Protective role of c-Jun NH2-terminal kinase-associated leucine zipper protein (JLP) in curcumin-induced cancer 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.…”

“…The pLVTH lentivirus plasmid vectors for two short hairpin RNAs (shRNAs) against human JLP, shJLP#1 and shJLP#2, as well as for scramble shRNA (shScr), were described previously (14). The JLP deletion mutants, including those that lacking the dynactin p150 Glued -binding domain (DBD), C-terminal and N-terminal half regions of JLP [amino acid residues 371-490, 657-1,321, and 1-646 of human JLP, respectively (RefSeq accession number NP_00112400)] were generated using overlapping PCR as described by Ito et al (15).…”

“…The JLP deletion mutants were expressed as hemagglutinin (HA)-tagged proteins using the pCL20c-CMV lentivirus plasmid vector (16). The pCL20c-CMV expression vectors for tandem fluorescent-tagged LC3, HA-tagged wild-type JLP, and JLP mutants lacking MAPK-binding domain (MBD) or kinesin-1 heavy chain (KHC)-binding domain (KBD) were previously described (10,14). To express the HA-JLP proteins at suboptimal levels in cells, a 405-bp deletion was made in the CMV promoter/enhancer region based on Morita et al (17), referred to as CMVΔ2.…”

“…It is known that JLP contains two domains, KBD and MBD, in its N-terminal region (10,14). We, therefore, examined whether KBD and/or MBD are involved in lysosome localization using HA-tagged shJLP#2-resistant JLP mutants lacking KBD (HA-JLP_ΔKBD) or MBD (HA-JLP_ΔMBD).…”

“…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.…”