Desmoplakin interacts with the coil 1 of different types of intermediate filament proteins and displays high affinity for assembled intermediate filaments

Abstract

The interaction of intermediate filaments (IFs) with the cell-cell adhesion complexes desmosomes is crucial for cytoskeletal organization and cell resilience in the epidermis and heart. The intracellular desmosomal protein desmoplakin anchors IFs to the cell adhesion complexes predominantly via its four last carboxy-terminal domains (C-terminus). However, it remains unclear why the C-terminus of desmoplakin interacts with different IF types or if there are different binding affinities for each type of IFs that may influence the stability of cell-specific adhesion complexes. By yeast three-hybrid and fluorescence binding assays, we found that the coiled-coil 1 of the conserved central rod domain of the heterodimeric cytokeratins (Ks) 5 and 14 (K5/K14) was required for their interaction with the C-terminus of desmoplakin, while their unique amino head- and C-tail domains were dispensable. Similar findings were obtained in vitro with K1/K10, and the type III IF proteins desmin and vimentin. Binding assays testing the C-terminus of desmoplakin with assembled K5/K14 and desmin IFs yielded an apparent affinity in the nM range. Our findings reveal that the same conserved domain of IF proteins binds to the C-terminus of desmoplakin, which may help explain the previously reported broad binding IF-specificity to desmoplakin. Our data suggest that desmoplakin high-affinity binding to diverse IF proteins ensures robust linkages of IF cytoskeleton and desmosomes that maintain the structural integrity of cellular adhesion complexes. In summary, our results give new insights into the molecular basis of the IF-desmosome association.

Fig 1. The coil 1 of K5/K14 is essential for their interaction with the C-terminus of desmoplakin in Y3H assays.

Summary of Y2/3H results obtained with the indicated constructs. Numbers in each protein show amino acid order. K domains are labeled; the coils are drawn as boxes. + or − means growth or no growth, respectively, on selection medium without histidine (His) or adenine (Ade).

Fig 2. The coil 1 of K5/K14 and K1/K10 contains the major binding site(s) for the C-terminus of desmoplakin in overlay assays.

(A) Nitrocellulose membranes, spotted as indicated with bovine serum albumin (BSA, control) and individual or mixed (/) K5 and K14, either full-length proteins or the fragments coil 1 (C1) and coil 2 to tail (C2-T) (3 pmol/spot), were stained with amido black or overlaid with soluble extracts of HEK 293T cells expressing either EGFP-DSP C-terminus (64 ± 29 nM, total concentration) or EGFP (≥ 70 nM) and scanned for fluorescence. (B) Quantified fluorescence signals are relative to the normalized results obtained with the mixture (/) of full-length K5/K14. K5 + K14 and K5-C1 + K14-C1 correspond to the sum of fluorescence signals obtained with the individual proteins (not mixed). Mean ± SD, n = 3; ANOVA-Tukey test; ** and ****, residual P < 0.01 and < 0.0001, respectively. (C) and (D) as for (A) and (B) with K1 and K10.

Fig 3. The C-terminus of desmoplakin binds to the coil 1 of desmin and vimentin.

(A) Purified vimentin, desmin and SUMO (negative control) (4 μg/lane), or whole extracts from E. coli expressing full-length vimentin or desmin, the rod, coil 1 and coil 2 of these proteins or no exogenous protein (none), as indicated, were size-fractionated on 15% SDS-PAGE and transferred to WB membranes that were stained with Ponceau-S and overlaid with soluble extracts from HEK 293T cells expressing EGFP-DSP C-terminus or EGFP (both at 67 ± 17 nM, total concentration). M, markers. (B) Quantified fluorescence signals are relative to the normalized results obtained with full-length vimentin, mean ± SD, n≥4; ANOVA-Tukey test; *** and ****, residual P < 0.001 and < 0.0001, respectively.

Fig 4. Binding of the C-terminus of desmoplakin to K5/K14 and desmin IFs under equilibrium condition.

Variable concentrations of active EGFP-DSP C-terminus (blue, dots) in soluble HEK 293T cell extracts were mixed with a constant concentration of in vitro assembled IFs (25 μg/mL). Binding of the control EGFP (red, inverted triangles) was measured at the highest concentration of total (active + inactive) EGFP-DSP C-terminus. Representative binding curves from three independent experiments are shown (RFU, relative fluorescence unit).