A FRET-based approach suggests allosteric activation of Mixed Lineage Kinases by mutant huntingtin

CARINA WEISSMANN; GERARDO MORFINI; KATHY GALLO; OSVALDO DANIEL UCHITEL; LUDOVIC D AURIA

Buenos Aires

Congreso; Congreso FALAN 2016; 2016

FALAN

Aberrant activation of c-Jun amino-terminal kinases (JNKs) represents a well-established pathogenic event in Huntington?s disease (HD), a devastating neurodegenerative disorder that results from mutations in huntingtin (Htt) protein. However, mechanisms underlying JNK activation by mutant Htt (mHtt) remain unknown.Mixed Lineage Kinases (MLKs 1, 2 and 3) are MAP3 kinases that promote stimulus-specific activation of JNKs. MLKs feature an Src homology-3 (SH3) at their amino terminus, which interacts intramolecularly with an internal PXXP sequence, rendering these kinases inactive. Disruption of this interaction has been proposed to represent a mechanism for allosteric activation of MLKs. Interestingly, mHtt features multiple PXXP motifs and interact with MLKs, but whether this interaction modulates MLK activity remains unknown. A FRET-based approach for the evaluation of MLK activation in cells using a sequential acceptor photobleaching technique showed changes in protein conformations. Functional MLK3 constructs tagged with CFP and YFP were expressed in HEK cells, and FRET experiments performed to evaluate intramolecular interactions between the SH3 and PXXP domains. Additionally, western blots were used to detect the phosphorylation of endogenous MLKs and substrates by these constructs.Significantly, preliminary results in different cell lines suggest that mHtt expression would lead to increased phosphorylation and activation of MKKs, endogenous MLK substrates responsible for JNK activation. Collectively, results from these experiments hint to a novel molecular mechanism of abnormal activation of the JNK pathway in HD.