Title

Elucidating The Role of Phosphorylation on Trafficking of Menkes Protein in AtT-20 Wild Type Cells

Date of Award

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Tami Steveson, PhD

Second Advisor

Raymond Larsen, PhD (Committee Member)

Third Advisor

Lee Meserve, PhD (Committee Member)

Fourth Advisor

Ana Maria Oyarce, PhD (Committee Member)

Fifth Advisor

Roudabeh Jamasbi, PhD (Committee Member)

Abstract

Copper, an essential trace element, is a cofactor for many reactions catalyzed by cuproenzymes. However, a deficiency or excess of copper can be detrimental to cells, thus copper homeostasis is carefully maintained. The Menkes protein (MNK), a copper transporter, plays a dual role, supplying copper to essential cuproenzymes and effluxing excess copper extracellularly. This efflux occurs by MNK trafficking between the trans-Golgi network and plasma membrane. Thus, the overall goal of this study was to begin to understand the localization and trafficking of MNK in anterior pituitary and anterior-pituitary derived (AtT-20) cells. Immunofluorescence microscopy studies using MNK and organelle-specific antibodies were performed to determine MNK localization and trafficking. In addition, MNK trafficking was examined using the secretagogues, phorbol myristate acetate (PMA) and corticotrophin releasing hormone (CRH), which act as external stimulatory signals. These studies showed MNK localization to the TGN and distinct punctate vesicles, indicative of secretory granules, which move towards the plasma membrane following cellular stimulation. After incubating the cells in high copper levels for two hours, MNK traffics to the plasma membrane. To determine if MNK expression is regulated by external signals, the AtT-20 cells were incubated with PMA or CRH and MNK expression determined by Western blot using MNK and beta-tubulin antibodies. Following incubation, an increase in MNK expression was observed. To determine the role of phosphorylation and begin to identify kinases involved in MNK trafficking in AtT-20 cells, immunofluorescence microscopy using protein kinase inhibitors was performed. Following incubation with PKA and PKC inhibitors, MNK was localized to the TGN and did not traffic to the plasma membrane as expected when incubated in high copper levels. Overall, these results demonstrate that MNK is localized to the TGN and secretory granules in anterior pituitary cells and that the kinases, PKA and PKC, play a role in trafficking MNK towards the plasma membrane in high copper levels. Furthermore, MNK expression is regulated by an external signal, suggesting that MNK expression is under the control of hypothalamic-releasing hormones that regulate the pituitary. This exciting find will lead to a greater understanding of how MNK normally functions in the body.