The role of the 90 kDa palladin in the regulation of actin filaments
Albraiki, Sharifah E.
AdvisorBeck, Moriah R.
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Metastasis is the most clinically significant step in cancer progression. Migration and metastasis are not fully understood, but it is clear that the actin cytoskeleton plays an essential role. Palladin is specifically involved in metastasis of cancer cells, but also co-localizes with actin stress fibers in normal cells. The 90 kDa palladin is the only ubiquitously expressed isoform and contains three Ig domains and one proline-rich region. This proline-rich region has been shown to bind directly to the actin-regulating protein VASP. In a previous paper, our lab showed that the Ig3 domain of palladin is the minimal binding site for F-actin. In this work we wanted to compare functions of the 90 kDa palladin to the isolated actin binding domain. Our hypothesis was that the 90 kDa palladin may be autoinhibited and may thereby block the binding site for monomeric actin. To understand the mechanism of action for how palladin can influence actin assembly, we used fluorescence spectroscopy to monitor pyrene actin polymerization. By using site-directed mutagenesis via PCR we were able to mutate the putative VASP binding site within the prolinerich region of the 90 kDa palladin. We then examined binding between VASP and WT or mutant palladin using a pulldown assay and far Western blot. Both palladin and VASP proteins are involved in the regulation of actin filaments and understanding the fundamental mechanism of these proteins may help us eliminate the progression of cancer invasion and metastasis. In addition, we sought to determine how palladin and VASP are involved in actin assembly required for cell motility. A facultative intracellular pathogen, Listeria monocytogenes, has been used to study the regulation of palladin in the regulation of actin dynamics. Our aim is to test the hypothesis that palladin promotes the nucleation, elongation and the stabilization of actin-based structure during cell motility. Understanding the role of palladin in actin cytoskeleton may help us prevent cancer cells from reaching the metastasis stage of cancer progression.
Thesis (Ph.D.)-- Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry