All ETDs from UAB

Advisory Committee Chair

Jaideep V Thottassery

Advisory Committee Members

Selvarangan Ponnazhagan

Andra R Frost

William B Parker

Yancey G Gillespie

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Cks1 is a crucial cell cycle regulator which plays pleiotropic roles in cancer cell growth and is highly expressed at both the protein, and mRNA levels, in cancer cells. In contrast, normal cells exhibit barely detectable levels of this protein, even though they express substantial Cks1 mRNA. One reason for high Cks1 protein in cancers appears to be its stabilization in these cells. Cks1 is known to be ubiquitinated and degraded by the proteasome. Coordinated ubiquitination dependent proteasomal degradation of cell cycle proteins is a major mechanism that regulates their activity. Therefore aberrant accumulation of Cks1, frequently observed in cancer, could arise due to defects in its regulated proteasomal clearance. Ubiquitination dependent proteasomal turnover is regulated by different mechanisms. In particular, recognition of degradation signals in the target protein is followed by covalent attachment of ubiquitin at specific lysines by ubiquitin ligases. These degradation signals or "degrons" are commonly generated or masked by phosphorylation. Phosphoproteomic studies have shown that Cks1 gets phosphorylated on highly conserved tyrosine residues. However the biological significance of this modification is not known. Since crosstalk between tyrosine phosphorylation and ubiquitination on lysines is one regulatory mechanism governing proteasomal degradation, we conducted site directed mutagenesis of tyrosine and lysine residues of Cks1. We found that substitution of tyrosine residues 8, 12 and 19 in Cks1 with phenylalanine resulted in increased instability. On the other hand, substitution of lysine residues 4, 26, 30 and 34 with arginine resulted in decreased Cks1 instability. Another crucial regulatory mechanism that determines stability is the chaperone assisted folding of proteins. Recently it was shown that Cks1 interacts with Hsp90, a component of a multichaperone machinery that guides proteins to achieve their native folded states within cells. We find that pharmacological inhibition of Hsp90 in cancer cells destabilizes Cks1 by inducing its ubiquitination and proteasomal depletion. This was accompanied by concomitant loss of Skp2 and Cdk1 and cell cycle blockade in different cancer cell lines. These findings point toward a previously unrecognized mechanism of Hsp90 mediated regulation of Cks1 stability. Further mechanistic studies of this regulation could allow therapeutic intervention in Cks1 overexpressing cancers. Keywords - Proteasomal turnover, Cks1, Ubiquitination, Hsp90, Site-directed Mutagenesis, Phosphorylation

Share

COinS