Syk and the regulation of the microtubule cytoskeleton in breast cancer cells
Abstract
Breast cancer is the second highest killer of women and the second most diagnosed. The cytoskeleton plays an important role in maintaining critical cell-cell junctions needed for epithelial tissue integrity and for the prevention of tumor metastasis. The spleen tyrosine kinase, Syk, is a well known signaling protein in the hematopoietic system but has also been identified as a tumor and metastasis suppressor in breast cancer. The expression of Syk is lost in invasive breast cancer cell lines due to hypermethylation of the promoter. Syk suppresses cell motility, prevents metastasis formation and negatively regulates the cell cycle when transfected into Syk deficient breast cancer cell lines. Studies in our lab have shown that Syk stabilizes microtubules against depolymerization caused by nocodazole and cold. I found that Syk also promotes the formation of or strengthens existing adherens junctions as Syk expression increases the amount of cell aggregates and causes vinculin to localize to cell-cell contacts. The relocalization of vinculin from focal adhesions to adherens junctions is reversed with depolymerization of microtubules showing that Syk has an effect on the microtubule cytoskeleton. I also found that Syk increases the expression of γ-tubulin which is necessary for microtubule nucleation. Syk and γ-tubulin interact in an immunoprecipitation as well. The Rho family of GTPases regulates the cytoskeleton. The amount of active Rac1 was less in Syk expressing cells than in cells not expressing Syk. It is possible that Syk also is involved in the RhoA pathway. Vav3 is a guanine nucleotide exchange factor for RhoA and Rac1. Vav3 and Syk interact in an immunoprecipitation, and the phosphorylation of Y173, which activates Vav3, is increased with activated Syk. Interestingly, Syk overexpression results in a multinucleated phenotype as does the overexpression of both Syk and Vav3. This could also be a result of the regulation of the microtubule cytoskeleton as microtubules are the main component of the spindle apparatus which controls choromsome segregation. My results suggest that Syk has a role in the regulation of the microtubule cytoskeleton through its relationship with Vav3, Rho/Rac and γ-tubulin.
Degree
M.S.
Advisors
Geahlen, Purdue University.
Subject Area
Molecular biology
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