Selective Inhibition of Mitochondrial JNK Signaling Achieved Using Peptide Mimicry of the Sab Kinase Interacting Motif-1 (KIM1)

Abstract

The c-jun N-terminal kinases (JNKs) are responsive to stress stimuli leading to activation of proapoptotic proteins and transcription. Additionally, JNK mitochondrial localization has been reported. To selectively target mitochondrial JNK signaling, we exploited JNKs interaction with its mitochondrial scaffold, Sab, using small interfering RNAs (siRNAs) and a cell permeable peptide corresponding to the KIM1 domain of Sab. Gene silencing and peptide interference of this interaction disrupted JNK translocation to the mitochondria and reduced phosphorylation of Bcl-2 without significant impact on c-Jun phosphorylation or AP-1 transcription. In contrast, the JNK inhibitory peptide (TI-JIP1) prevented these three functions. Tat-SabKIM1 selectivity was also demonstrated in anisomycin-stressed HeLa cells where Tat-SabKIM1 prevented Bcl-2 phosphorylation, cell death, loss of mitochondrial membrane potential, and superoxide generation, but not c-Jun phosphorylation. Conversely, TI-JIP1 prevented all aforementioned stress-induced events. This probe introduces a means to evaluate JNK-mediated events on the mitochondria without intervening in nuclear functions of JNK.

Blocking c-Jun N-terminal Kinase (JNK) Translocation to the Mitochondria Prevents 6-Hydroxydopamine-induced Toxicity in Vitro and in Vivo

“…Mitochondrial Isolation-Purification of mitochondria was conducted according to the protocol described in our previous research (6,21). Briefly, 2.5 ϫ 10 8 SHSY5Y cells were grown on 150-mm 2 tissue culture plates as described above.…”

“…Gene Silencing-Knockdown of JNK and Sab was conducted as described in our previous publications (6,21). Briefly, SHSY5Y cells were grown to 60% confluency in the apparatus required for specific assays.…”

“…Cell Viability-Cell viability was monitored by TUNEL assay (Millipore) and Cell Titer-Glo (Promega) assay as described in our previous works (6,21). For these 96-well plate assays, 4 ϫ 10 4 cells were seeded in a 96-well plate and then treated as described in the experiments.…”

“…The JNK-interacting protein-1 is most well studied, and a cell-permeable version of this peptide has been shown to be efficacious in numerous efficacy models ranging from PD (13) to cerebral ischemia (19) and diabetes (20). Recently, we designed a retro-inverso peptide containing the HIV-Tat sequence along with 20 residues from the Sab KIM1 domain (Tat-Sab KIM1 ), which could also be used for in vivo purposes (21). Utilizing both JNK and Sab siRNA silencing, along with peptide mimicry with Tat-Sab KIM1 , we demonstrated that prevention of JNK translocation to the mitochondria was protective against reactive oxygen species (ROS) generation, dissipation of the mitochondrial membrane potential, and cytotoxicity induced by anisomycin in HeLa cells, and this occurred in a nucleus-independent fashion.…”

“…Utilizing both JNK and Sab siRNA silencing, along with peptide mimicry with Tat-Sab KIM1 , we demonstrated that prevention of JNK translocation to the mitochondria was protective against reactive oxygen species (ROS) generation, dissipation of the mitochondrial membrane potential, and cytotoxicity induced by anisomycin in HeLa cells, and this occurred in a nucleus-independent fashion. Indeed, this peptide has been shown to be highly selective for the JNK pathway over the p38 * This work was supported, in whole or in part, by National Institutes of Health pathway (6,21). These results, coupled with our demonstration that highly selective JNK inhibitors, such as SR-3306 that do not inhibit p38, PI3K, Akt, and over 300 other kinases (10), are efficacious in protecting dopaminergic neurons and providing behavioral benefit in 6-OHDA-lesioned rats (11), led us to hypothesize that Tat-Sab KIM1 could provide the same benefits in neuronal cells and in rats.…”