Abstract
Type I keratins K18 and K19 undergo caspase-mediated degradation during apoptosis. Two known K18 caspase cleavage sites are aspartates in the consensus sequences VEVDA and DALDS, located within the rod domain and tail domain, respectively. Several K14 (another type I keratin) mutations within the caspase cleavage motif have been described in patients with epidermolysis bullosa simplex. Here we use extensive mutational analysis to show that K19 and K14 are caspase substrates and that the ability to undergo caspase-mediated digestion of K18, K19, or K14 is highly dependent on the location and nature of the mutation within the caspase cleavage motif. Caspase cleavage of K14 occurs at the aspartate of VEMDA, a consensus sequence found in type I keratins K12-17 with similar but not identical sequences in K18 and K19. For K18, apoptosis-induced cleavage occurs sequentially, first at (393)DALD and then at (234)VEVD. Hyperphosphorylation of K18 protects from caspase-3 in vitro digestion at (234)VEVD but not at (393)DALD. Hence, keratins K12-17 are likely caspase substrates during apoptosis. Keratin hyperphosphorylation, which occurs early in apoptosis, protects from caspase-mediated K18 digestion in a cleavage site-specific manner. Mutations in epidermolysis bullosa simplex patients could interfere with K14 degradation during apoptosis, depending on their location.
Highlights
Whereas their type II partner (i.e. K8) manifests remarkable resistance to apoptotic degradation
Most keratin mutations that have been identified in patients with epidermal blistering keratin diseases are located at the N-terminal region of the rod IA subdomain [12, 13], at least four K14 mutations have been described within the linker 1–2 (L1–2) region (14 –17) in close proximity to the caspase recognition motif (VEMDA, referred to as the caspase box)
Given the conserved nature of the rod domain motif (X1E/DX2DX3; X1-X3, aliphatic residues with caspase cleavage occurring at the aspartate between X2 and X3; Fig. 1A) and the presence of K14 mutations at the aspartate of VEMD (D3 G) and at X1 (V3 M), X2 (M3 R), and X3 (A3 D) in patients with epidermolysis bullosa simplex (EBS), we asked whether these mutations have an effect on type I keratin fragmentation during apoptosis
Summary
Vol 276, No 29, Issue of July 20, pp. 26792–26798, 2001 Printed in U.S.A. Effect of Mutation and Phosphorylation of Type I Keratins on Their Caspase-mediated Degradation*. Several K14 (another type I keratin) mutations within the caspase cleavage motif have been described in patients with epidermolysis bullosa simplex. Keratin hyperphosphorylation, which occurs early in apoptosis, protects from caspase-mediated K18 digestion in a cleavage site-specific manner. The proximity of these mutations to the caspase digestion site raises the possibility that the phenotype of the keratin disease in these instances may be associated with perturbations in keratin degradation. If so, this could potentially impact the disease pathophysiology in patients with epidermolysis bullosa simplex (EBS), who harbor K14 L1–2 region mutations, and may offer more directed therapeutic strategies. The VEMDA caspase box, which is found in many type I keratins (K12–17) is a suitable caspase substrate, as shown here for K14
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