The fate of desmosomal proteins in apoptotic cells.

Activation of caspases results in the disruption of structural and signaling networks in apoptotic cells. Recent biochemical and cell biological studies have shown that components of the cadherin-catenin adhesion complex in epithelial adherens junctions are targeted by caspases during apoptosis. In epithelial cells, desmosomes represent a second type of anchoring junctions mediating strong cell-cell contacts. Using antibodies directed against a set of desmosomal proteins, we show that desmosomes are proteolytically targeted during apoptosis. Desmogleins and desmocollins, representing desmosome-specific members of the cadherin superfamily of cell adhesion molecules, are specifically cleaved after onset of apoptosis. Similar to E-cadherin, the desmoglein-3 cytoplasmic tail is cleaved by caspases. In addition the extracellular domains of desmoglein-3 and desmocollin-3 are released from the cell surface by a metalloproteinase activity. In the presence of caspase and/or metalloproteinase inhibitors, both cleavage reactions are almost completely inhibited. As reported previously, the desmosomal plaque protein plakoglobin is cleaved by caspase-3 during apoptosis. Our studies now show that plakophilin-1 and two other major plaque proteins, desmoplakin-1 and -2, are also cleaved by caspases. Immunofluorescence analysis confirmed that this cleavage results in the disruption of the desmosome structure and thus contributes to cell rounding and disintegration of the intermediate filament system.

Plakophilin 1 interferes with plakoglobin binding to desmoplakin, yet together with plakoglobin promotes clustering of desmosomal plaque complexes at cell-cell borders.

Desmosomes are adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. These junctions play an important role in providing strength to tissues that experience mechanical stress such as heart and epidermis. The basic structural elements of desmosomes are similar to those of the better-characterized adherens junctions, which anchor actin-containing microfilaments to cadherins at the plasma membrane. This linkage of actin to classic cadherins is thought to occur through an indirect mechanism requiring the associated proteins, alpha- and beta-catenin. In the case of desmosomes, both linear and lateral interactions have been proposed as playing an important role in formation of the plaque and linkage to the cytoskeleton. However, the precise nature of these interactions and how they cooperate in desmosome assembly are poorly understood. Here we employ a reconstitution system to examine the assembly of macromolecular complexes from components found in desmosomes of the differentiated layers of complex tissues. We demonstrate the existence of a Triton-soluble complex of proteins containing full length desmoplakin (DP), the arm protein plakoglobin, and the cytoplasmic domain of the desmosomal cadherin, desmoglein 1 (Dsg1). In addition, full length DP, but not an N-terminal plakoglobin binding domain of DP, co-immunoprecipitated with the Dsg1 tail in the absence of plakoglobin in HT1080 cells. The relative roles of the arm proteins plakoglobin and plakophilin 1 (PKP1) were also investigated. Our results suggest that, in the Triton soluble pool, PKP1 interferes with binding of plakoglobin to full length DP when these proteins are co-expressed. Nevertheless, both plakoglobin and PKP1 are required for the formation of clustered structures containing DP and the Dsg1 tail that ultrastructurally appear similar to desmosomal plaques found in the epidermis. These findings suggest that more than one armadillo family member is required for normal assembly and clustering of the desmosomal plaque in the upper layers of the epidermis.

The function of plakophilin 1 in desmosome assembly and actin filament organization.

Plakophilin 1, a member of the armadillo multigene family, is a protein with dual localization in the nucleus and in desmosomes. To elucidate its role in desmosome assembly and regulation, we have analyzed its localization and binding partners in vivo. When overexpressed in HaCaT keratinocytes, plakophilin 1 localized to the nucleus and to desmosomes, and dramatically enhanced the recruitment of desmosomal proteins to the plasma membrane. This effect was mediated by plakophilin 1's head domain, which interacted with desmoglein 1, desmoplakin, and keratins in the yeast two-hybrid system. Overexpression of the armadillo repeat domain induced a striking dominant negative phenotype with the formation of filopodia and long cellular protrusions, where plakophilin 1 colocalized with actin filaments. This phenotype was strictly dependent on a conserved motif in the center of the armadillo repeat domain. Our results demonstrate that plakophilin 1 contains two functionally distinct domains: the head domain, which could play a role in organizing the desmosomal plaque in suprabasal cells, and the armadillo repeat domain, which might be involved in regulating the dynamics of the actin cytoskeleton.

Expression of secreted frizzled related proteins 3 and 4 in human ventricular myocardium correlates with apoptosis related gene expression.

OBJECTIVE: Overload-induced heart failure is associated with myocyte apoptosis induced by unknown mechanisms. Wnt genes encode secreted signaling molecules that bind to frizzled receptors and stabilize cytosolic beta-catenin which is translocated into the nucleus, acts as transcriptional activator and imparts an apoptosis resistant phenotype. This signaling pathway is antagonized by secreted frizzled related proteins (sFRPs) which modulate apoptosis susceptibility in cell culture models. On the basis of these considerations, the present investigation compares myocardial mRNA expression of sFRPs and the level of soluble beta-catenin in tissue samples from nonfailing and failing hearts. METHODS: Nonischemic transmural samples from human failing left ventricles and from nonfailing donor ventricles were used in the present study. The mRNA concentration of the Wnt-antagonists sFRP 1-4 were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). The myocardial localization of sFRP 3 and 4 expression was investigated using in situ RT-PCR. The pool of soluble beta-catenin was quantified by Western blot analysis of protein extracts. RESULTS: The mRNA levels of proapoptotic sFRPs 3 and 4 but not of sFRP 1 and 2 were elevated in failing ventricles compared to donor hearts. There was no significant difference between patients suffering from a dilated cardiomyopathy or a coronary heart disease. sFRPs 3 and 4 were expressed in cardiomyocytes and their expression correlated with the mRNA expression of the proapoptotic Fas/Fas-antagonist ratio, but inversely with the mRNA levels of the antiapoptotic bcl-xL. The size of the pool of 0.1% Triton soluble beta-catenin tended to decrease in myocardial samples with high sFRP 3 and 4 expression levels. CONCLUSIONS: The results support the hypothesis that in failing human myocardium the Wnt/beta-catenin pathway is attenuated by enhanced expression of two endogenous Wnt-antagonists. This might contribute to an apoptosis susceptible phenotype of overloaded human myocardium.

p0071, a member of the armadillo multigene family, is a constituent of sarcomeric I-bands in human skeletal muscle.

p0071 is a member of the armadillo gene family that is expressed in a wide variety of mammalian tissues and cell types with a prominent cell-cell contact association in epithelial cells. Here, we report the expression and localization patterns of p0071 in differentiating human skeletal muscle cells and in normal and diseased human skeletal muscle tissues. Northern blots revealed expression of p0071 mRNA in adult skeletal muscle tissue. RT-PCR analysis and Western blotting experiments identified two differentially spliced isoforms of p0071. The balance between these isoforms shifted during in vitro differentiation of isolated muscle cells from predominant expression of the short variant to a preponderance of the larger variant from day 6 onwards. Immunolocalization studies in mature skeletal muscle tissue revealed that p0071 is a constituent of myofibrils with a distinct localization at the level of sarcomeric N2-lines. During myofibrillogenesis, p0071 was not detected in non-striated nascent myofibrils, but became apparent shortly after the development of compact Z-discs in early myotubes. Furthermore, we studied the expression of p0071 in a wide variety of neuromuscular disorders by indirect immunofluorescence. Here, the myofibrillar staining of p0071 was preserved in all the disease entities included in our study. Our results provide the first evidence that a member of the armadillo multigene family is a constituent of the contractile apparatus in human skeletal muscle. The localization of p0071 at the level of I-bands and the timepoint of its integration into developing myofibrils suggest a possible role in the organization of thin filaments.

Molecular map of the desmosomal plaque.

Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the 'a' form of desmocollin overlaps with plakoglobin and desmoplakin, the shorter 'b' form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.

The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes. Implications for cutaneous disease.

The contribution of desmosomes to epidermal integrity is evident in the inherited blistering disorder associated with the absence of a functional gene for plakophilin-1. To define the function of plakophilin-1 in desmosome assembly, interactions among the desmosomal cadherins, desmoplakin, and the armadillo family members plakoglobin and plakophilin-1 were examined. In transient expression assays, plakophilin-1 formed complexes with a desmoplakin amino-terminal domain and enhanced its recruitment to cell-cell borders; this recruitment was not dependent on the equimolar expression of desmosomal cadherins. In contrast to desmoplakin-plakoglobin interactions, the interaction between desmoplakin and plakophilin-1 was not mediated by the armadillo repeat domain of plakophilin-1 but by the non-armadillo head domain, as assessed by yeast two-hybrid and recruitment assays. We propose a model whereby plakoglobin serves as a linker between the cadherins and desmoplakin, whereas plakophilin-1 enhances lateral interactions between desmoplakin molecules. This model suggests that epidermal lesions in patients lacking plakophilin-1 are a consequence of the loss of integrity resulting from a decrease in binding sites for desmoplakin and intermediate filaments at desmosomes.

The armadillo family of structural proteins.

The armadillo gene is a segment polarity gene of Drosophila involved in signal transduction through wingless. Since the mid-1980s, a growing number of related proteins have been identified based on sequence homologies. These proteins share a central domain that is composed of a series of imperfect 45 amino acid repeats. Armadillo family members reveal diverse cellular locations reflecting their diverse functions. A single protein exerts several functions through interactions of its armadillo repeat domain with diverse binding partners. The proteins combine structural roles as cell-contact and cytoskeleton-associated proteins and signaling functions by generating and transducing signals affecting gene expression. The study of armadillo family members has made it increasingly clear that a distinction between structural proteins on the one hand and signaling molecules on the other is rather artificial. Instead armadillo family members exert both functions by interacting with a number of distinct cellular-binding partners.

Tyrosine phosphorylation and src family kinases control keratinocyte cell-cell adhesion.

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that beta-catenin, gamma-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of alpha-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of alpha-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of beta- and gamma-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn-/- mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of beta-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.

Protein-protein interactions between keratin polypeptides expressed in the yeast two-hybrid system.

Keratin filaments are obligatory heteropolymers of type I and type II keratin polypeptides. Specific type I/type II pairs are coexpressed in vivo. In contrast, all type I/type II pairs assemble into filaments in vitro, but the different pairs have different stabilities as demonstrated by treatment with increasing concentrations of urea. We have used the yeast two-hybrid system to analyse type I/type II interactions in a cellular context. We measured interactions between two different keratin pairs and we confirm the findings that K6+K17 form very stable heterodimers whereas K8+K18 interactions were weaker. The deletion of head domains did not reduce the strength of type I/type II interactions. Rather, the affinities were increased and the differences between the two pairs were retained in headless mutants. These findings argue against a major role of the head domains in directing heterodimer interactions and in defining heterodimer stabilities.

Cloning and characterization of a new armadillo family member, p0071, associated with the junctional plaque: evidence for a subfamily of closely related proteins.

Cell contacts of the adherens type are organized around transmembrane proteins of the cadherin family. Whereas the extracellular domains mediate homophilic interactions between cadherins of neighbouring cells the cytoplasmic domains organize a set of proteins into the junctional plaque. Among these junctional plaque proteins are members of the armadillo gene family, beta-catenin, plakoglobin (gamma-catenin), B6P/plakophilin and p120. These proteins are assumed to play a key role in cell cell signalling through intercellular junctions. Here we report cloning of a cDNA encoding a new armadillo family member, p0071, closely related to p120 and B6P/plakophilin and more distantly related to armadillo, plakoglobin, beta-catenin and other members of the gene family. The deduced amino acid sequence encodes a basic protein of 1,211 amino acids with a central armadillo repeat region which is conserved in sequence and organization of its ten individual motifs between p120, B6P/plakophilin and p0071. In contrast the end domains of the three proteins are variable in size and sequence. The RNA coding for p0071 is expressed in all tissues examined. Using antibodies generated against the armadillo repeat region of the protein we show that p0071 is localized at cell-cell borders and is expressed in the desmosomal plaque of some cultured epithelial cells. The protein seems to be an accessory component of the desmosomal plaque as well as of other adhesion plaques and might be involved in regulating junctional plaque organization and cadherin function. Our data provide evidence for a subfamily of armadillo related proteins that share not only structural features but also have in common their localisation in the junctional plaque. We therefore suggest that family members exert similar functions and might be involved in cell signalling through cell contacts.

Band 6 protein, a major constituent of desmosomes from stratified epithelia, is a novel member of the armadillo multigene family.

Desmosomes are intercellular adhering junctions characteristic of epithelial cells. Several constitutive proteins--desmoplakin, plakoglobin and the transmembrane glycoproteins desmoglein and desmocollin--have been identified as fundamental constituents of desmosomes in all tissues. A number of additional and cell type-specific constituents also contribute to desmosomal plaque formation. Among these proteins is the band 6 polypeptide (B6P). This positively charged, non-glycosylated protein is a major constituent of the plaque in stratified and complex glandular epithelia. Using an overlay assay we show that purified keratins bind in vitro to B6P. Thus B6P may play a role in ordering intermediate filament networks of adjacent epithelial cells. To characterize the structure of B6P in the desmosome we have isolated cDNA clones representing the entire coding sequence. The predicted amino acid sequence of human B6P shows strong sequence homology with a murine p120 protein, which is a substrate of protein tyrosine kinase receptors and of p60v-src. P120 and B6P show amino-terminal domains differing distinctly in length and sequence. These are followed in both proteins by 460 residues that display a series of imperfect repeats corresponding to the repeats in the cadherin binding proteins armadillo, plakoglobin and beta-catenin. Over this repeat region B6P and p120 share 33% sequence identity (54% similarity). These sequence characteristics define B6P as a novel member of the armadillo multigene family and raise the question of whether the structural proteins B6P, plakoglobin, beta-catenin and armadillo share some function. Since armadillo, plakoglobin, beta-catenin and p120 seem involved in signal transduction this may also hold for B6P. The amino-terminal region of B6P (residues 1 to 263) shows no significant homology to any known protein sequence. It may therefore be involved in unique functions of B6P.

Function of type I and type II keratin head domains: their role in dimer, tetramer and filament formation.

To examine the role of the keratin head region and its subdomains in filament assembly we constructed several deletion mutants of type I and type II keratins and analysed their in vitro IF forming capacity. The delta K8 (1-74) and delta K18 (1-56), mutants formed only soluble oligomers, predominantly tetramers with their heterotypic partners. K8 mutants that retained either the entire (delta K8 (1-64)) or nearly the entire (delta K8 (1-66)) H1 subdomain formed some short and irregular IF-like structures with K18. However, filaments never reached the normal length and more protofilamentous material was observed. Analysis of the soluble complexes in 2 M guanidine-HCl indicated that tetramer formation was impaired in the truncated molecules. The length of the deletion correlated with the degree of tetramer destabilization. These results suggest that the head domain--specifically the H1 subdomain of type II keratins-plays a direct role in IF assembly. Its functions include a stabilization of the tetramer molecule, suggesting a role in directing the alignment of dimers as well as in elongation. We also analysed whether both head domains are required or if either type I or type II head domains alone are sufficient for IF formation. Hybrid molecules carrying their partner keratins head domains (K18 (8 head) and K8 (18 head)) were combined with their wild-type partners and tested for IF-forming ability. Both combinations formed filaments distinct from normal IF. The effect of the 'replaced' head domains was not compensated when both hybrid molecules were combined. Taken together, the results indicate that complete removal of the head domains of either K8 or K18 arrested IF assembly at the state of soluble oligomers. Replacement of the head domains by head domains of the complementary partner partly compensated for the effect. However, regular IF formation could not take place when either the head domain was missing or it was replaced by the partner's keratin head.

In vitro assembly properties of vimentin mutagenized at the beta-site tail motif.

The intermediate filament (IF) proteins vimentin, desmin and peripherin share a 9-residue sequence motif (beta-site) located near the end of their COOH-terminal tail domain. Peptide inhibition experiments have previously suggested that the beta-site is involved in interactions that limit the lateral growth of IFs and prevent inappropriate filament-filament associations. To investigate this question further, we have constructed and expressed, in Escherichia coli, hamster vimentin bearing different mutations in the beta-site. We show here that a single exchange of glycine 450 with a valine residue, or an internal deletion of amino acids 444-452, strongly interferes with the normal assembly of IFs under in vitro conditions. These mutants polymerize into irregular fibrils that have a strong tendency to anastomose and laterally aggregate under isotonic conditions. In contrast, a non-conservative substitution of arginine 448 for glutamic acid does not significantly interfere with filament structure and yields subunits that polymerize into long, smooth filaments that show a slight aberration in thickness. All mutant proteins are soluble in low salt and form oligomers similar to the ones formed by wild-type vimentin. On the basis of these findings and on related observations, we propose that the tail domain of type III IF proteins contains important structural elements involved in lateral protofilament-protofilament interactions.

Truncation of recombinant vimentin by ompT. Identification of a short motif in the head domain necessary for assembly of type III intermediate filament proteins.

Recombinant vimentin expressed in E. coli JM 101 cells is cleaved after cell lysis between arginines 11 and 12. The truncated vimentin is assembly incompetent. Expression of the same cDNA construct in BL21 cells, which lack the protease ompT, provides intact and polymerization-competent vimentin. The ompT cleavage site is contained in a short sequence motif (YRRMF) shared by the head domains of type III and IV intermediate filament (IF) proteins. We propose that a related motif present in the N-terminal 32 residues of lambda CII accounts for the known IF formation of a fusion protein formed with a truncated GFAP.

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.

Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain.

All known intermediate filament (IF) proteins display -8 -4 -1 a consensus sequence TYRKLLEGE at the carboxyl end of the rod domain. To analyse the contribution of this sequence to the formation of IF we have changed two of the invariant positions of this motif by site-directed mutagenesis. We produced three mutant keratins, each containing a single point mutation. Tyrosine at position -8 was changed to alanine in keratin K8 (K8Y----A-8) and keratin K18 (K18Y----A-8) and leucine at position -4 was changed to glycine in keratin K18 (K18L----G-4). Mutant keratins were expressed in Escherichia coli, purified and analysed for their filament-forming capacity in vitro using either the complementary wild-type keratin or the corresponding mixture of mutant keratins. In standard filament buffer (50 mM Tris-HCl, pH7.5), assembly involving any of the mutants leads to large electron-dense aggregates instead of normal IF. In order to explain this effect, we studied the process of filament formation in more detail. Whereas the formation of tetramers in buffers containing 4M urea is unaffected, the elongation process seems slowed down. In buffer of lower ionic strength (10 mM Tris-HCl, pH7.5) mutant keratins K8Y----A-8 plus K18Y----A-8 become able to form long filaments, although short filaments and protofilamentous material are still detected. The filaments formed differ from normal keratin IF by their remarkable tendency to aggregate into thick cables. Assemblies involving K18L----G-4 can only form short IF lengths. The dense aggregates formed in standard filament buffer are able to dissociate into IF and their fragments upon dialysis into 10 mM Tris-HCl, pH7.5. The results show that the consensus sequence is needed for IF formation under normal conditions and that already one mutation per heterodimer affects the assembly.

Tailless keratins assemble into regular intermediate filaments in vitro.

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.

The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression.

Recombinant DNA technology has been used to analyze the first step in keratin intermediate filament (IF) assembly; i.e., the formation of the double stranded coiled coil. Keratins 8 and 18, lacking cysteine, were subjected to site specific in vitro mutagenesis to change one amino acid in the same relative position of the alpha-helical rod domain of both keratins to a cysteine. The mutations lie at position -36 of the rod in a "d" position of the heptad repeat pattern, and thus air oxidation can introduce a zero-length cystine cross-link. Mutant keratins 8 and 18 purified separately from Escherichia coli readily formed cystine homodimers in 2 M guanidine-HCl, and could be separated from the monomers by gel filtration. Heterodimers with a cystine cross-link were obtained when filaments formed by the two reduced monomers were allowed to oxidize. Subsequent ion exchange chromatography in 8.5 M urea showed that only a single dimer species had formed. Diagonal electrophoresis and reverse phase HPLC identified the dimer as the cystine containing heterodimer. This heterodimer readily assembled again into IF indistinguishable from those obtained from the nonmutant counterparts or from authentic keratins. In contrast, the mixture of cystine-stabilized homodimers formed only large aberrant aggregates. However, when a reducing agent was added, filaments formed again and yielded the heterodimer after oxidation. Thus, the obligatory heteropolymer step in keratin IF assembly seems to occur preferentially at the dimer level and not during tetramer formation. Our results also suggest that keratin I and II homodimers, once formed, are at least in 2 M guanidine-HCl a metastable species as their mixtures convert spontaneously into heterodimers unless the homodimers are stabilized by the cystine cross-link. This previously unexpected property of homodimers explains major discrepancies in the literature on the keratin dimer.

Phosphorylation in vitro of vimentin by protein kinases A and C is restricted to the head domain. Identification of the phosphoserine sites and their influence on filament formation.

The in vitro phosphorylation of vimentin, the intermediate filament protein of mesenchymal cells, by kinases A and C is serine-specific and involves only the N-terminal head domain. In oligomeric protofilament units each kinase recognizes five sites, which have been identified by sequence analysis. Kinase C introduces 1.5 mol phosphate/mol vimentin, while kinase A treatment results in 4 mol phosphate/mol. Kinase-A-treated oligomers do not polymerize in standard assays whereas kinase C treatment has no inhibitory effect. Filaments exposed to kinase A remain stable and incorporate only 1.7 mol phosphate/mol vimentin. These phosphates are essentially restricted to two of the five kinase A sites found in protofilament units. Thus the head domain, previously related to in vitro assembly competence and filament stability, changes in accessibility between the oligomeric and polymeric state. We discuss the possibility that in vivo phosphorylation of vimentin filaments by kinase A may not necessarily be accompanied by an extensive depolymerization. It could instead involve a dynamic change of the filament surfaces, which could alter the interaction of the filaments with other cellular structures.