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Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD.

Inada H, Izawa I, Nishizawa M, Fujita E, Kiyono T, Takahashi T, Momoi T, Inagaki M - J. Cell Biol. (2001)

Bottom Line: We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein.Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF.We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan.

ABSTRACT
Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

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Identification of TRADD as a K18-interacting protein. (A) Domain organization of human TRADD protein. Death domain (DD) is indicated. Position of the original clone encoding the COOH-terminal region of TRADD (TRADD-C) is also indicated. Numbers refer to amino acid position. (B) Interactions of TRADD-C or full-length TRADD (TRADD-F) with K18 or other IFs in two- hybrid system. Y190 cells cotransformed with various pGBD-C1-IFs and pGAD-C1-TRADD-F or pGAD-C1-TRADD-C were selected in minus tryptophan (Trp)-leucine (Leu) media and subjected to β-galactosidase filter assay. The numbers of plus signs represent the relative rates at which the transformed yeast colonies turned blue after incubation at 30°C on filters, +++, <3 h; ++, 3–8 h; +, >8 h. Minus sign represents colonies remaining white at 24 h. (C) Identification of the coil Ia region of K18 that is sufficient for binding to TRADD. Y190 cells cotransformed with various pGBD-C1-K18 deletion mutants and pGAD-C1-TRADD-F or pGAD-C1-TRADD-C were selected in minus Trp-Leu media and subjected to β-galactosidase filter assay. Plus and minus signs are the same as in B. Numbers refer to amino acid position.
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fig1: Identification of TRADD as a K18-interacting protein. (A) Domain organization of human TRADD protein. Death domain (DD) is indicated. Position of the original clone encoding the COOH-terminal region of TRADD (TRADD-C) is also indicated. Numbers refer to amino acid position. (B) Interactions of TRADD-C or full-length TRADD (TRADD-F) with K18 or other IFs in two- hybrid system. Y190 cells cotransformed with various pGBD-C1-IFs and pGAD-C1-TRADD-F or pGAD-C1-TRADD-C were selected in minus tryptophan (Trp)-leucine (Leu) media and subjected to β-galactosidase filter assay. The numbers of plus signs represent the relative rates at which the transformed yeast colonies turned blue after incubation at 30°C on filters, +++, <3 h; ++, 3–8 h; +, >8 h. Minus sign represents colonies remaining white at 24 h. (C) Identification of the coil Ia region of K18 that is sufficient for binding to TRADD. Y190 cells cotransformed with various pGBD-C1-K18 deletion mutants and pGAD-C1-TRADD-F or pGAD-C1-TRADD-C were selected in minus Trp-Leu media and subjected to β-galactosidase filter assay. Plus and minus signs are the same as in B. Numbers refer to amino acid position.

Mentions: To identify proteins interacting with K8/18, we screened a human liver cDNA library, using the yeast two-hybrid procedure with full-length K8 or K18 as a bait. In the library screening for K18, we earlier found that human Mrj, a DnaJ/heat shock protein 40 (hsp40) family protein, directly interacted with K18 as a cochaperone to regulate K8/18 filament organization (Izawa et al., 2000). We have also identified a clone that encodes the COOH-terminal region of TRADD as a K18-interacting protein. TRADD is a 34-kD protein that interacts specifically with TNFR1 (Hsu et al., 1995). In the initial step of TNFR1 signaling, TNF binds to the extracellular domain of TNFR1 and induces receptor trimerization (Banner et al., 1993). Next, the death domain of TNFR1 recruits the adaptor protein TRADD (Hsu et al., 1995). TRADD, in turn, binds Fas-associated death domain (FADD), TNFR-associated factor 2, and RIP, and activates the downstream signaling pathway leading to apoptosis (Hsu et al., 1996; Yeh et al., 1997), JNK/SAPK activation (Hsu et al., 1996; Liu et al., 1996; Yeh et al., 1997), and nuclear factor-κB (NF-κB) activation (Hsu et al., 1996; Kelliher et al., 1998). To determine the role of K8/18 in TNF signaling, we further analyzed the TRADD–K18 interaction. The human TRADD cDNA encodes a 312-aa protein, and the clone derived from the liver library contained residues 245 to 312 of TRADD, hereafter termed TRADD-C (Fig. 1 A). The TRADD-C fragment is the COOH-terminal part of the death domain of TRADD mediating the interaction between TRADD and the death domain of TNFR1, FADD, and RIP. To confirm that full-length TRADD (TRADD-F) interacts with K18 and to determine if TRADD can bind to other IF proteins, we examined the interaction of TRADD-F and TRADD-C with K5, K8, K14, K18, and type III IF proteins in the two-hybrid system (Fig. 1 B). TRADD-C strongly interacted with K14 and K18, type I (acidic) keratins, but did not interact with K5, K8, vimentin, glial fibrillary acidic protein (GFAP), or desmin. In the same fashion, TRADD-F specifically interacted with K14 and K18. We next analyzed the region of K18 that contained the TRADD interaction site. For this, the binding between a series of truncations of K18 and TRADD-F or TRADD-C was determined using the two-hybrid system (Fig. 1 C). The coil Ia region of the rod domain of K18 specifically interacted with both TRADD-F and TRADD-C.


Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD.

Inada H, Izawa I, Nishizawa M, Fujita E, Kiyono T, Takahashi T, Momoi T, Inagaki M - J. Cell Biol. (2001)

Identification of TRADD as a K18-interacting protein. (A) Domain organization of human TRADD protein. Death domain (DD) is indicated. Position of the original clone encoding the COOH-terminal region of TRADD (TRADD-C) is also indicated. Numbers refer to amino acid position. (B) Interactions of TRADD-C or full-length TRADD (TRADD-F) with K18 or other IFs in two- hybrid system. Y190 cells cotransformed with various pGBD-C1-IFs and pGAD-C1-TRADD-F or pGAD-C1-TRADD-C were selected in minus tryptophan (Trp)-leucine (Leu) media and subjected to β-galactosidase filter assay. The numbers of plus signs represent the relative rates at which the transformed yeast colonies turned blue after incubation at 30°C on filters, +++, <3 h; ++, 3–8 h; +, >8 h. Minus sign represents colonies remaining white at 24 h. (C) Identification of the coil Ia region of K18 that is sufficient for binding to TRADD. Y190 cells cotransformed with various pGBD-C1-K18 deletion mutants and pGAD-C1-TRADD-F or pGAD-C1-TRADD-C were selected in minus Trp-Leu media and subjected to β-galactosidase filter assay. Plus and minus signs are the same as in B. Numbers refer to amino acid position.
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Related In: Results  -  Collection

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fig1: Identification of TRADD as a K18-interacting protein. (A) Domain organization of human TRADD protein. Death domain (DD) is indicated. Position of the original clone encoding the COOH-terminal region of TRADD (TRADD-C) is also indicated. Numbers refer to amino acid position. (B) Interactions of TRADD-C or full-length TRADD (TRADD-F) with K18 or other IFs in two- hybrid system. Y190 cells cotransformed with various pGBD-C1-IFs and pGAD-C1-TRADD-F or pGAD-C1-TRADD-C were selected in minus tryptophan (Trp)-leucine (Leu) media and subjected to β-galactosidase filter assay. The numbers of plus signs represent the relative rates at which the transformed yeast colonies turned blue after incubation at 30°C on filters, +++, <3 h; ++, 3–8 h; +, >8 h. Minus sign represents colonies remaining white at 24 h. (C) Identification of the coil Ia region of K18 that is sufficient for binding to TRADD. Y190 cells cotransformed with various pGBD-C1-K18 deletion mutants and pGAD-C1-TRADD-F or pGAD-C1-TRADD-C were selected in minus Trp-Leu media and subjected to β-galactosidase filter assay. Plus and minus signs are the same as in B. Numbers refer to amino acid position.
Mentions: To identify proteins interacting with K8/18, we screened a human liver cDNA library, using the yeast two-hybrid procedure with full-length K8 or K18 as a bait. In the library screening for K18, we earlier found that human Mrj, a DnaJ/heat shock protein 40 (hsp40) family protein, directly interacted with K18 as a cochaperone to regulate K8/18 filament organization (Izawa et al., 2000). We have also identified a clone that encodes the COOH-terminal region of TRADD as a K18-interacting protein. TRADD is a 34-kD protein that interacts specifically with TNFR1 (Hsu et al., 1995). In the initial step of TNFR1 signaling, TNF binds to the extracellular domain of TNFR1 and induces receptor trimerization (Banner et al., 1993). Next, the death domain of TNFR1 recruits the adaptor protein TRADD (Hsu et al., 1995). TRADD, in turn, binds Fas-associated death domain (FADD), TNFR-associated factor 2, and RIP, and activates the downstream signaling pathway leading to apoptosis (Hsu et al., 1996; Yeh et al., 1997), JNK/SAPK activation (Hsu et al., 1996; Liu et al., 1996; Yeh et al., 1997), and nuclear factor-κB (NF-κB) activation (Hsu et al., 1996; Kelliher et al., 1998). To determine the role of K8/18 in TNF signaling, we further analyzed the TRADD–K18 interaction. The human TRADD cDNA encodes a 312-aa protein, and the clone derived from the liver library contained residues 245 to 312 of TRADD, hereafter termed TRADD-C (Fig. 1 A). The TRADD-C fragment is the COOH-terminal part of the death domain of TRADD mediating the interaction between TRADD and the death domain of TNFR1, FADD, and RIP. To confirm that full-length TRADD (TRADD-F) interacts with K18 and to determine if TRADD can bind to other IF proteins, we examined the interaction of TRADD-F and TRADD-C with K5, K8, K14, K18, and type III IF proteins in the two-hybrid system (Fig. 1 B). TRADD-C strongly interacted with K14 and K18, type I (acidic) keratins, but did not interact with K5, K8, vimentin, glial fibrillary acidic protein (GFAP), or desmin. In the same fashion, TRADD-F specifically interacted with K14 and K18. We next analyzed the region of K18 that contained the TRADD interaction site. For this, the binding between a series of truncations of K18 and TRADD-F or TRADD-C was determined using the two-hybrid system (Fig. 1 C). The coil Ia region of the rod domain of K18 specifically interacted with both TRADD-F and TRADD-C.

Bottom Line: We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein.Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF.We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan.

ABSTRACT
Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

Show MeSH
Related in: MedlinePlus