טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentWasserman Tanya
SubjectCharacterization of WDR62: a Novel JNK-Scaffold Protein
Involved in Cellular Stress and Mitosis
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 role in signal transmission and regulation. A novel JNK-binding protein was isolated in a cDNA expression library screen, 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.WDR62 is a ubiquitously expressed heat-sensitive 175 kDa protein that was shown to specifically associate with JNK but not with ERK and p38. WDR62 potentiates JNK kinase activity; however it inhibits AP-1 transcription through recruitment of JNK to non-nuclear compartment. This recruitment is mediated via a conserved D-Box domain in WDR62. Mammalian cells transfected with WDR62 display cytoplasmic granular localization. Over-expression of stress granule (SG) resident proteins results in the recruitment of endogenous WDR62 and activated JNK to SG. In addition, cell treatment with arsenite results in recruitment of WDR62 to SG and activated JNK to processing bodies(PB). JNK inhibition results in reduced size of SG and PB,as well as de-stabilization of WDR62 protein.

WDR62 was recently identified as a centrosomal protein involved in neuro-developmental defect of microcephaly. Similar to active JNK, we have confirmed, WDR62 localization to centrosome during all mitotic stages from prophase to telophase. WDR62-depleted epidermal cells and a lymphoblast cell line derived from a microcephaly patient have shown a reduced proliferative capacity. Interestingly, a point mutation in WDR62 in patient lymphoblasts, resulted in loss of WDR62 expression, thus mimicking a knock-down phenotype in these cells. Further analysis of patient lymphoblast cells by FACS revealed their slight accumulation in G2/M stage of the cell cycle and a significant increase in tetraploid cell number. Additionally, immuno-fluorescence experiments have shown defects in mitotic spindle organization and reduced recruitment of α- and γ-tubulin to centrosomes. Taken together, this data suggests WDR62 is required for proper spindle organization and coordinated cell cycle. However, we have not found any link, so far, for JNK-WDR62 regulation to this process.

Collectively, we propose that WDR62 is a novel JNK-scaffold protein. Coordinated function of WDR62 and JNK may regulate the dynamic interplay between polysomes, SG and PB, thereby mediating mRNA fate following stress. Further research is needed in order to fully establish WDR62 role in the mitotic process of neurogenesis and investigate whether JNK-mediated regulation by WDR62 is essential to this process.