

Download Full-text

Tailless keratins assemble into regular intermediate filaments in vitro

Journal of Cell Science ◽

10.1242/jcs.97.2.317 ◽

1990 ◽

Vol 97 (2) ◽

pp. 317-324

Author(s):

M. Hatzfeld ◽

K. Weber

Keyword(s):

Intermediate Filaments ◽

Consensus Sequence ◽

In Vitro Mutagenesis ◽

Type I ◽

Type Ii ◽

Tail Domain ◽

Filament Formation ◽

Wild Type ◽

Keratin 8

To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.

Download Full-text

Identification of a novel caspase cleavage site in huntingtin that regulates mutant huntingtin clearance

10.1101/232827 ◽

2017 ◽

Author(s):

D.D.O. Martin ◽

M. E. Schmidt ◽

Y. T. Nguyen ◽

N. Lazic ◽

M. R. Hayden

Keyword(s):

Cleavage Site ◽

Motor Coordination ◽

Amino Acid Position ◽

Mutant Huntingtin ◽

Post Translational Modifications ◽

Caspase Cleavage ◽

Caspase 1 ◽

Cell Death Pathways ◽

Caspase Cleavage Site ◽

Caspase 6

ABSTRACTHuntington disease (HD) is a progressive neurodegenerative disease that initially affects the striatum leading to changes in behavior and loss of motor coordination. It is caused by an expansion in the polyglutamine repeat at the N-terminus of huntingtin (HTT) that leads to aggregation of mutant HTT. The loss of wildtype function, in combination with the toxic gain of function mutation, initiates various cell death pathways. Wildtype and mutant HTT are regulated by different post-translational modifications that can positively or negatively regulate their function or toxicity. In particular, we have previously shown that caspase cleavage of mutant HTT at amino acid position aspartate 586 (D586) by caspase-6 is critical for the pathogenesis of the disease in an HD mouse model. Herein, we describe the identification of a new caspase cleavage site at position D572 that is mediated by caspase-1. Inhibition of caspase-1 also inhibits cleavage at D586 through inhibition of caspase-6. Inhibition of caspase cleavage at D572 significantly decreases mutant HTT aggregation and significantly increased the turnover of soluble mutant HTT. This suggests that caspase-1 may be a viable target to inhibit caspase cleavage of mutant HTT at both D572 and D586 to promote mutant HTT clearance.

Download Full-text

The role of the N-terminal caspase cleavage site in the nucleoprotein of influenza A virus in vitro and in vivo

Archives of Virology ◽

10.1007/s00705-007-0003-8 ◽

2007 ◽

Vol 153 (3) ◽

pp. 427-434 ◽

Cited By ~ 10

Author(s):

A. S. Lipatov ◽

H.-L. Yen ◽

R. Salomon ◽

H. Ozaki ◽

E. Hoffmann ◽

...

Keyword(s):

Influenza A Virus ◽

Cleavage Site ◽

Influenza A ◽

Caspase Cleavage ◽

Caspase Cleavage Site

Download Full-text

Type II Keratins Are Phosphorylated on a Unique Motif during Stress and Mitosis in Tissues and Cultured Cells

Molecular Biology of the Cell ◽

10.1091/mbc.01-12-0591 ◽

2002 ◽

Vol 13 (6) ◽

pp. 1857-1870 ◽

Cited By ~ 57

Author(s):

Diana M. Toivola ◽

Qin Zhou ◽

Luc S. English ◽

M. Bishr Omary

Keyword(s):

Cultured Cells ◽

Uv Light ◽

Ex Vivo ◽

Psoriatic Skin ◽

Type I ◽

Type Ii ◽

Intermediate Filament Proteins ◽

Phosphatase Inhibitors

Epithelial cell keratins make up the type I (K9–K20) and type II (K1–K8) intermediate filament proteins. In glandular epithelia, K8 becomes phosphorylated on S73 (71LLpSPL) in human cultured cells and tissues during stress, apoptosis, and mitosis. Of all known proteins, the context of the K8 S73 motif (LLS/TPL) is unique to type II keratins and is conserved in epidermal K5/K6, esophageal K4, and type II hair keratins, except that serine is replaced by threonine. Because knowledge regarding epidermal and esophageal keratin regulation is limited, we tested whether K4–K6 are phosphorylated on the LLTPL motif. K5 and K6 become phosphorylated in vitro on threonine by the stress-activated kinase p38. Site-specific anti-phosphokeratin antibodies to LLpTPL were generated, which demonstrated negligible basal K4–K6 phosphorylation. In contrast, treatment of primary keratinocytes and other cultured cells, and ex vivo skin and esophagus cultures, with serine/threonine phosphatase inhibitors causes a dramatic increase in K4–K6 LLpTPL phosphorylation. This phosphorylation is accompanied by keratin solubilization, filament reorganization, and collapse. K5/K6 LLTPL phosphorylation occurs in vivo during mitosis and apoptosis induced by UV light or anisomycin, and in human psoriatic skin and squamous cell carcinoma. In conclusion, type II keratins of proliferating epithelia undergo phosphorylation at a unique and conserved motif as part of physiological mitotic and stress-related signals.

Download Full-text

Purification of developmentally regulated avian 400-kDa intermediate filament associated protein. Molecular interactions with intermediate filament proteins and other cytoskeleton components

Biochemistry and Cell Biology ◽

10.1139/o95-072 ◽

1995 ◽

Vol 73 (9-10) ◽

pp. 651-657 ◽

Cited By ~ 5

Author(s):

Marie Duval ◽

Xiaoying Ma ◽

Jean-Paul Valet ◽

Michel Vincent

Keyword(s):

Chick Embryo ◽

Intermediate Filaments ◽

Intermediate Filament ◽

Specific Binding ◽

Developmentally Regulated ◽

Intermediate Filament Proteins ◽

In Vitro Recombination ◽

Associated Proteins ◽

Structural Lattice

IFAPa-400, a 400-kDa developmentally regulated protein thought to be associated with intermediate filaments, has been purified from chick embryo hearts to investigate its interaction with vimentin and other IF proteins and to identify other cellular components to which this cytoskeletal protein associates. Previous studies suggested that this protein was associated with the vimentin-containing intermediate filament lattice of myoblasts and neuroblasts before their terminal differentiation, providing these cells with a particular intermediate filament cytoskeleton that could satisfy specific mechanical requirements during their intense morphogenetic activities. Although IFAPa-400 partially reassociated with vimentin and desmin in disassembly–reassembly experiments using crude IF preparations from chick embryo hearts, in vitro recombination of purified IFAPa-400 with vimentin and desmin failed to demonstrate any direct association. When purified IFAPa-400 was used as a probe in blot overlay assays, however, specific binding to vimentin and desmin was observed, providing the first evidence of a physical association between IFAPa-400 and intermediate filament proteins. The blot overlay experiments also demonstrated that IFAPa-400 binds to two unidentified polypeptides of 19 and 32 kDa. These results are thus consistent with the hypothesis that a structural lattice requiring a vimentin–IFAPa-400 combination constitutes the intermediate filament system of myogenic and neurogenic cells.Key words: cytoskeleton, intermediate filaments, intermediate filament associated proteins, vimentin, IFAPa-400.

Download Full-text

Neurofilaments are obligate heteropolymers in vivo

The Journal of Cell Biology ◽

10.1083/jcb.122.6.1337 ◽

1993 ◽

Vol 122 (6) ◽

pp. 1337-1350 ◽

Cited By ~ 256

Author(s):

MK Lee ◽

Z Xu ◽

PC Wong ◽

DW Cleveland

Keyword(s):

Intermediate Filaments ◽

Intermediate Filament ◽

Cultured Cells ◽

Dna Transfection ◽

Tail Domain ◽

In Vitro Assembly ◽

Mature Neurons ◽

Filament Network

Neurofilaments (NFs), composed of three distinct subunits NF-L, NF-M, and NF-H, are neuron-specific intermediate filaments present in most mature neurons. Using DNA transfection and mice expressing NF transgenes, we find that despite the ability of NF-L alone to assemble into short filaments in vitro NF-L cannot form filament arrays in vivo after expression either in cultured cells or in transgenic oligodendrocytes that otherwise do not contain a cytoplasmic intermediate filament (IF) array. Instead, NF-L aggregates into punctate or sheet like structures. Similar nonfilamentous structures are also formed when NF-M or NF-H is expressed alone. The competence of NF-L to assemble into filaments is fully restored by coexpression of NF-M or NF-H to a level approximately 10% of that of NF-L. Deletion of the head or tail domain of NF-M or substitution of the NF-H tail onto an NF-L subunit reveals that restoration of in vivo NF-L assembly competence requires an interaction provided by the NF-M or NF-H head domains. We conclude that, contrary to the expectation drawn from earlier in vitro assembly studies, NF-L is not sufficient to assemble an extended filament network in an in vivo context and that neurofilaments are obligate heteropolymers requiring NF-L and NF-M or NF-H.

Download Full-text

The nucleoporin Nup88 is interacting with nuclear lamin A

Molecular Biology of the Cell ◽

10.1091/mbc.e10-05-0463 ◽

2011 ◽

Vol 22 (7) ◽

pp. 1080-1090 ◽

Cited By ~ 25

Author(s):

Yvonne C. Lussi ◽

Ilona Hügi ◽

Eva Laurell ◽

Ulrike Kutay ◽

Birthe Fahrenkrog

Keyword(s):

Nuclear Lamina ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Lamin A ◽

Xenopus Laevis Oocyte ◽

Tail Domain ◽

Intermediate Filament Proteins ◽

Nuclear Lamin ◽

In Cells

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and mediate bidirectional nucleocytoplasmic transport. Their spatial distribution in the NE is organized by the nuclear lamina, a meshwork of nuclear intermediate filament proteins. Major constituents of the nuclear lamina are A- and B-type lamins. In this work we show that the nuclear pore protein Nup88 binds lamin A in vitro and in vivo. The interaction is mediated by the N-terminus of Nup88, and Nup88 specifically binds the tail domain of lamin A but not of lamins B1 and B2. Expression of green fluorescent protein–tagged lamin A in cells causes a masking of binding sites for Nup88 antibodies in immunofluorescence assays, supporting the interaction of lamin A with Nup88 in a cellular context. The epitope masking disappears in cells expressing mutants of lamin A that are associated with laminopathic diseases. Consistently, an interaction of Nup88 with these mutants is disrupted in vitro. Immunoelectron microscopy using Xenopus laevis oocyte nuclei further revealed that Nup88 localizes to the cytoplasmic and nuclear face of the NPC. Together our data suggest that a pool of Nup88 on the nuclear side of the NPC provides a novel, unexpected binding site for nuclear lamin A.

Download Full-text

Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins.

The Journal of Cell Biology ◽

10.1083/jcb.106.3.723 ◽

1988 ◽

Vol 106 (3) ◽

pp. 723-733 ◽

Cited By ~ 100

Author(s):

R Foisner ◽

F E Leichtfried ◽

H Herrmann ◽

J V Small ◽

D Lawson ◽

...

Keyword(s):

Immunoelectron Microscopy ◽

Intermediate Filaments ◽

Intermediate Filament ◽

Solid Phase ◽

Chinese Hamster ◽

Structural Element ◽

Intermediate Filament Proteins ◽

Rat Glioma ◽

Branching Points

The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.

Download Full-text