The dynamic phosphorylation of the human intermediate filament keratin 1 chain

The major sites of phosphorylation have been determined on the human keratin intermediate filament keratin 1 (type II) chain expressed in terminally differentiating epidermis. A total of nine phosphate sites were found, involving 1 threonine and 8 serine residues, and were localized to end domain sequences. The sites identified corresponded to major sites of phosphorylation as determined by direct quantitation of O-phosphoserine. Since the tissue was cultured with [32 P] orthophosphate only briefly, labeling occurred primarily by turnover, so that information on the dynamics of phosphorylation was also obtained. The degrees and specific activities (that is, turnover rates) of phosphorylation of these sites varied widely between different isoelectric variants (phosphate isomers) of keratin 1 chains and correlated with their locations on the chain: those sites on the more exposed E1 and E2 subdomains were fully phosphorylated and turning over at high rates, while a site near the end of the rod domain in a presumably more confined location was only slightly phosphorylated and turning over at low rate. The nature of the sequences around the phosphorylated residues indicates that cAMP-dependent and probably other protein kinase activities operate simultaneously in intact normal epidermal tissue. The correlation between the degrees and rates of turnover of phosphorylation with the locations on the chain may have an important bearing on the functional role of phosphorylation of the keratin intermediate filaments in this tissue.     

The nonhelical tail domain of keratin 14 promotes filament bundling and enhances the mechanical properties of keratin intermediate filaments in vitro.

Keratin filaments arise from the copolymerization of type I and II sequences, and form a pancytoplasmic network that provides vital mechanical support to epithelial cells. Keratins 5 and 14 are expressed as a pair in basal cells of stratified epithelia, where they occur as bundled arrays of filaments. In vitro, bundles of K5-K14 filaments can be induced in the absence of cross-linkers, and exhibit enhanced resistance to mechanical strain. This property is not exhibited by copolymers of K5 and tailless K14, in which the nonhelical tail domain has been removed, or copolymers of K5 and K19, a type I keratin featuring a short tail domain. The purified K14 tail domain binds keratin filaments in vitro with specificity (kD approximately 2 microM). When transiently expressed in cultured cells, the K14 tail domain associates with endogenous keratin filaments. Utilization of the K14 tail domain as a bait in a yeast two-hybrid screen pulls out type I keratin sequences from a skin cDNA library. These data suggest that the tail domain of K14 contributes to the ability of K5-K14 filaments to self-organize into large bundles showing enhanced mechanical resilience in vitro.     

The cDNA sequence of a human epidermal keratin: divergence of sequence but conservation of structure among intermediate filament proteins

We have determined the DNA sequence of a cloned cDNA that is complementary to the mRNA for the 50 kilodalton (kd) human epidermal keratin. This provides the first amino acid sequence for a cytoskeletal keratin. Comparison of this sequence with those of other keratins reveals an evolutionary relationship between the cytoskeletal and the microfibrillar keratins, but shows no homology to matrix or feather keratins. The 50 kd keratin shares 28%-30% homology with partial sequences of other intermediate filament proteins, which suggests that keratins may be the most distantly related members of this class of fibrous proteins. Our computer analyses predict that the 50 kd keratin contains two long alpha-helical domains separated by a cluster of helix-inhibitory residues in the middle of the protein. These findings indicate that despite major sequence divergence among intermediate filament proteins, they retain sequences compatible with secondary structural features that appear to be common to all of them.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Overlapping sequences providing the complete sequence

The complete amino acid sequence of the alpha chain of human fibrinogen has been determined. It contains 610 amino acid residues and has a calculated molecular weight of 66,124. The chain has 10 methionines, and fragmentation with cyanogen bromide yields 11 peptides [Doolittle, R.F., Cassman, K.G., Cottrell, B.A., Friezner, S.J., Hucko, J.T., & Takagi, T. (1977) Biochemistry 16, 1703]. The arrangement of the 11 fragments was determined by the isolation of peptide overlaps from plasmic and staphylococcal protease digests of fibrinogen and/or alpha chains. In addition, certain of the cyanogen bromide fragments, preliminary reports of whose sequences have appeared previously, have been reexamined in order to resolve several discrepancies. The alpha chain is homologous with the beta and gamma chains of fibrinogen, although a large repetitive segment of unusual composition is absent from the latter two chains. The existence of this unusual segment divides the sequence of the alpha chain into three zones of about 200 residues each that are readily distinguishable on the basis of amino acid composition alone.     

Amphioxus type I keratin cDNA and the evolution of intermediate filament genes.

We report the cloning of an intermediate filament (IF) cDNA from the cephalochordate amphioxus that encodes a protein assignable to the type I keratin group. This is the first type I keratin reported from an invertebrate. Molecular phylogenetic analyses reveal that amphioxus also possesses a type II keratin, and that the genes encoding short-rod IF proteins underwent different patterns of duplication in vertebrates and their closest relatives, the cephalochordates. Extensive IF gene duplication and divergence may have facilitated the origin of new specialised cell types in vertebrates.     

Amino acid sequences of mouse and human epidermal type II keratins of Mr 67,000 provide a systematic basis for the structural and functional diversity of the end domains of keratin intermediate filament subunits

From the nucleotide sequences of specific cDNA clones, we present partial amino acid sequences (75-90% of the total) of 67-kDa type II keratin subunits expressed in terminally differentiating mouse and human epidermis. Analysis of the sequence information reveals that their secondary structures conform to the pattern common for all intermediate filament (IF) subunits. Together with the previously published sequence of the mouse 59-kDa type I keratin (Steinert, P. M., Rice, R. H., Roop, D. R., Trus, B. L., and Steven, A. C. (1983) Nature 302, 794-800) these data allow us to make comparisons between two keratins which are coexpressed in an epithelial cell type and which coassemble into the same IF. Moreover, these comparisons suggest a systematic plan for the general organization of the end domains of other keratin subunits. We postulate that each end domain consists of a set of subdomains which are distributed with bilateral symmetry with respect to the central alpha-helical domain. Type II (but not type I) keratins contain short globular sequences, H1 and H2, immediately adjacent to the central domain, that have been conserved in size and sequence and which account for most of the difference in mass between coexpressed type II and type I keratins. These are flanked by subdomains V1 and V2 that are highly variable in both length and sequence, often contain tandem peptide repeats, and are conspicuously rich in glycines and/or serines. At the termini are strongly basic subdomains (N and C, respectively) that are variable in sequence. Among keratins of a given type, their variability in mass appears to reside in the size of their V1 and V2 subdomains. However, coexpressed type I and type II keratins have generally similar V1 and/or V2 sequences. By virtue of the ease with which large portions of these subdomain sequences can be removed from intact keratin IF by limited proteolysis, we hypothesize that they lie on the periphery of the IF where they participate in interactions with other constituents of epithelial cells.     

The intermediate filament protein keratin 8 is a novel cytoplasmic substrate for c-Jun N-terminal kinase.

Keratins 8 (K8) and 18 are the primary intermediate filaments of simple epithelia. Phosphorylation of keratins at specific sites affects their organization, assembly dynamics, and their interaction with signaling molecules. A number of keratin in vitro and in vivo phosphorylation sites have been identified. One example is K8 Ser-73, which has been implicated as an important phosphorylation site during mitosis, cell stress, and apoptosis. We show that K8 is strongly phosphorylated on Ser-73 upon stimulation of the pro-apoptotic cytokine receptor Fas/CD95/Apo-1 in HT-29 cells. Kinase assays showed that c-Jun N-terminal kinase (JNK) was also activated with activation kinetics corresponding to that of K8 phosphorylation. Furthermore, K8 was also phosphorylated on Ser-73 by JNK in vitro, yielding similar phosphopeptide maps as the in vivo phosphorylated material. In addition, co-immunoprecipitation studies revealed that part of JNK is associated with K8 in vivo, correlating with decreased ability of JNK to phosphorylate the endogenous c-Jun. Taken together, K8 is a new cytoplasmic target for JNK in Fas receptor-mediated signaling. The functional significance of this phosphorylation could relate to regulation of JNK signaling and/or regulation of keratin dynamics.     

Conformational changes in the rod domain of human keratin 8 following heterotypic association with keratin 18 and its implication for filament stability

Keratin intermediate filaments are heteropolymers of type I and type II polypeptides that constitute the bulk of the epithelial cytoskeleton. We microinjected seven keratin monoclonal antibodies into human epithelial cells, and two of them, only A45-B/B3 and LP3K, caused the formation of keratin aggregates. The keratin filaments in human epithelial cells were also disrupted by a monovalent A45-B/B3 Fab fragment, suggesting that the binding of the antibody, rather than cross-linking, collapses the filaments. Immunoblotting and ELISA experiments suggested that the antibody reacted weakly with recombinant K8 but did not react with recombinant K18 at all. However, the antibody reactivity increased substantially when a mixture of the two keratin polypeptides, either recombinant or derived from MCF-7, was used. The epitopes of 15 monoclonal antibodies recognizing human K8 were characterized by their reactivity with recombinant fragments of K8. Reactivity of antibody A45-B/B3 with fragments of K8 in the presence of K18 revealed that the antibody recognizes an epitope in the rod domain of K8, between residues 313 and 332, on the amino-terminal side of the stutter in helix 2B, which is involved in heterotypic association. The data suggest that this region of K8 undergoes a conformational change following interaction with the complementary K18 either to expose the epitope or to increase its affinity for the antibody. Taken together, the data highlight the role of this epitope in heterotypic association and in filament stabilization.     

A synthetic peptide representing the consensus sequence motif at the carboxy-terminal end of the rod domain inhibits intermediate filament assembly and disassembles preformed filaments

All intermediate filament (IF) proteins share a highly conserved sequence motif at the COOH-terminal end of their rod domains. We have studied the influence of a 20-residue peptide, representing the consensus motif on filament formation and stability. Addition of the peptide at a 10-20-fold molar excess over keratins K8 plus K18 had a severe effect on subsequent IF assembly. Filaments displayed a rough surface and variable diameters with a substantial amount present in unravelled form. At higher peptide concentration (50-100-fold molar excess), IF formation was completely inhibited and instead only loose aggregates of "globular" particles were formed. The peptide also influenced performed keratin IF in a dose-dependent manner. While a three-fold molar excess was sufficient to cause partial fragmentation of IF, a 50-fold molar excess caused complete disassembly within 5 min. Loosely associated protofibrils, short needlelike IF fragments, and aggregates of globular particles were detected. The motif peptide also caused the disassembly of filaments formed by desmin, a type III IF protein. Peptide concentrations and incubation times required for complete disassembly were somewhat higher than for the filaments containing K8 plus K18. A 50-fold molar excess was sufficient to cause complete disassembly within 1 h. Peptides unrelated in sequence to the motif did not interfere with filament formation or stability even when present for more than 12 h at a 100-fold molar excess. The results suggest that the motif sequence normally binds to a specific acceptor site for which the motif peptide can successfully compete. Taken together with current models of IF structure the results indicate that normal binding of the motif sequence to its acceptor must play an essential role in IF formation, possibly by directing the proper alignment of neighboring tetramers or protofilaments. Finally we show that in vitro formed IF are much more sensitive and dynamic strutures than previously thought.     

The amino acid sequence of chicken muscle desmin provides a common structural model for intermediate filament proteins.

The complete amino acid sequence of muscle desmin reported here is the first for an intermediate filament protein. Alignment with partial data available for vimentin, glial fibrillary acid protein, neurofilament 68 K, two wool alpha-keratins, and a recently described DNA clone covering 90% of an epidermal keratin shows that all seven proteins have extensive homologies and therefore form a complex multigene family, the intermediate filament proteins. The hard alpha-keratins of wool appear to be a special subset of epithelial keratins. The sequence information reveals, as the dominant structural principle, a rod-like middle domain arising from several alpha-helical segments able to form interchain coiled-coil elements. The proposed helices are separated by short spacers, which like the two terminal domains seem built from non-alpha-helical material. Attention is drawn to the sometimes very striking sequence homologies along the rod and the high sequence variability in the terminal domains. Finally, chemical cross-linking experiments performed on the isolated desmin rod show that intermediate filament structure seems not to be based on triple-stranded coiled-coils as currently thought, but rather reflects protofilament units built as a dimer of normal interchain double-stranded coiled-coils.     

The amino acid sequence of chicken and muscle desmin provides a common structural model for intermediate filament proteins

[This corrects the article on p. 1649 in vol. 1.].     

The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments

The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two "pairs" (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily. While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro. These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments.     

Three-dimensional modelling of interchain sequence similarities and differences in the coiled-coil segments of keratin intermediate filament heterodimers highlight features important in assembly

Using structural data derived from crystal fragments of vimentin, three-dimensional models have been constructed for the major coiled-coil segments (1A, 1B and 2B) in epidermal and hair keratin intermediate filament molecules. Similarity and difference distributions arising from the heterodimer nature of the keratin molecules have been calculated, colour-coded for ease of observation and represented as movie clips. This approach has enabled the spatial distributions of the charged and apolar residues to be visualized along the seam between the chains and on the surface of the molecule, thus providing new insights into the features of the IF molecule that are important in assembly. An observation of note is that one face of both segment 1A and segment 1B is predominantly apolar and, furthermore, contains the bulk of the differences in the charged residues that occur between the two chains. The face rotated by 180 degrees contains far fewer apolar residues. This suggests the likely internal face of segments 1A and 1B and, hence, those sequence and spatial features that are important in assembly. In addition, the similarity distributions of the acidic and basic residues display a period of about 19 residues over much of each of the two faces of segment 1B. The two 19-residue periods are out of phase with respect to one another, however, thus leading to the previously recorded 9.51 residue period in the axial distributions of the acidic and the basic residues. The apparent doubling of the period arises because 9.51 residues corresponds to a non-integral number of turns of alpha-helical coiled coil.     

Importance of sequence specificity for predicting protein folding pathways: perturbed Gaussian chain model

Recent experimental and theoretical studies suggest that rates and pathways of protein folding are largely decided by topology of the native structures, at least for small proteins. However, some exceptions are known; for example, protein L and protein G have the same topology, but exhibit different characteristics of the TSE. Thus, folding pathways of some proteins are critically affected by detailed information on amino acid sequences. To investigate the sequence specificity, we calculate folding pathways of 20 small proteins using the perturbed Gaussian chain model developed by Portman et al. (Phys Rev Lett 1998;81:5237-5240; J Chem Phys 2001;114:5069-5081). Characteristics of the TSE predicted by the model are in good agreement with experimental phi-value data for many proteins at coarse-grained level. Especially, estimation of folding TSE for protein G and protein L based on both topology and additional sequence information are consistent with experimental phi-value data. With only topology information, however, the model predicts the TSE of protein G incorrectly. Moreover, the model that uses topology and sequence information describes free energy profiles of two-state and three-state folders consistently with experiment, whereas the topology only model predicts free energy profiles of some proteins incorrectly. This indicates that sequence specificity also has critical roles in determining the folding pathways for some proteins.     

Distribution of the intermediate filament proteins vimentin,keratin, and desmin in the bovine ovary

The distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co-localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3–7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunore-active, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small (<3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin-positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport. © 1995 Wiley-Liss, Inc.     

The human mid-size neurofilament subunit: a repeated protein sequence and the relationship of its gene to the intermediate filament gene family.

We report the isolation and sequence of cDNA and genomic clones for one of the two large subunits of human neurofilament, NF-M. Analysis of the sequence has allowed us to investigate the structure of the carboxy-terminal tail of this protein, and to compare it to that of the small neurofilament as well as to other intermediate filaments. The carboxy-terminal region of the protein contains a 13 amino acid proline- and serine-rich sequence repeated six times in succession. Within each repeat unit are two smaller repeats of the sequence Lys-Ser-Pro-Val. The four amino acid repeat may represent a kinase recognition site in a region of the protein that is known to be highly phosphorylated. We also note the presence of an additional heptad repeat at the extreme carboxy terminus of the protein. This region of 60 amino acids may be involved in coiled-coil interactions similar to those that facilitate the filament formation in the rod region. The human gene contains only two introns. Their positions correspond to two of the three introns found in the small neurofilament of the mouse. Thus, two of the three neurofilament genes of mammals have similar structures which are quite different from those of the other intermediate filaments. This finding suggests a common origin of the neurofilament subunits, whose evolutionary relationship to other intermediate filament genes is uncertain.     

The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin.

Overlapping human erythroid alpha-spectrin cDNA clones were isolated from lambda gt11 libraries constructed from cDNAs of human fetal liver and erythroid bone marrow. The composite 8001-base pair (bp) cDNA nucleotide sequence contains 187-bp 5'- and 528-bp 3'-untranslated regions and has a single long open reading frame of 7287 bp that encodes a polypeptide of 2429 residues. As previously described (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180), spectrin is composed largely of homologous 106-amino acid repeat units. From the amino acid sequence deduced from the cDNA, alpha-spectrin can be divided into 22 segments. Segments 1-9 and 12-19 are homologous and can therefore be considered repeats; the average number of identical residues in pairwise comparisons of these repeats is 22 out of 106, or 21%. Of these 17 repeats, 11 are exactly 106 amino acids in length, whereas five others differ from this length by a single residue. Segments 11, 20, and 21, although less homologous, appear to be related to the more highly conserved repeat units. The very N-terminal 22 residues, segment 10, which is atypical both in length and sequence, and the C-terminal 150 residues in segment 22 appear to be unrelated to the conserved repeat units. The sequence of the erythroid alpha-spectrin polypeptide chain is compared to that of human alpha-fodrin and chicken alpha-actinin to which it is related. alpha-Spectrin is more distantly related to dystrophin.     

Amino acid sequences and homopolymer-forming ability of the intermediate filament proteins from an invertebrate epithelium.

Intermediate filaments (IF) isolated from the oesophagus epithelium of the snail Helix pomatia contain two polypeptides of mol. wt 66,000 (A) and 52,000 (B), which we have now characterized by in vitro self-assembly studies and by protein sequences. A and B can each form morphologically normal IF and share extended regions of sequence identity. All A-specific sequences seem to locate to an extension of the carboxyl-terminal domain. Although the Helix protein(s) reveal the IF-consensus sequences at the ends of the coiled-coil, the remainder of the rod domain shows conservation of sequence principles rather than extended homology, when compared with any subtype of vertebrate IF proteins. Interestingly, the Helix proteins have the longer coil 1b domain found in nuclear lamins and not in cytoplasmic IF proteins of vertebrates. They lack, however, the karyophilic signal sequence typical for lamins. Obvious implications for IF evolution and structure are discussed.     

Detection of a hidden folding intermediate in the focal adhesion target domain: Implications for its function and folding

The focal adhesion target (FAT) domain of focal adhesion kinase has a four-helix bundle structure. Based on a hydrogen exchange-constrained computer simulation study and some indirect experimental results, it has been suggested that a partially unfolded state of the FAT domain with the N-terminal helix unfolded plays an important role in its biological function. Here, using a native-state hydrogen exchange method, we directly detected an intermediate with the N-terminal helix unfolded in a mutant (Y925E) of the FAT domain. In addition, kinetic folding studies on the FAT domain suggest that this intermediate exists on the native side of the rate-limiting transition state for folding. These results provide more direct evidence of the existence of the proposed intermediate and help to understand the folding mechanism of small single domain proteins.