Date of Graduation

2008

Document Type

Dissertation/Thesis

Abstract

The actin filament associated protein of 110 kDA (AFAP-110) is a well-characterized protein composed of amino-terminal binding motifs that function upstream of a carboxy-terminal actin binding domain. In vitro studies demonstrate that the amino-terminal pleckstrin homology 1 (PH1) domain interacts with activated PKCα, relaying signals that activate Src family kinases via SH3 and SH2 binding. Serving as an adaptor protein, AFAP-110 relays signals that result in the assembly of motility structures. These findings could be of physiological as well pathological significance as AFAP-110 colocalizes with SFKs in the developing brain as well as in the glial-derived tumor, glioblastoma multiforme. More recently, AFAP-110 and c-Src expression were analyzed in ovarian cancer. While c-Src exhibits a diffuse expression pattern, AFAP-110 localized focally. Interestingly, colocalization between AFAP-110 and c-Src always occurs in desmoplastic regions of the tumor. In order to determine if a genetic variant of AFAP-110 exists, a PCR analysis of AFAP cDNA derived from ovarian cancer cell lines, ovarian tumor sections, as well as normal myometrium revealed a nucleotide change of G1426C resulting in an amino-acid change of serine (S) to cysteine (C) at position 403 of AFAP-110 (AFAP-110403S/C). This SNP was found to exist in approximately 25% of the samples. Molecular modeling predicts that the tertiary structure of AFAP-110403S/C could be contorted in a manner that facilitates altered protein binding. This is supported by GST-absorption studies that suggest that AFAP-110403S/C may differ from AFAP WT in its ability to self-associate. Altered self-association could present AFAP in a conformation that facilitates c-Src activation. In a model system that mimics the level of c-Src over-expression in ovarian cancer, transient transfection of AFAP-110403S/C results in the activation of c-Src and the formation of podosomes. This could be of clinical significance as the generation of invasive motility structures could facilitate tumor progression. In addition, the efficient activation of c-Src by AFAP-110403S/C could contribute to rapid development of paclitaxel resistance. Therefore, the genetic variant of AFAP-110 could be used in a gene assay to predict those most likely to exhibit rapid tumor progression as well as those most likely to develop chemotherapy-resistance.

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