Interaction of the Bullous Pemphigoid Antigen 1 (BP230) and Desmoplakin with Intermediate Filaments Is Mediated by Distinct Sequences within Their COOH Terminus

Abstract

The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.

Characterization of human epiplakin: RNAi-mediated epiplakin depletion leads to the disruption of keratin and vimentin IF networks

“…Furthermore, the linker and the B domain might have different roles in the binding to IFs. It was reported that the linker, B and C subdomains of desmoplakin and BP230 play an important role in the specificity and interaction with IFs (Fontao et al, 2003). We observed preferential binding of epiplakin fragments to different type of keratins.…”

“…Expression, purification of keratins, filaments assembly and dot blot overlay assays The expression and purification of human keratins were the same as described by Fontao et al (Fontao et al, 2003) except that the proteins were extracted from final pellets by urea buffer (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole, 8 M urea). After an additional centrifugation at 37,500 g, the supernatants were collected and the urea concentration was adjusted to 6 M. Keratins were further purified using a 5 ml Hi-Trap Ni-NTA column (Pharmacia) and eluted by a gradient of 5-500 mM imidazole.…”

Desmosomes and Intermediate Filaments: Their Consequences for Tissue Mechanics

“…Keratins interact with the plectin and DSP carboxy termini through segments of their rod domain (Meng et al 1997;Fontao et al 2003;Bouameur et al 2014). Association with keratins is regulated by serine phosphorylation and arginine methylation of the DSP tail, involving a coordinated activity of glycogen synthase kinase 3 (GSK3) and protein arginine methyltransferase 1 (PRMT1).…”

Spectraplakin family proteins – cytoskeletal crosslinkers with versatile roles

“…In addition, BPAG1e also binds to the epidermal cytokeratin network that is formed by keratins 5 and 14 (K5, K14) in basal keratinocytes and thereby connects them to the extracellular matrix (Fontao et al, 2003;Guo et al, 1995). In the epidermis, loss of BPAG1e leads to a selective fragility of the base of the columnar basal cells, resulting in blisters and compromised wound healing (Guo et al, 1995).…”