Characterization of JNK Binding Proteins: A Dissertation

Abstract

The JNK signal transduction pathway mediates a broad, complex biological process in response to inflammatory cytokines and environmental stress. These responses include cell survival and apoptosis, proliferation, tumorigenesis and the immune response. The divergent cellular responses caused by the JNK signal transduction pathway are often regulated by spatial and cell type contexts, as well as the interaction with other cellular processes. The discovery of additional components of the JNK signal transduction pathway are critical to elucidate the stress response mechanisms in cells. This thesis first discusses the cloning and characterization of two novel members of the JNK signal transduction pathway. JIP1 and JMP1 were initially identified from a murine embryo library through a yeast Two-Hybrid screen to identify novel JNK interacting proteins. Full length cDNAs of both genes were cloned and analyzed. JIP1 represents the first member of the JIP group of JNK scaffold proteins which were characterized. The JNK binding domain (JBD) of JIP1 matches the D-domain consensus of other JNK binding proteins, and it demonstrates JNK binding both in vitro and in vivo. This JNK binding was demonstrated to inhibit JNK signal transduction and over-expression of JIP1 inhibits the JNK mediated pre-B cell transformation by bcr-abl. Over-expressed JIP1 also sequesters JNK in the cytoplasm, which may be a mechanism of the inhibition of JNK signaling. A new, high-resolution digital imaging microscopy technique using deconvolution demonstrated the absence of JNK1 in the nucleus of co-transfected JIP1 and JNK1 cells. The other protein discussed in this thesis is JMP1, a novel JNK binding, microtubule co-localized protein. There is a JBD in the JMP1 carboxyl end and a consensus D-domain within this region. The JMP1 JBD demonstrates an increased association with phospho-JNK from UV irradiated cells compared to un-irradiated cells in vivo. JMP1 also has 12 WD-repeat motifs in its amino terminal end which are required for microtubule co-localization. JMP1 demonstrates a cell cycle specific localization at the mitotic spindle poles. This co-localization is dependent on intact microtubules and the amino-terminal WD-repeats are required for this localization. JMP1 mRNA is highly expressed in testis tissues. Immunocytochemistry on murine testis sections using an affinity purified anti-JMP1 antibody demonstrates JMP1 protein in the lumenal compartment of the seminiferous tubules. JMP1 protein is expressed in primary and secondary spermatocytes, cells which are actively undergoing meiosis. The results obtained from the localization of JMP1 in meiotic spermatocytes led to an investigation of the roles of JNK signal transduction in the testis. The testis is an active region of cellular proliferation, apoptosis and differentiation, which make it an appealing model for studying JNK signal transduction. However, the roles JNK signaling have in the testis are poorly understood. I investigated the reproduction capability of Jnk3-/- male mice and discovered older Jnk3-/- males had a reduced capacity to impregnate females compared to younger animals and age-matched wild type controls. The testis morphology and sperm motility of these animals were similar to wild-type animals, and there was no alteration of apoptosis in the testis. The final section of this thesis involves the study of this breeding defect and investigating for cellular defects that might account for this age-related Jnk3-/- phenotype.