טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentCohen Katsenelson Ksenya
SubjectBiochemical Characterization of WDR62 - a Novel JNK-binding
Protein
DepartmentDepartment of Medicine
Supervisor Professor Ami Aronheim
Full Thesis textFull thesis text - English Version


Abstract

The c-Jun N-terminal kinase (JNK) is part of a mitogen-activated protein kinase (MAPK) signaling cascade. Scaffold proteins simultaneously associate with various components of the MAPK signaling pathway and play a crucial role in signal transmission and regulation. The precise mechanism by which scaffold proteins function is still lacking. A novel JNK-binding protein was isolated in our lab using the yeast Ras recruitment system. This JNK binding protein has no sequence homology to any known protein and corresponds to a genebank predicted open reading frame of WDR62. We hypothesize that WDR62 is a JNK scaffold protein that plays a role in JNK signaling and activity. We aimed to biochemically characterize the novel protein, identify its protein partners, map its protein association domain, study its function and mode of regulation. We demonstrate that WDR62 is a ubiquitously expressed 175-kDa protein that specifically associates with JNK but not with ERK and p38. WDR62 associates with endogenous and overexpressed proteins of both JNK2 and the JNK2-activating kinase MKK7. Furthermore, these associations occur via direct protein-protein interactions. We mapped the docking domain of WDR62 responsible for the association with JNK. WDR62 interacts with all JNK isoforms through a conserved D domain motif located at the C-terminus. WDR62 association with JNK2 requires both the JNK CD and ED domains, and the binding requisite is distinct from that of the previously described JNK2 association with JNK interacting protein 1. Next, we characterized the association between WDR62 and MKK7. WDR62 associates directly with the MKK7b1 isoform independently of JNK binding, but fails to interact with MKK7a1. Recently it was found that recessive mutations within WDR62 result in severe brain malformations. One of the WDR62 mutant proteins found in a patient with microcephaly encodes a C-terminal truncated protein that preserves both JNK and MKK7 docking domains yet fails to associate with them. We show that this WDR62 C-terminal region that is lacking in this mutant protein consists of a novel dimerization domain. A minimal domain of 122 amino acids is necessary and sufficient for WDR62 dimerization. It is also necessary for JNK and MKK7 association. Importantly, fusion of the WDR62 dimerization mutant to a functional heterologous dimerization motif was able to reconstitute WDR62-JNK association but not the association of MKK7, demonstrating that WDR62 dimerization is critical for its scaffolding function. Bioinformatic analysis suggests the existence of three a-helices that possibly form a dimerization platform.