title: Kallikrein-related peptidase-7 regulates caspase-14 maturation during keratinocyte terminal differentiation by generating an intermediate form.
abstract: The maturation and activation mechanisms of caspases are generally well understood, except for those of caspase-14, which is activated at the onset of keratinocyte terminal differentiation. We investigated the possible involvement of epidermal proteases expressed in the late stage of differentiation, and found that the chymotrypsin-like serine protease kallikrein-related peptidase-7 (KLK7) cleaved procaspase-14 at Tyr(178), generating an intermediate form that consists of a large (20 kDa) and a small subunit (8 kDa). We prepared an antibody directed to this cleavage site (h14Y178 Ab), and confirmed that it recognized a 20-kDa band formed when procaspase-14 was incubated with chymotrypsin or KLK7. We then constructed a constitutively active form of the intermediate, revC14-Y178. The substrate specificity of revC14-Y178 was completely different from that of caspase-14, showing broad specificity for various caspase substrates except WEHD-7-amino-4-trifluoromethylcoumarin (AFC), the preferred substrate of active, mature caspase-14. K(m) values for VEID-AFC, DEVD-AFC, LEVD-AFC, and LEHD-AFC were 0.172, 0.261, 0.504, and 0.847 μM, respectively. We confirmed that the mature form of caspase-14 was generated when procaspase-14 was incubated with KLK7 or revC14-Y178. Expression of constitutively active KLK7 in cultured keratinocytes resulted in generation of both the intermediate form and the mature form of caspase-14. Immunohistochemical analysis demonstrated that the intermediate form was localized at the granular layer. Our results indicate that regulation of procaspase-14 maturation during terminal differentiation is a unique two-step process involving KLK7 and an activation intermediate of caspase-14.
authors: Shiseido Research Center, 2-12-1 Fukuura, Kanazawa-ku, Yokohama 236-8643, Japan.
authors: Mami
authors: M
authors: Yamamoto
authors: Masashi
authors: M
authors: Miyai
authors: Yuuko
authors: Y
authors: Matsumoto
authors: Ryoji
authors: R
authors: Tsuboi
authors: Toshihiko
authors: T
authors: Hibino
mesh_terms: D020169:Caspases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000737:chemistry; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D007610:Kallikreins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D008297:Male; D059748:Proteolysis; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D004792:Enzyme Precursors; C082472:KLK7 protein, human; D007610:Kallikreins; D002918:Chymotrypsin; C506446:CASP14 protein, human; D020169:Caspases