The regulation by Syk of the calpain-calpastatin proteolyrtic system in human breast cancer cells
Abstract
Syk is a 72 kDa non-receptor tyrosine kinase that is best characterized in hematopoietic cells. Recently, Syk has been reported as a negative regulator of cell growth in human breast epithelial cells. I found that both pervanadate (PV) treatment and the stable expression of Syk in MCF7 cells protected the RelA subunit of NF-kappaB from calpain-mediated proteolysis. While PV treatment significantly inhibited calpain activity in both cell lysates and living cells, Syk only moderately decreased it in cell lysates. In contrast, calpain activity was dramatically enhanced by Syk in living cells, which could be the result of the increased calcium influx induced by Syk. To explore the mechanisms by which Syk decreased calpain activity in cell lysates, I investigated the effect of Syk on calpastatin (CAST), the endogenous inhibitor of calpain. The protein level of CAST was elevated in Syk-positive breast cancer cells, particularly in the cytostol. CAST was also found to become phosphorylated on tyrosine in a Syk-dependent manner in MDA-MB-231 cells. Conversely, calpain negatively regulated several Syk-mediated signaling pathways. I found that inhibition of calpain enhanced both TNF-alpha; induced NF-kappaB activation and integrin stimulated tyrosine phosphorylation in a Syk-dependent manner. Overall, the negative regulation by Syk of the calpain-CAST system provides a novel insight into the tumor suppressing function of Syk in human breast epithelial cells. Another part of my project was focused on the development of a tool for the study of protein ubiquitination. To reduce the background noise produced by trypsin digestion of multiple ubiquitination proteins, I constructed a series of FLAG-tagged ubiquitin mutants that could be selectively cleaved by factor Xa. The cleaved ubiquitin chains can be removed, leaving the substrates with a 22 amino acid ubiquitin fragment. This system is designed to aid in identifying proteins that become ubiquitinated in response to various stimuli, and their sites of ubiquitination. Mutations replacing one or more lysine(s) on ubiquitin with arginine(s) can be also introduced to help characterize specific types of ubiquitination.
Degree
Ph.D.
Advisors
Geahlen, Purdue University.
Subject Area
Molecular biology|Oncology
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