Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that can activate the c-Jun N-terminal kinase and the p38 signaling pathways. It plays a critical role in cytokine-and stressinduced apoptosis. To further characterize the mechanism of the regulation of the ASK1 signal, we searched for ASK1-interacting proteins employing the yeast twohybrid method. The yeast two-hybrid assay indicated that mouse glutathione S-transferase Mu 1-1 (mGSTM1-1), an enzyme involved in the metabolism of drugs and xenobiotics, interacted with ASK1. We subsequently confirmed that mGSTM1-1 physically associated with ASK1 both in vivo and in vitro. The in vitro binding assay indicated that the C-terminal portion of mGSTM1-1 and the N-terminal region of ASK1 were crucial for binding one another. Furthermore, mGSTM1-1 suppressed stress-stimulated ASK1 activity in cultured cells. mG-STM1-1 also blocked ASK1 oligomerization. The ASK1 inhibition by mGSTM1-1 occurred independently of the glutathione-conjugating activity of mGSTM1-1. Moreover, mGSTM1-1 repressed ASK1-dependent apoptotic cell death. Taken together, our findings suggest that mGSTM1-1 functions as an endogenous inhibitor of ASK1. This highlights a novel function for mGSTM1-1 insofar as mGSTM1-1 may modulate stress-mediated signals by repressing ASK1, and this activity occurs independently of its well-known catalytic activity in intracellular glutathione metabolism.

Hsp72, a major inducible member of the heat shock protein family, can protect cells against many cellular stresses including heat shock. In our present study, we observed that pretreatment of NIH 3T3 cells with mild heat shock (43 degrees C for 20 min) suppressed UV-stimulated c-Jun N-terminal kinase 1 (JNK1) activity. Constitutively overexpressed Hsp72 also inhibited JNK1 activation in NIH 3T3 cells, whereas it did not affect either SEK1 or MEKK1 activity. Both in vitro binding and kinase studies indicated that Hsp72 bound to JNK1 and that the peptide binding domain of Hsp72 was important to the binding and inhibition of JNK1. In vivo binding between endogenous Hsp72 and JNK1 in NIH 3T3 cells was confirmed by co-immunoprecipitation. Hsp72 also inhibited JNK-dependent apoptosis. Hsp72 antisense oligonucleotides blocked Hsp72 production in NIH 3T3 cells in response to mild heat shock and concomitantly abolished the suppressive effect of mild heat shock on UV-induced JNK activation and apoptosis. Collectively, our data suggest strongly that Hsp72 can modulate stress-activated signaling by directly inhibiting JNK.

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Glutamine has been known to be an apoptosis suppressor, since it blocks apoptosis induced by heat shock, irradiation, and c-Myc overexpression. Here, we demonstrated that HeLa cells were susceptible to Fas-mediated apoptosis under the condition of glutamine deprivation. Fas ligation activated apoptosis signalregulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK; also known as stress-activated protein kinase (SAPK)) in Gln-deprived cells but not in normal cells, suggesting that Gln might be involved in the activity control of ASK1 and JNK/SAPK. As one of the possible mechanisms for the suppressive effect of Gln on ASK1, we investigated the molecular interaction between human glutaminyl-tRNA synthetase (QRS) and ASK1 and found the Gln-dependent association of the two molecules. While their association was enhanced by the elevation of Gln concentration, they were dissociated by Fas ligation within 5 min. The association involved the catalytic domains of the two enzymes. The ASK1 activity was inhibited by the interaction with QRS as determined by in vitro kinase and transcription assays. Finally, we have shown that QRS inhibited the cell death induced by ASK1, and this antiapoptotic function of QRS was weakened by the deprivation of Gln. Thus, the antiapoptotic interaction of QRS with ASK1 is controlled positively by the cellular concentration of Gln and negatively by Fas ligation. The results of this work provide one possible explanation for the working mechanism of the antiapoptotic activity of Gln and suggest a novel function of mammalian ARSs.