Date of Award

5-2005

Degree Type

Thesis

Degree Name

MS Biology

Department

Biology

Advisor

Allan Blake

Committee Member

Jane Ko

Committee Member

Linda Hsu

Committee Member

Sulie Chang

Keywords

Biology, Phosphotyrosine phosphatases, AtT-20 murine corticotroph cell line

Abstract

Somatostatin (somatotropin release inhibitory factor; SRIF) is a peptide-signaling molecule that activates a family of heterotrimeric guanine nucleotide binding protein (G­ protein) coupled receptors (sst.-sst-). SRIF receptors control essential intracellular signaling events and, by reducing cyclic nucleotide levels, ion concentrations and protein phosphorylation, ultimately controlling cell proliferation and secretion. In the current study, we investigated the intracellular phosphatase activity present in the AtT-20 cell, as well as whether these enzymes were under direct SRIF receptor control. AtT-20 cells retain many of the properties of anterior pituitary corticotrophs, yet are an established cell line that expresses at least two SRIF receptor subtypes (sst, and sst.). Both SRIF receptor subtypes potently suppress hormonally induced adrenocorticotropic hormone (ACTH) secretion from AtT-20 cells. Since intracellular protein kinase activation plays a crucial role in ACTH release from AtT-20 cells, identifying the corresponding protein phosphatases will provide valuable information on corticotroph function. The novel fluorescent phosphatase substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiF­ MUP) was employed to identify soluble or membrane-associated phosphatase activities. Treatment with a selective tyrosine phosphatase inhibitor (sodium vanadate; Na3 V04) attenuated DiF-MUP phosphatase activity by 75%, suggesting that the dominant activity detected in AtT-20 cells is a tyrosine phosphatase. Na3V04 inhibition was potent as concentration response studies demonstrated an inhibitory concentration for 50% activity nM. (ICso) of 90 A serine/threonine phosphatase inhibitor failed todecrease phosphatase activity, indicating that the dominant phosphatase activity present is due to protein tyrosine phosphatases. Immunoprecipitation studies, in conjunction with sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-P AGE) and immunoblotting identified two intracellular phosphotyrosine phosphatases: Src homology phosphatase- I (SHP-1) and Src homology phosphotyrosine phosphatase-2 (SHP-2). Immunoblotting with phospho­ specific antisera confirmed the presence of SHP-1 and SHP-2, while also demonstrating that neither enzyme appears to be under SRIF control in AtT-20 cells. Together, these results show that AtT-20 murine corticotrophs contain sodium vanadate sensitive, soluble tyrosine phosphatase activity that is independent of SRIF control.

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