JLP-JNK signaling protects cancer cells from reactive oxygen species-induced cell death

Functional role of c-Jun NH₂-terminal kinase-associated leucine zipper protein (JLP) in lysosome localization and autophagy

“…U87, HeLa, HT1080, and HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Wako, Tokyo, Japan) as described previously (13,14). Amino acid-free DMEM was obtained from Wako.…”

“…JLP (also known as SPAG9 or JIP4) was first identified as a scaffold protein in the mammalian mitogen-activated protein kinase (MAPK) signaling pathway (5,6), and subsequently was found to also function as an adaptor protein that links cargoes to kinesin and/or dynein motors (7)(8)(9). Recent studies have shown that JLP is a multifunctional protein involved in axonal transport, cytokinesis, and oxidative stressinduced cell death (10)(11)(12)(13)(14). In this study, we explored the role of JLP in lysosome localization and autophagy, and propose a novel, TMEM55B-independent mechanism of intracellular lysosomal trafficking.…”

c-Jun NH₂-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) attenuates curcumin-induced cell death differently from its family member, JNK-associated leucine zipper protein (JLP)

“…Human colon carcinoma HCT116 and DLD-1 cells were cultured in Dulbecco's Modified Eagle Medium (Wako, Tokyo, Japan) and Roswell Park Memorial Institute 1640 (Wako), respectively, as described previously (20). Curcumin, puromycin, 4',6-diamidino-2-phenylindole (DAPI), and chloroquine were obtained from Sigma-Aldrich (St. Louis, MO, USA).…”

“…The pLVTH expression vectors for scramble shRNA (pLVTH-shScr) and JLP shRNA (pLVTH-shJLP), and the pCL20c expression vectors for tandem monomeric red fluorescent protein (mRFP) and green fluorescent protein (GFP)-tagged microtubule-associated protein 1 light chain 3 (LC3) (pCL20c-CMV-mRFP-GFP-LC3) and hemagglutinintagged wild-type human JLP (pCL20c-CMVΔ4-HA-JLP) were described previously (4). A human JSAP1 cDNA (RefSeq accession number NM_001040439) encoding the full-length JSAP1 protein was prepared as for the human JLP cDNA (20), and inserted into pCL20c-HA (21) to generate pCL20c-CMV-HA-JSAP1. To express the HA-JSAP1 at suboptimal levels in cells, the CMV promoter/enhancer region was deleted and the resulting plasmid, named pCL20c-ΔCMV-HA-JSAP1, was used for the analysis in Figure 2C.…”

“…Total cell lysates were prepared and analyzed by immunoblotting as previously described ( 22), using anti-ATG5 (12994), anti-Phospho-JNK (9251), anti-Phospho-p38 (4631) (each diluted 1:1,000, from Cell Signaling, Boston, MA, USA), anti-HA (1:1,000; 11867423001), anti-α-tubulin (1:3,000; T5168) (two from Sigma-Aldrich) antibodies. Anti-JLP and anti-JSAP1 antibodies were affinity purified as described previously (20), and used for immunoblotting at 0.25 μg/mL and 0.33 μg/mL, respectively.…”