Evolution of tissue-specific keratins as deduced from novel cDNA sequences of the lungfish Protopterus aethiopicus

Lungfishes are possibly the closest extant relatives of the land vertebrates (tetrapods). We report here the cDNA and predicted amino acid sequences of 13 different keratins (ten type I and three type II) of the lungfish Protopterus aethiopicus. These keratins include the orthologs of human K8 and K18. The lungfish keratins were also identified in tissue extracts using two-dimensional polyacrylamide gel electrophoresis, keratin blot binding assays and immunoblotting. The identified keratin spots were analyzed by peptide mass fingerprinting which assigned seven sequences (inclusively Protopterus K8 and K18) to their respective protein spot. The peptide mass fingerprints also revealed the fact that the major epidermal type I and type II keratins of this lungfish have not yet been sequenced. Nevertheless, phylogenetic trees constructed from multiple sequence alignments of keratins from lungfish and distantly related vertebrates such as lamprey, shark, trout, frog, and human reveal new insights into the evolution of K8 and K18, and unravel a variety of independent keratin radiation events.     

Type II keratins are phosphorylated on a unique motif during stress and mitosis in tissues and cultured cells

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 ((71)LLpSPL) 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.     

Sequence and expression of a type II keratin, K5, in human epidermal cells.

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.     

cDNA sequences of the authentic keratins 8 and 18 in zebrafish

From the zebrafish Danio rerio, we have cDNA cloned and sequenced a novel type II and a novel type I keratin, termed DreK8 and DreK18, respectively. We identified DreK8/18 as the true orthologs of the human keratin pair K8/18 as follows: (i) MALDI-MS assignment to the biochemically identified K8 and K18 candidates that are co-expressed in simple epithelia and absent in epidermal keratinocytes; (ii) multiple sequence alignments and phylogenetic tree analysis, showing that DreK8, within the phylogenetic tree of type II keratins, forms a highly bootstrap-supported branch together with K8 from goldfish and rainbow trout, whereas DreK18, within the phylogenetic tree of type I keratins, groups with the K18 sequences from all other vertebrates studied; (iii) presence of a conserved motif in the tail domain of DreK8 (VxKxxETxDGxxVSESSxV) that is typical for all hitherto sequenced K8 orthologs. Moreover, several zebrafish type II keratin sequences published by other authors have now been assigned to epidermal keratins, previously identified biochemically.     

The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines

We have deleted cDNA sequences encoding portions of the carboxy-terminal end of a human type I epidermal keratin K14, and examined the molecular consequences of forcing the expression of these mutants in simple epithelial and squamous cell carcinoma lines. To follow the expression of our mutant products in transfected cells, we have tagged the 3' end of the K14 coding sequence with a sequence encoding an antigenic domain of the neuropeptide substance P. Using DNA transfection and immunohistochemistry (with an antibody against substance P), we have identified a collection of mutants that have a wide range of morphological effects on the endogenous keratin filament networks of transfected cells. Mutants that are missing most of the nonhelical carboxy-terminal domain of K14 incorporate into the endogenous keratin filaments without any visible perturbations on the network. In contrast, mutants that are missing as few as 10 of the 310 amino acids of the central alpha-helical domain of the polypeptide cause gross alterations in the keratin network. In some cases, the entire cytoskeletal network of keratins was disrupted, leaving no evidence of 8-nm filaments. These results reveal the existence of a dynamic exchange between newly synthesized subunits and preexisting keratin filaments.     

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution- binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.     

New epidermal keratin genes from Xenopus laevis: hormonal and regional regulation of their expression during anuran skin metamorphosis

Xenopus larval keratin (XLK) was isolated by gel electrophoresis of proteins of tadpole skin. Screening of an expression cDNA library of tail tissues by specific polyclonal antibodies against XLK produced XLK cDNA (xlk). Its complete nucleotide and predicted amino acid sequences revealed that XLK was a new member of type II keratin. Screening of a cDNA library of adult Xenopus skin using an oligonucleotide probe which had been designed from well-conserved N-terminal amino acid sequences of the rod domain of type I keratin produced two cDNAs, xak-a and xak-b, which were found to be new members of type I keratin gene. Northern blot analysis showed that xlk was expressed exclusively in the larval skin whereas xak-a and xak-b were expressed exclusively in the adult skin. Their expression level was regulated in a region- and metamorphic stage- dependent manner during larval skin development. mRNA in situ hybridization experiments identified the cells that expressed xlk, and xak-a and xak-b as larva- specific epidermal cells (skein cells and basal cells), and adult suprabasal epidermal cells, respectively. These three genes were found to be late responsive to thyroid hormone. Phylogenetic relationships of these keratins with known ones are discussed.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

Sequence, evolution and tissue expression patterns of an epidermal type I keratin from the shark Scyliorhinus stellaris

From the shark Scyliorhinus stellaris we cloned and sequenced a cDNA encoding a novel type I keratin, termed SstK10. By MALDI-MS peptide mass fingerprinting of cytoskeletal proteins separated on polyacrylamide gels, we assigned SstK10 to a 46-kDa protein which is the major epidermal type I ("IE") keratin in this fish and is specifically expressed in stratified epithelia. In a phylogenetic tree based on type I keratin sequences and with lamprey keratins applied as outgroup, SstK10 branches off in a rather basal position. This tree strongly supports the concept that teleost keratins and tetrapod keratins resulted from two independent gene radiation processes. The only exception is human K18 because its orthologs have been found in all jawed vertebrates (Gnathostomata) studied; in the tree, they form a common, most early branch, with the shark version, SstK18, in the most basal position. Thus, the sequences of SstK10 and SstK18 also favor the classical view of vertebrate evolution that considers the cartilaginous fishes as the most ancient living Gnathostomata. To determine the overall expression patterns of epidermal ("E") and simple epithelial ("S") keratins in this shark, we furthermore tested a panel of monoclonal anti-keratin antibodies by immunofluorescence microscopy of frozen tissue sections, and in immunoblots of cytoskeletal preparations, demonstrating that immunodetection of specific keratins is a convenient method to characterize epithelial tissues in shark.     

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.     

Expression of mutant keratin cDNAs in epithelial cells reveals possible mechanisms for initiation and assembly of intermediate filaments

We have deleted cDNA sequences encoding portions of the amino- and carboxy-terminal end of a human type I epidermal keratin K14, and examined the molecular consequences of forcing the expression of these mutants in simple epithelial and squamous cell carcinoma lines. To follow the expression of our mutant products in transfected cells, we have tagged the 3' end of the K14 coding sequence with a sequence encoding an antigenic domain of the neuropeptide substance P. Using DNA transfection and immunohistochemistry (with an antibody against substance P), we have defined the limits of K14 sequence necessary to incorporate into a keratin filament network in vivo without disrupting its architecture. We have also uncovered major differences in the behavior of carboxy- and amino-terminal alpha-helical mutants which do perturb the cytoskeletal network of IFs: whereas carboxy terminal mutants give rise to aggregates of keratin in the cytoplasm, amino-terminal mutants tend to produce aggregates of keratins which seem to localize at the nuclear surface. An examination of the phenotypes generated by the carboxy and amino-terminal mutants and the behavior of cells at late times after transfection suggests a model whereby initiation of filament assembly occurs at discrete sites on the nuclear envelope and filaments grow from the nucleus toward the cytoplasm.     

Three cDNA sequences of mouse type I keratins. Cellular localization of the mRNAs in normal and hyperproliferative tissues

We have constructed cDNA libraries with poly(A)+ RNA from normal mouse footpad epidermis and from a squamous cell carcinoma of mouse back skin. Both libraries were screened for type I keratin clones. We present sequence data of three keratin cDNA clones which selected mRNAs coding for two 52-kDa proteins (clones pke 52 and pkSCC 52) as well as for a 50-kDa protein (clone pkSCC50). According to their carboxyl-terminal sequences, the two 52-kDa keratin proteins belong to a group of keratins with serine-rich subdomains adjacent to the alpha-helix, whereas the short carboxyl-terminus of the 50-kDa protein lacks a distinct substructure. Sequentially the two 52-kDa keratins are more closely related to each other than to any other mouse type I keratin. However, in situ hybridization with specific subclones reveals a distinctly different pattern of expression in mouse epithelia. Clone pkSCC 52 contains sequence information for a 52-kDa keratin present in basal cells of epidermis and other stratified epithelia, whereas the pke 52 cDNA encodes a keratin which is predominantly expressed in suprabasal cells of nonepidermal tissues. In terms of nucleotide sequence identities, it cannot precisely be decided which of the two mouse 52-kDa proteins is the equivalent of the human epidermal 50-kDa keratin protein (Hanukoglu, I., and Fuchs, E. (1982) Cell 31, 243-252). In the case of the bovine keratin VII, however (Jorcano, J.L., Rieger, M., Franz, J.K., Schiller, D.L., Moll, R., and Franke, W.W. (1984) J. Mol. Biol. 179, 257-281) the sequence identity values speak for an equivalence with the mouse ke 52 keratin. Obviously, in situ hybridization experiments would best be suited to unravel the precise interspecies relationship between the four highly similar keratins. The discriminatory efficacy of this technique is further emphasized by the demonstration that the mRNA for a 50-kDa keratin is present not only in hyperproliferative epithelia, but also in normal cells of hair follicles.     

Amino acid sequence diversity between bovine epidermal cytokeratin polypeptides of the basic (type II) subfamily as determined from cDNA clones

The nucleotide sequences of four cDNA clones, each representing the carboxyterminal portion of a bovine epidermal cytokeratin of the "basic" (type II) subfamily, were determined, i.e., components Ia (Mr 68,000), Ib (Mr 68,000), III (Mr 60,000), and IV (Mr 59,000). The comparison of the sequences with each other and with the human type-II cytokeratin of Mr 56,000 reported by Hanukoglu and Fuchs [24] allows the following conclusions: The four major epidermal keratins of the basic (type II) subfamily, which are co-expressed in keratinocytes of the bovine muzzle, exhibit a high homology (greater than 90%) in the alpha-helical portion, but differ considerably in their nonhelical carboxy-terminal regions. The nonhelical carboxyterminal regions of all four cytokeratins are exceptionally rich in glycine and serine. Within the extrahelical tail, three different domains can be distinguished. The consensus sequence TYR(X)LLEGE which demarcates the end of the alpha-helical rod in all intermediate filaments is followed by a relatively short (22-27 amino acids) intercept rich in hydroxy amino acids and valine (carboxyterminal tail domain C1). This is followed by a long region that is variable in size and sequence, rich in glycine di-, tri-, and tetrapeptides, and contains diverse repeated sequences (domain C2). This is followed by another short (20 residues) hydroxy-amino-acid-rich intercept (domain C3) that ends with a conspicuously basic sequence of approximately four to six carboxyterminal amino acids. The first half of domain C1 is also homologous in all four keratins, suggesting that this region also assumes a common conformation and/or serves a special common function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization

When human epidermal cells were seeded on floating rafts of collagen and fibroblasts, they stratified at the air-liquid interface. The suprabasal cells synthesized the large type II (K1) and type I (K10/K11) keratins characteristic of terminal differentiation in skin. At earlier times in culture, expression of the large type II keratins appeared to precede the expression of their type I partners. At later times, all suprabasal cells expressed both types, suggesting that the accumulation of a critical level of K1 keratin may be a necessary stimulus for K10 and K11 expression. Expression of the terminal differentiation-specific keratins was completely suppressed by adding retinoic acid to the culture medium, or by submerging the cultures in normal medium. In submerged cultures, removal of vitamin A by delipidization of the serum restored the keratinization process. In contrast, calcium and transforming growth factor-beta did not influence the expression of the large keratins in keratinocytes grown in the presence of retinoids, even though they are known to induce certain morphological features of terminal differentiation. Retinoic acid in the raft medium not only suppressed the expression of the large keratins, but, in addition, induced the synthesis of two new keratins not normally expressed in epidermis in vivo. Immunofluorescence localized one of these keratins, K19, to a few isolated cells of the stratifying culture. In contrast, the other keratin, K13, appeared uniformly in a few outer layers of the culture. Interestingly, K13 expression correlated well with the gradient of retinoid-mediated disruptions of intercellular interactions in the culture. These data suggest that K13 induction may in some way relate to the reduction in either the number or the strength of desmosomal contacts between suprabasal cells of stratified squamous epithelial tissues.     

Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells.

Human keratin 18 (K18) and the homologous mouse protein, Endo B, are intermediate filament subunits of the type I keratin class. Both are expressed in many simple epithelial cell types including trophoblasts, the first differentiated cell type to appear during mouse embryogenesis. The K18 gene was identified and cloned from among the 15 to 20 similar sequences identified within the human genome. The identity of the cloned gene was confirmed by comparing the sequence of the first two exons to the K18 cDNA sequence and transfecting the gene into various murine cell lines and verifying the encoded protein as K18 by immunoprecipitation and partial peptide mapping. The transfected K18 gene was expressed in mouse HR9 parietal endodermal cells and mouse fibroblasts even though the fibroblasts fail to express endogenous Endo B. S1 nuclease protection analysis indicated that mRNA synthesized from the transfected K18 gene is initiated at the same position as authentic K18 mRNA found in both BeWo trophoblastoma cells and HeLa cells. Pulse-chase experiments indicated that the human K18 protein is stable in murine parietal endodermal cells (HR9) which express EndoA, a complementary mouse type II keratin. Surprisingly, however, K18 was degraded when synthesized in cells which lack a type II keratin. This turnover of K18 may be an important mechanism by which epithelial cells maintain equal molar amounts of both type I and II keratins. In addition, the levels of the endogenous type I Endo B in parietal endodermal cells were compensatingly down regulated in the presence of the K18 protein, while the levels of the endogenous type II Endo A were not affected in any of the transfected cell lines.     

Cytoskeletal disorders in human keratinocytes--epidermolysis bullosa simplex]

The cytoskeletons possibly related to pathogenesis in skin disease may be limited to keratin intermediate filaments (KIF) in epidermal keratinocytes. Keratins are divided into two subclasses; 11 acidic (type I) keratins and 8 basic (type II) keratins. Combination of equimolar amounts of type I and type II can form KIF. KIFs in human epidermal basal cells consist of a pair of type I and type II keratins specifically synthesized in the basal cells, and those in spinous cells contain two pairs of keratin; a pair of basal cell keratin and another pair of keratin specific for suprabasal cells. In the first section, molecular biology and differentiation of keratins are reviewed. In the second section, epidermolysis bullosa simplex (EBS) was introduced from the view point of abnormal organization of KIFs. In the epidermis of EBS, clefts are induced in the basal cells by minor trauma or frictions consequently to produce bullae. Electron microscopy reveals small spherical aggregations of tonofilaments (KIFs) in the basal cells. In biopsies, these KIF aggregations might be caused by artifacts during procedures for biopsies, so that, in order to avoid these artifacts, we studied the KIF organization in cultured keratinocytes from a patient by immunofluorescence using anti-keratin antibodies and electron microscopy. Anti-keratin antibodies revealed a formation of small droplet-like aggregations of KIFs in many cultured cells adhering to the culture bottles, which were also suggested by electron microscopy. From these observations, it is suggested that the abnormal organization (droplets) of KIFs might be one of intrinsic factors for the pathogenesis of EBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type II keratin cDNAs from the rainbow trout: implications for keratin evolution

From a teleost fish, the rainbow trout Oncorhynchus mykiss, we have cloned and sequenced cDNAs encoding five different type II keratins. The corresponding protein spots, as separated by 2D-PAGE of trout cytoskeletal preparations, have been identified by peptide mass mapping using MALDI mass spectrometry. Three of the sequenced keratins are expressed in the epidermis (subtype IIe), and two in simple epithelia and mesenchymal cells (subtype IIs). The IIs keratins are both orthologs of human K8. This leaves unsequenced only the trace component S3 of the biochemically established trout keratin catalog. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new keratin sequences together with type II sequences from other vertebrates such as shark, zebrafish, and human; lamprey K8 (recently sequenced in our laboratory) has been used as outgroup. This tree suggests, in a highly bootstrap-supported manner, that the teleost IIe keratins diversified independently from the mammalian IIe keratins. In contrast, all the species investigated express K8-like keratins, suggesting that the different IIe branches evolved from K8-like progenitors. The tree also indicates that the published zebrafish sequences represent IIe keratins and that the biochemically identified K8 ortholog in zebrafish has not yet been sequenced.