Recent studies have demonstrated that ASK1 activity is positively or negatively regulated by its interacting partners, including tumor necrosis factor receptor-associated factor (28), Daxx (1), JNK/stress-activated protein kinase-associated protein 1 (JSAP1)/JNK-interacting protein 3 (JIP3) (29), thioredoxin (3,4), glutaredoxin (7), HSP72 (6), Raf-1 (3), Akt/PKB (30), PP5 (5), and 14-3-3 (8). both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H2O2-mediated apoptosis by enhancing the ASK1 activity through Thr838phosphorylation. These results suggest that MPK38 physically interacts with ASK1in vivoand acts as a positive upstream regulator of ASK1. Apoptosis signal-regulating kinase 1 (ASK1)2is one of the mitogen-activated protein kinase kinase kinases (MAPKKK) that is stimulated in response to various cellular stresses, including reactive oxygen species, tumor necrosis factor (TNF-), Fas, ischemia insult, and anti-tumor agents. ASK1 stimulation leads to activation of the c-Jun NH2-terminal kinase (JNK)/p38 signaling cascade by phosphorylating and activating mitogen-activated protein kinase kinases (MAPKK) such as MKK3, -4, -6, and -7 (13). Emerging evidence indicates that ASK1 activity is regulated by its interaction with several cellular partners (38), including thioredoxin (Trx), glutaredoxin, heat shock protein 72 (Hsp72), 14-3-3, and protein serine/threonine phosphatase 5 (PP5). For example, Trx and glutaredoxin bind to the NH2- and COOH-terminal domains of ASK1, respectively, and inhibit ASK1 kinase activity, and Hsp72 inhibits ASK1 activation through direct interaction. These findings suggest that other ASK1-interacting proteins could be involved in the regulation of ASK1 activity. Murine protein serine/threonine kinase 38 (MPK38), also known as maternal embryonic leucine zipper kinase (Melk), is a member of the AMP-activated protein kinase-related serine/threonine kinase family (9,10). MPK38 was originally identified as a murine counterpart for its human homolog, HPK38/hMelk/KIAA175, that may be involved in the proliferation of interleukin-4-induced normal human keratinocytes (9). Lupeol The importance of MPK38 in oncogenesis is also underscored by the finding that MPK38 expression is increased in tumor-derived progenitor cells as well as in cancers of nondifferentiated cells (1113). However, the physiological regulation and functions of MPK38 have remained unclear. To explore a functional link between ASK1 and MPK38 signaling pathways, we investigated the effect of MPK38 on ASK1 and its downstream targets. We demonstrated that MPK38 physically interacts with ASK1. MPK38 phosphorylates Thr838(corresponding to Thr845in mice) within the activation loop of human ASK1, which subsequently leads to the stimulation of ASK1 kinase activity. Moreover, this interaction results in the enhancement of JNK-mediated transactivation and H2O2-induced apoptosis. == MATERIALS AND METHODS == Cell Culture, Plasmids, Reagents, and Cell Line ConstructionHEK293, 293T, HaCaT, and SK-N-BE(2)C cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen). HA-tagged wild-type and kinase-dead ASK1 were kindly provided by Dr. H. Ichijo (Tokyo Medical and Dental University, Tokyo, Japan). HA-tagged ASK1-K and ASK1-C were a gift from Dr. S. Cho (Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea). The activator protein 1 (AP-1)-Luc reporter, pSuper vector, and c-Fos were the kind gifts from Dr. Y. Yeom (Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea). GlutathioneS-transferase (GST)-tagged MKK6(K82A) and p38 were a gift from Dr. E.-J. Choi (Korea University, Seoul, Korea). To generate the kinase-dead MPK38(K40R) construct, site-directed mutagenesis was carried out using the QuickChange II site-directed mutagenesis kit (Stratagene, La Jolla, Rabbit polyclonal to LYPD1 CA). PCR was performed using the full-length Lupeol MPK38 cDNA cloned into pBluescript KS (Stratagene) as the template in the presence of forward (5-GAGATGGTAGCTATACGCATCATGGATAAGAAT-3) and reverse (5-ATTCTTATCCATGATGCGTATAGCTACCATCTC-3) primers. The amplified PCR products were cut with ClaI plus NotI and cloned into pEBG vectors to generate the GST-MPK38(K40R) construct. pEBG-MPK38 (wobble) was generated by QuickChange II site-directed mutagenesis kit using KS-MPK38 as the template and the following mutant primers containing alterations in the nucleotide sequence (5-CAGGCAGACAAUGGAGGAUTT-3, amino acids 297303) of MPK38 siRNA, sense 5-CAGCAGACAAACCATGGAGGATTTA-3, and antisense 5-TAAATCCTCCATGGTTTGTCTGCTG-3 (14). The identities of all the PCR products were confirmed by nucleotide sequencing analysis on both strands. Anti-FLAG (M2), anti-His, and anti–actin antibodies were from Sigma; the anti-hemagglutinin (HA), anti-ASK1, anti-MKK3, anti-ATF2, and anti-poly(ADP-ribose) polymerase (PARP) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); the anti-phospho-MKK3/6(S189/207), anti-p38, anti-phospho-p38(T180/Y182), anti-phospho-ATF2(T71), and anti-phospho-ASK1 (T845) Lupeol antibodies were from Cell Signaling Technology (Danvers, MA); and the anti-caspase-3 antibody was from Calbiochem. Anti-MPK38 and anti-GST antibodies were described previously (15,16). Isopropyl -d-thiogalactopyranoside, ionomycin, thapsigargin,N-acetyl-l-cysteine (Nac), dithiothreitol (DTT), aprotinin, puromycin, and phenylmethylsulfonyl fluoride were from Sigma. TNF- was purchased from R & D Systems. Polyvinylidene difluoride membrane was from Millipore Corp. (Bedford, MA). [-32P]ATP was from PerkinElmer Life Sciences. To prepare HaCaT cells stably expressing MPK38-specific shRNA (MPK38(KD)), double-stranded oligonucleotide (forward, 5-GATCCCCCAGGCAGACAATGGAGGATTTCAAGAGAATCCTCCATTGTCTGCCTGTTTTTGGAAA-3; reverse, 5-AGCTTTTCCAAAAACAGGCAGACAATGGAGGATTCTCTTGAAATCCTCCATTGTCTGCCTGGGG-3; MPK38 sequence underlined) was cloned into the pSuper vector. MPK38(KD) cells were screened in the presence of 1.5 g/ml puromycin until all control parental HaCaT cells died. Construction of ASK1(K709R) and ASK1-N MutantsASK1(K709R) mutants were generated by PCR as described previously (17). In brief, HA-ASK1(K709R) was used as the template for amplification with either the ASK1 forward 5-GCGTCGACATGAGCACGGAGGCGGAC-3 (SalI site underlined) or reverse 5-GCGCGGCCTCAAGTCTGTTTGTTTCG-3 (NotI.