Roles of TRP14, a Thioredoxin-related Protein in Tumor Necrosis Factor-α Signaling Pathways

Abstract

The possible roles of a 14-kDa human thioredoxin (Trx)-related protein (TRP14) in TNF-␣ signaling were studied in comparison with those of Trx1 by RNA interference in HeLa cells. Depletion of TRP14 augmented the TNF-␣-induced phosphorylation and degradation of I B␣ as well as the consequent activation of NF-B to a greater extent than did Trx1 depletion. Deficiency of TRP14 or Trx1 enhanced TNF-␣-induced activation of caspases and subsequent apoptosis by a similar extent. The TNF-␣-induced activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), however, was promoted by depletion of TRP14 but not by that of Trx1. Unlike Trx1, TRP14 neither associated with nor inhibited the kinase activity of apoptosis signal-regulating kinase-1 (ASK1), an upstream activator of JNK and p38. In combination with the results in the accompanying paper that TRP14 did not reduce the known substrates of Trx1, these results suggest that TRP14 modulates TNF-␣ signaling pathways, provably by interacting with proteins distinct from the targets of Trx1. In an effort to identify target proteins of TRP14, a mutant of TRP14, in which the active site cysteine (Cys 46 ) was substituted with serine, was shown to form a disulfide-linked complex with LC8 cytoplasmic dynein light chain. The complex was detected in HeLa cells treated with H 2 O 2 or TNF-␣ but not in untreated cells, suggesting that LC8 cytoplasmic dynein light chain is a possible substrate of TRP14.The cellular redox state is affected not only by normal respiration and metabolism but also by the transient production of reactive oxygen species (ROS) 1 that results from the ligation of various cell surface receptors (1-3). Tumor necrosis factor-␣ (TNF-␣) was one of the first receptor ligands shown to generate ROS in nonphagocytic cells and is among the ligands whose signaling pathways have been studied most in relation to ROS production (4, 5). The binding of TNF-␣ to TNF receptor 1 (TNFR1) generates both proapoptotic and prosurvival signals by inducing the formation of a multiprotein signaling complex at the cell membrane. This complex in turn triggers the caspase cascade, the activation of specific kinases such as the I B kinase (IKK) complex and mitogen-activated protein kinases (MAPKs), and the transcription of various genes mediated by nuclear factor-B (NF-B) and activator protein-1 (AP-1) (6 -10). Many TNF-␣ signaling pathways are activated by ROS (7, 11-16) and attenuated by Trx overexpression (17-25).In the accompanying study (26), we identified and characterized a 14-kDa cytosolic protein, designated Trx-related protein 14 (TRP14). Here we studied the possible roles of TRP14 in TNF-␣ signaling in comparison with those of Trx1 by RNA interference (RNAi) in HeLa cells. Partial depletion of TRP14 or Trx1 by RNAi resulted in enhancement of TNF-␣-induced activation of caspases and NF-B. The TNF-␣-induced activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK, however, was augmented by depletion of TRP14 but not by that of Trx1. Furthermore, unl...

Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-β receptor tyrosine kinase signaling

“…5). TRP14 was originally described as a dynein light chain LC8-interacting protein that inhibits NF-κB signaling upon TNF-α stimulation of HeLa cells (37). Although TRP14 shares many structural features with Trx1, this redoxin cannot reduce classic Trx1 protein disulfide substrates, such as ribonucleotide reductase, peroxiredoxins, or methionine sulfoxide reductases (36,55).…”

“…The most prominent and ubiquitously expressed components of the Trx system are the cytosolic TrxR1 (encoded by the Txnrd1 gene) and its main substrate Trx1 (34,35). Another less characterized substrate of TrxR1 is TRP14 (36), discovered as a TrxR1-dependent oxidoreductase affecting TNF-α-induced NF-κB activation, which displays a more restricted substrate specificity than Trx1 (37,38).…”

Selenoproteins: Molecular Pathways and Physiological Roles

“…Similar to Trx1, TRP14 serves as a disulfide reductase, although it acts on a distinct set of protein targets. TRP14 has been shown to activate NF-B by stimulating TNF-␣ signaling (162). In addition to Trx1 and Trp14, TR1 can reduce nondisulfide substrates such as hydroperoxides, vitamin C, and selenite.…”

Disulfide Bond-mediated Multimerization of Ask1 and Its Reduction by Thioredoxin-1 Regulate H2O2-induced c-Jun NH2-terminal Kinase Activation and Apoptosis

“…The cells were transfected 24 h after plating with 0.01 to 4.0 g DNA plasmid by calcium phosphate precipitation as described previously (Landry et al, 1989). Small interfering RNA (siRNA) targeting human Trx1 was based on nucleotides 256-276 (Jeong et al, 2004) and obtained from Dharmacon RNA Technologies (Lafayette, CO). The nontargeting siRNA, siCONTROL #1 (Dharmacon RNA Technologies), was used as a negative control.…”