Murine Protein Serine/Threonine Kinase 38 Activates Apoptosis Signal-regulating Kinase 1 via Thr838 Phosphorylation

Abstract

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/ threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H 2 O 2 or tumor necrosis factor ␣ treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr 838 phosphorylation and enhanced ASK1-mediated signaling to 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 H 2 O 2 -mediated apoptosis by enhancing the ASK1 activity through Thr 838 phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.Apoptosis signal-regulating kinase 1 (ASK1) 2 is 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 NH 2 -terminal kinase (JNK)/p38 signaling cascade by phosphorylating and activating mitogen-activated protein kinase kinases (MAPKK) such as MKK3, -4, -6, and -7 (1-3). Emerging evidence indicates that ASK1 activity is regulated by its interaction with several cellular partners (3-8), 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 NH 2 -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). 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 (11-13). However, the physiological regulation and functions of MPK38 have r...

Serine-Threonine Kinase Receptor-associated Protein Inhibits Apoptosis Signal-regulating Kinase 1 Function through Direct Interaction

“…To prepare HCT116 cells stably expressing a STRAP-specific short hairpin RNA (shRNA) (STRAP(KD)), double-stranded oligonucleotides (forward, 5Ј-GATCCCCTTACGCATATAT-GACTTGATTCAAGAGATCAAGTCATATATGCGTAAT-TTTTGGAAA-3Ј; reverse, 5Ј-AGCTTTTCCAAAAATTAC-GCATATATGACTTGATCTCTTGAATCAAGTCATATA-TGCGTAAGGG-3Ј; STRAP sequence underlined) were cloned into the pSuper vector. STRAP(KD) cells were screened in the presence of 1.5 g/ml puromycin until all control parental HCT116 cells died, as described previously (11). The presence of endogenous and exogenous STRAP proteins was confirmed by immunoblot analysis.…”

“…Anti-phospho-PDK1(Ser 241 ) antibody was from Cell Signaling Technology (Danvers, MA). Wild-type and kinase-dead ASK1, ASK1-N, ASK1-K, ASK1-C, wild-type STRAP and its mutants (WD1-3, WD4 -6, WD4, C152S, WD5, WD6, C270S, C152S/ C270S, and CT), activator protein 1 (AP-1)-Luc reporter, pSuper vector, GST-tagged MKK6(K82A) and p38, and c-fos were also previously described (11,25).…”

“…Antibodies and Plasmids-Anti-FLAG (M2), anti-His, antihemagglutinin (HA), anti-ASK1, anti-MPK38, anti-GST, anti-MKK3, anti-ATF2, anti-poly(ADP-ribose) polymerase (PARP), anti-caspase-3, anti-phospho-MKK3/6(Ser 189 /Ser 207 ), antip38, anti-phospho-p38(Thr 180 /Tyr 182 ), anti-phospho-ATF2 (Thr anti-phospho-ASK1(Ser 967 ), and anti-␤-actin antibodies were described previously (11). Anti-PDK1, anti-phospho-AKT- (Thr 308 ), anti-AKT, anti-phospho-Bad (Ser 136 ), anti-Bad, and antiphospho-Ser/Thr antibodies were described previously (21,24).…”

“…The double-stranded oligonucleotides (forward, 5Ј-TCGAGGTT-ACGCATATATGACTTGATTCAAGAGATCAAGTCATA-TATGCGTAACTTTTTTA-3Ј; reverse, 5Ј-AGCTTAAAAA-AGTTACGCATATATGACTTGATCTCTTGAATCAAGT-CATATATGCGTAACC-3Ј; STRAP sequence underlined) were cloned into the XhoI/HindIII sites of pSingle-tTS-shRNA vector (Clontech). HEK293 cells were transfected with pSingletTS-shRNA harboring STRAP-specific shRNA, pSingle-tTSshRNA harboring scrambled shRNA as a nonspecific control (24), or pSingle-tTS-shRNA empty vector using WelFect-Ex TM Plus (11). Inducible STRAP shRNA stable clones were screened in the presence of 450 g/ml G418 for 14 days until all control parental HEK293 cells had died.…”

“…Cell Culture and Stable Cell Line Constructions-HEK293, NIH 3T3, HeLa, HCT116, and 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) as described previously (11). HCT116 cells stably expressing pcDNA3-His-STRAP plasmid (STRAP(OE)) were screened in the presence of 850 g/ml G418 until control parental HCT116 cells completely died.…”