Date of Award

2004

Degree Name

Chemistry

College

College of Science

Type of Degree

M.S.

Document Type

Thesis

First Advisor

William Price

Second Advisor

Beverly Delidow

Third Advisor

Michael Norton

Abstract

Beta-catenin is an essential cell adhesion and signaling protein, associated with high prolactin levels in rat pituitary tumor cells. It has been shown that phosphorylation affects the location and activity of b-catenin. Glycogen synthase kinase (GSK3-b) is a serine-threonine kinase that phosphorylates b-catenin on N-terminal residues, targeting it for proteasomal degradation. Studies have shown that C-terminal tyrosine phosphorylation decreases the association of b-catenin with cadherin. In 235-1 rat pituitary tumor cells, our lab has shown that the glucocorticoid analog dexamethasone (Dex) decreases the half- life of b-catenin while increasing the activity of GSK3-b. The current study was undertaken to examine whether active GSK3-b is necessary for the regulation of b-catenin in Dex-treated cells. Duplicate cultures of 235-1 cells were grown for four days in the presence of 100 nM Dex. To inhibit the activity of GSK3-b, control and Dex-treated cells were treated with 20 mM LiCl, a known inhibitor of GSK3- b activity. Cell lysates were collected and analyzed by Western blotting. Phosphorylation-specific antibodies were used to detect levels of phosphorylated b- catenin (Ser 33/37/Thr 41) and (Ser 45/Thr41), active GSK3-b (Tyr 216) and inactive GSK3-b (Ser 9). Additional experiments examined tyrosine phosphorylated b-catenin through immunoprecipitation us ing a phosphotyrosine specific antibody, and by using a LAR tyrosine phosphatase which removes phosphates from ß-catenin. Using l- phosphatase, phosphates were removed nonspecifically from Ser, Thr, and Tyr residues in b-catenin to evaluate total phospho rylation. After four days of treatment with Dex, b- catenin levels were reduced by approximately 50%. Levels of active GSK3-b (Tyr 216) were increased in Dex-treated cells and levels of inhibited GSK3-ß (Ser 9) were decreased in Dex-treated cells. This shows that Dex treatment alters activation of GSK3- ß and likely functions in the regulation of b-catenin in 235-1 cells. Tyrosine C-terminal phosphorylation of ß-catenin leads to a loss of cell- to-cell adhesion, which would lead to morphological changes. Microscopic examination of Dex-treated cells showed the presence of more rounded cells than control cells. Therefore, phosphorylation studies were conducted to examine changes in tyrosine C-terminal phosphorylation. These studies demonstrated no detectable alterations in tyrosine C-terminal phosphorylation; however, the changes in cellular morphology suggest a loss of the cadherin-catenin adhesion complex in Dex-treated cells. In Dex-treated cells l-Phosphatase removed phosphates in one major step. However, in control cells an additional step was needed to remove phosphates from b-catenin, indicating that there is an alteration in phosphorylated b-catenin in Dex-treated cells. Dex-treated cells showed an increase in the amount of phosphorylated (Ser 45/Thr41 and Ser33/37/Thr41) ß-catenin. After a 24 hour treatment with LiCl, there was a decrease in the amount of Ser33/37/Thr41 phosphorylated ßcatenin. Phosphorylation on Ser 45 was still present after treatment with LiCl indicating that two separate kinases phosphorylate ß-catenin. Furthermore, ß-catenin levels were restored, even though there was no apparent change in the level of active GSK3-b. These data suggest that the presence of Dex caused an increase in the degradation rate of b- catenin by stimulating GSK3-b activity. Supported by NSF grant #IBN-9810327 to BCD.

Subject(s)

Pituitary gland -- Tumors -- Research.

Glucocorticoids.

Biochemistry.

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