M.Sc Thesis

M.Sc StudentHadad Miriam
SubjectBiochemical Characterization of the Interaction
of WDR62, C-Jun N-Terminal Kinase Scaffold
Protein, with MAP3K Proteins
DepartmentDepartment of Medicine
Supervisor PROF. Ami Aronheim
Full Thesis textFull thesis text - English Version



Mitogen-activated protein kinases (MAPKs) form a kinase tier module in which MAPK, MAP2K and MAP3K are held by scaffold proteins. The scaffold proteins serve as a protein platform for selective and spatial kinase activation. The precise mechanism by which the scaffold proteins function has not yet been fully explained. WDR62 is a novel scaffold protein of the c-Jun N-terminal kinase (JNK) pathway. WDR62 is a 1523 a.a. long protein with no significant sequence homology to a known gene. Recessive mutations in the WDR62 gene cause of a wide spectrum of severe cerebral cortical malformations including microcephaly and pachygyria. Previously, WDR62 was shown to associate with JNK and MKK7 in a modular fashion. The first aim of the thesis was to study the interaction between WDR62 with MAP3K proteins. We focused on MAP3K from the mixed lineage kinase (MLK) family and more specifically on MLK3. We used cell lysates derived from HEK-293T cells transfected with plasmids encoding for proteins or protein fragments fused to various tags such as HA, Myc and GST. Immuno-precipitation and GST-pull down experiments were performed to identify protein-protein interaction. We conclude that WDR62 is able to associate with multiple members of the MAP3K of the mixed lineage kinase family. We focused on MLK3 as a paradigm and mapped WDR62-MLK3 interacting domains. Two separable domains were identified on both WDR62 and MLK3 proteins that can cross associate. MLK3 association with WDR62 is independent of JNK and MKK4/7 domains and activities. CDC42 is a small GTPase of the Rho-subfamily, which regulates signaling pathways and activated MLK3. We have found that CDC42 activation disrupts WDR62-MLK3 association independent of MLK3 kinase activity. WDR62-association preserves MLK3 in an un-phosphorylated inactive form. The second aim of the thesis, was to map JNK phosphorylation sites on WDR62. We used bacterially expressed purified recombinant protein fragments of WDR62 as substrate in an in vitro kinase assay. We mapped the phosphorylation site of WDR62 by JNK to serine 1330. Rabbit polyclonal Phospho-specific antibodies that recognize phospho-serine 1330, were generated. Activation of the JNK tier by MLK3 overexpression potentiates WDR62 S1330 phosphorylation in a JNK dependent manner. Further experiments are needed to determine the role of Serine 1330 phosphorylation site and WDR62 function. Collectively, WDR62 is a novel JNK-scaffold phospho-protein interacting independently with all three components of the JNK tier. The biological role of WDR62 in health and disease is yet to be identified.