We are interested how docking motifs contribute to substrate recognition by JNK, a Ser/Thr protein kinase and member of the Mitogen-Activated Protein Kinase (MAPK) family. The activation of JNK signaling pathways and concomitant phosphorylation of a range of JNK substrates has been implicated with regulation of critical events, including cell growth and proliferation, cell division and cell death. Specifically, we are defining the structural and functional properties of the microtubule-associated protein doublecortin (DCX) as a JNK substrate. DCX is unusual with its evolutionary conserved DC domains, rather than a typical linear basic/hydrophobic motif, reported to mediate its interaction with JNK. Mutations on DCX clustering on the DC domains cause abnormal neuronal migration during brain development leading to X-linked lissencephaly in males and subcortical band heterotopia (SBH) in females. In particular, we focus on Arginine mutations on the surface of N-terminal DC domain (NDC) which have been shown to disrupt the DCX-tubulin interface. Through extensive biochemical and biophysical analyses, we investigated the effects of these pathogenic mutations on phosphorylation and interactions with JNK. Mass spectrometry analysis had identified a novel JNK-mediated phosphorylation site on the DCX protein, which was confirmed by site-directed mutagenesis and detection by phosphospecific antibodies. This studies were further complemented with the use of NMR approaches to gain new structural insights into DCX with the ultimate aim to identify the features of the JNK-DC domain interaction interface that support this non-conventional mode of JNK-substrate recognition.