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Induction of adipose conversion in 3T3-L1 by bezafibrate has been previously shown to be enhanced by dibutyryl cAMP and to be associated with an early phosphorylation of a 60 kDa acidic protein [Biochim. Biophys. Acta 1054 (1990) 219-224; FEBS Lett. 285 (1991) 63-65]. We describe here the isolation and sequencing of two peptides of the protein concerned. Both appear to be homologous with two respective amino acid sequences of the mouse intermediate filament protein vimentin.

Tropomodulin (Tmod) is an actin filament pointed end capping protein found in the membrane skeleton of lens fiber cells. We demonstrate that Tmod4 is able to bind the lens-specific intermediate filament protein, filensin, in either co-sedimentation or solid phase binding assays in a saturable fashion, but with low affinity and stoichiometry. Furthermore, Tmod4 does not bind the 53 kDa rod domain of filensin, nor to CP49, the obligate assembly partner of filensin. Finally, the binding of filensin to Tmod4 does not inhibit the actin capping activity of Tmod4 in vitro, suggesting that the two functions are not mutually exclusive.

The low ionic strength structures of the type III intermediate filament (IF) proteins desmin and glial fibrillary acidic protein (GFAP) have been studied by transient electric birefringence measurements. Flexible dimers with a length of around 45 nm, particles with a length of 68 ± 6 nm (presumably tetramers and hexamers) and larger aggregates of 108 ± 19 nm are found. GFAP has an increased tendency to aggregate upon lowering of the pH. The aggregation state of desmin does not change in the pH range studied. The results are compared with previous results on vimentin.

A selective tyrosine fluorescence quenching is found on interaction of vimentin with poly(dT) and poly(rA). However, addition of poly(dA) does not result in tyrosine quenching. The number of nucleotides covered by vimentin upon binding (n) of poly(dT) (50 +/- 4) appeared to be approximately the same as for poly(rA) (44 +/- 4), while the apparent binding constant (K(app)) of the latter is slightly larger (5.0 +/- 2.0 x 10(7) M-1.cm-1 vs. 2.5 +/- 0.5 x 10(7) M-1.cm-1). The finding that there exists a specific strong interaction between vimentin and nucleic acids could help in the search for cellular functions of intermediate filament proteins.

The intermediate filament protein vimentin was phosphorylated with cAMP-dependent protein kinase under conditions that induce filament disassembly. Digestion of phosphorylated vimentin with lysine-specific endoprotease and subsequent tryptic peptide mapping indicated that a 12 kDa N-terminal fragment contained all the phosphorylation sites found in the intact molecule. Analysis of cyanogen bromide digests indicated that two phosphorylated peptides were produced, with the major 32 P-labeled species representing amino acid position 14–72, and a minor 32 P-labeled peptide representing amino acid positions 1–13. These results demonstrate that phosphorylation of sites within the N-terminal head domain of vimentin are associated with phosphorylation induced filament disassembly.

Filaments formed by desmin, the myogenic intermediate filament protein, were crosslinked with the lysine specific crosslinker DST (disuccinimidyl tartrate; 0.64 nm span) and three DST crosslinked peptides were characterized. Two correspond to crosslinks previously obtained with the longer crosslinker EGS (ethylene glycol bis(succinimidylsuccinate), 1.61 nm span) which defined the antiparallel on-stagger relationship of neighbouring coiled coils. The two DST crosslinks now provide the relative positions of the coiled coils within a limit of about 9 α-helical residues. The third DST crosslink most likely connecting two helices of a single coiled coil gives a direct measure of the distance spanned in DST crosslinks.

Keratin 8 and 18 are simple epithelial intermediate filament (IF) proteins, whose expression is differentiation- and tissue-specific, and is maintained during tumorigenesis. Vimentin IF is often co-expressed with keratins in cancer cells. Recently, IF have been proposed to be involved in signaling pathways regulating cell growth, death and motility. The PI3K/Akt pathway plays a pivotal role in these processes. Thus, we investigated the role of Akt (1 and 2) in regulating IF expression in different epithelial cancer cell lines. Over-expression of Akt1 increases K8/18 proteins. Akt2 up-regulates K18 and vimentin expression by an increased mRNA stability. To our knowledge, these results represent the first indication that Akt isoforms regulate IF expression and support the hypothesis that IFs are involved in PI3K/Akt pathway.

The mouse monoclonal antibody ME 101 raised against human peripherin, an intermediate filament protein (IFP) specific to well defined neuronal populations, recognizes all the major classes of vertebrate IFP in immunoblotting assays. Desmin, GFAP, vimentin, peripherin and the lightest neurofilament protein (NF-L) were cleaved into carboxy- and amino-terminal halves by N-chlorosuccinimide at their unique trytophan residue. Whereas the antibody directed against the epitope common to every IFP (intermediate filament antigen or IFA) and located on the carboxy-terminal end of the rod domain recognizes the carboxy-terminal half, the ME 101 antibody, as the present study illustrates, recognizes specifically the amino-terminal half. From the amino acid sequence data of IFP, it is deduced that the cognate epitope is localized on the amino-terminal part of coil la.

A novel, simple and relatively rapid method is described for the isolation of the intermediate-sized filament protein vimentin from eye lens tissue. Chromatofocusing is applied as the sole purification step. The apparent isoelectric point of the protein in 6 M urea and at 22 degrees C is 4.9. Electrophoretic mobility on one- and two-dimensional polyacrylamide gels, solubility in 6 M urea and amino acid composition were used for identification.

The amino-terminal 98 residues of porcine vimentin have been determined by amino acid sequence studies. Extensive overlap is seen with the corresponding region of the carboxyterminal 448 residues of hamster vimentin predicted from DNA sequence studies, which left the very amino-terminal region unknown. The combined data show that contrary to gel electrophoretic results, mammalian vimentin contains only about 467 residues, and that species-specific drift occurs mainly in the amino-terminal non-α-helical array. The results are discussed parallel to emerging concepts on intermediate filament protein diversity.

The expression of cytokeratins and vimentin was investigated in epithelial cells under conditions of varied cell spreading and cell-cell contact. When extensive cell-cell contact was achieved in dense monolayer cultures, or in suspension in multicellular aggregates, the cells synthesized high levels of cytokeratins and low levels of vimentin. In contrast, sparse monolayer and suspension cultures, with minimal cell-cell contact, synthesized low levels of cytokeratins and high levels of vimentin. The ratio of cytokeratin to vimentin synthesis was independent of the cell cycle and was also reflected at the level of mRNA translational activity in vitro. Thus control of cytokeratin synthesis involves cell-cell contact, while vimentin synthesis responds to cell shape changes.