Characterization of the Expression and Regulation of the Menkes Protein in an Adrenocorticotropic Tumor Cell Line and Rat Endocrine Tissues
Date of Award
Doctor of Philosophy (Ph.D.)
To ensure copper homeostasis, organisms express copper chaperones and the Menkes copper transporter (MNK). Mutations within the MNK gene result in MNK disease, characterized by neurodegeneration, cutis laxa, and hypopigmentation. These symptoms are attributed to a deficiency in copper delivery to cuproenzymes, such as peptidylglycine alpha-amidating monooxygenase (PAM). Fibroblasts transfected with MNK have copper-dependent MNK localization in the trans-Golgi network (TGN). Since MNK localization in neuroendocrine cells has not been investigated, this study examined MNK localization in adrenocorticotropic tumor wild type (AtT-20 WT) and PAM (AtT-20 PAM-1) cell lines by immunofluorescence microscopy, subcellular fractionation, and Western blot analyses. To analyze the effect of copper levels on MNK localization, cells were treated with CuCl2, the copper chelator, BCS, and BCS/CuCl2. Immunofluorescence microscopy confirmed that MNK localization is in the TGN in WT and PAM-1 control and BCS treated cells, while in CuCl2 treated cells, MNK showed TGN and diffuse staining throughout the cytoplasm. Sucrose density gradients from control cells showed MNK localization in TGN and secretory granules. A wider distribution of MNK into lighter fractions in these gradients was observed following copper treatments, indicating MNK trafficking is copper-dependent. In addition, developmental differences in MNK expression in rat brain and endocrine tissues also were examined by Western blot analyses and RT-PCR. In adrenals, MNK protein expression increased, whereas MNK mRNA remained unchanged. In the pituitary, hippocampus and cerebellum, MNK protein and mRNA decreased, while in the atria, they remained unchanged. Therefore, MNK mRNA was differentially expressed during development. Thus, little parallel between expression of MNK protein and mRNA was observed from postnatal day 3 to adult. Since secretagogues alter PAM expression and PAM activity requires copper delivery from MNK, this study also examined the MNK expression regulation by secretagogues. While MNK secretion was not stimulated, it was regulated by secretagogues. MNK expression was increased after treatment with PMA, CRH and cAMP, with a slight decrease after BaCl2 treatment. Overall, these studies have begun to shed light on how the endogenous MNK protein functions in cells expressing a cuproenzyme dependent on copper delivery from MNK.
Collaco, Anne, "Characterization of the Expression and Regulation of the Menkes Protein in an Adrenocorticotropic Tumor Cell Line and Rat Endocrine Tissues" (2006). Biology Ph.D. Dissertations. 7.