Biology Ph.D. Dissertations

Regulation of the Menkes Protein in neuroendocrine Cells in Response to Copper

Date of Award

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Tami Steveson

Second Advisor

Raymond Larsen (Committee Member)

Third Advisor

George Bullerjahn (Committee Member)

Fourth Advisor

Ana Maria Oyarce (Committee Member)

Fifth Advisor

Roudabeh Jamasbi (Committee Member)

Abstract

Copper is an essential trace element required as an integral component of several important enzymes including cytochrome c oxidase, superoxide dismutase, lysyl oxidase, tyrosinase, ascorbic acid oxidase, dopamine β-monooxygenase, and peptidylglycine α-amidating monooxygenase (PAM). The Menkes protein (MNK) is a copper-translocating P-type ATPase that plays a crucial role in copper homeostasis as well as the efflux of copper out of cells. MNK is localized to the trans-Golgi network in fibroblasts and contributes to the delivery of copper to the cuproenzymes listed above. Mutations in MNK are known to cause Menkes disease, a lethal X-linked recessive disorder characterized by neurodegeneration, connective tissue and bone abnormalities as well as early childhood death. Previous studies have reported that the expression and trafficking of MNK in response to changes in copper levels is primarily regulated by post-translational modifications including phosphorylation, and does not involve de novo protein synthesis in fibroblasts. We are studying the expression of the MNK protein in adrenocorticotropic derived tumor cells (AtT-20), which differ from fibroblasts in that they express MNK endogenously and have a regulated secretory pathway in addition to a constitutive pathway. Using Western blot analyses, we determined that MNK protein expression is altered in AtT-20 cells when cultured in varying levels of copper and undergoes de novo protein synthesis. We also determined that this alteration of MNK expression is specific to copper and not to other transition metal ions. Furthermore, we observed that PAM, a cuproenzyme, remains unchanged in its expression in AtT-20 cells in varying copper levels. Using the techniques of subcellular fractionation and Western blotting, we observed that MNK localizes to the TGN and secretory vesicles under steady-state conditions and in excess copper conditions appears more enriched in vesicles that may be secretory in nature. Moreover, using reverse transcriptase PCR, we did not observe change in the MNK transcript in AtT-20 cells in response to copper levels. Taken together, these studies suggest that MNK undergoes translational regulation in response to changes in copper levels in AtT-20 cells and that it may play a unique role in neuroendocrine cells.

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