c-Jun N-TERMINAL KINASE BINDING DOMAIN-DEPENDENT PHOSPHORYLATION OF MITOGEN-ACTIVATED PROTEIN KINASE KINASE 4 AND MITOGEN-ACTIVATED PROTEIN KINASE KINASE 7 AND BALANCING CROSS-TALK BETWEEN c-Jun N-TERMINAL KINASE AND EXTRACELLULAR SIGNAL-REGULATED KINASE PATHWAYS IN CORTICAL NEURONS

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor D-JNKI1 (D-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of D-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that D-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that D-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of D-JNKI1. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.