Figure 8.

A critical role for MPK38-mediated STRAP Ser¹⁸⁸ phosphorylation in STRAP-dependent cell death through ASK1/TGF-β/p53 signaling pathways. (A) HEK293 cells were treated with 1-chloro-2,4-dinitrobenzene (DNCB) (50 μM) for 30 min and further incubated with (+) or without (−) the following stimuli: H₂O₂ (5 mM, 30 min), TGF-β1 (100 ng/ml, 20 h), or 5-fluorouracil (5FU: 0.38 mM, 30 h). STRAP or Trx immunoprecipitates (IP) were subjected to immunoblot analyses with anti-MPK38, anti-phospho-STRAP(S188), and anti-phospho-Trx(T76) antibodies to assess the association between endogenous MPK38 and STRAP (or Trx) and the level of STRAP Ser¹⁸⁸ (or Trx Thr⁷⁶) phosphorylation. The kinase activity of MPK38 was determined by in vitro kinase assays with ZPR9 as a substrate (IP:α-MPK38). Results were expressed as mean ${\displaystyle \pm }$ SEM (*P < 0.05, **P < 0.01). (B) HEK293 cells expressing inducible MPK38 or scrambled shRNA were incubated with (+) or without (−) the stimuli described above in the presence or absence of doxycycline (Dox: 1 μg/ml, 72 h). To determine the endogenous association between MPK38 and STRAP (or Trx) and the level of STRAP Ser¹⁸⁸ (or Trx Thr⁷⁶) phosphorylation, STRAP or Trx immunoprecipitates were subjected to immunoblot analyses with anti-MPK38, anti-phospho-STRAP(S188), and anti-phospho-Trx(T76) antibodies. Sc, scrambled. Results were expressed as mean ${\displaystyle \pm }$ SEM (*P < 0.05). (C) STRAP-null or intact MEF cells were treated with DNCB and further incubated with (+) or without (−) the stimuli described in Fig. 8A. The levels of STRAP Ser¹⁸⁸ phosphorylation and MPK38 kinase activity were determined by immunoblot analyses and in vitro kinase assays. Apoptotic cell death under the same conditions was determined using the green fluorescent protein expression system (A–C). The relative levels of association between MPK38 and STRAP (or Trx), STRAP Ser¹⁸⁸ (or Trx Thr⁷⁶) phosphorylation, and MPK38 kinase activity were quantified by densitometric analyses. The fold increase relative to untreated parental HEK293, scrambled shRNA HEK293, or STRAP-intact MEF cells was calculated. Results were expressed as mean ${\displaystyle \pm }$ SEM (**P < 0.01, ***P < 0.001 versus STRAP-intact MEF). Sc, scrambled; P-ZPR9, phosphorylated ZPR9; P-MPK38, autophosphorylated MPK38.