Date of Graduation

5-2010

Document Type

Dissertation (PhD)

Program Affiliation

Genes and Development

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Pierre D. McCrea, Ph.D.

Committee Member

Michelle C. Barton, Ph.D.

Committee Member

Warren S. Liao, Ph.D.

Committee Member

Fernando R. Cabral, Ph.D.

Committee Member

Milan Jamrich, Ph.D.

Abstract

Xenopus ARVCF (xARVCF), a member of p120-catenin subfamily, binds cadherin cytoplasmic domains to enhance cadherin metabolic stability, or when dissociated, modulates Rho-family GTPases. We previously found that xARVCF binds directly to Xenopus KazrinA (xKazrinA), a widely expressed, conserved protein that bears little homology to established protein families. xKazrinA is also known to influence keratinocyte proliferation-differentiation and cytoskeletal activity. In my study, I first evaluated the expression pattern of endogenous Kazrin RNA and protein in Xenopus embryogenesis as well as in adult tissues. We then collaboratively predicted the helical structure of Kazrin’s coiled-coil domain, and I obtained evidence of Kazrin’s dimerization/oligomerization. In considering the intracellular localization of the xARVCF-catenin:xKazrin complex, I did not resolve xKazrinA in a larger ternary complex with cadherin, nor did I detect its co-precipitation with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin. This suggested a potential means by which xKazrinA localizes to cell-cell junctions, and indeed, biochemical assays confirmed a ternary xARVCF:xKazrinA:xβ2-spectrin complex. Functionally, I demonstrated that xKazrin stabilizes cadherins by negatively modulating the RhoA small-GTPase. I further revealed that xKazrinA binds to p190B RhoGAP (an inhibitor of RhoA), and enhances p190B’s association with xARVCF. Supporting their functional interaction in vivo, Xenopus embryos depleted of xKazrin exhibited ectodermal shedding, a phenotype that could be rescued with exogenous xARVCF. Cell shedding appeared to be caused by RhoA activation, which consequently altered actin organization and cadherin function. Indeed, I was capable of rescuing Kazrin depletion with ectopic expression of p190B RhoGAP. In addition, I obtained evidence that xARVCF and xKazrin participate in craniofacial development, with effects observed upon the neural crest. Finally, I found that xKazrinA associates further with delta-catenin and p0071-catenin, but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin sub-family. Taken together, my study supports Kazrin’s essential role in development, and reveals KazrinA’s biochemical and functional association with ARVCF-catenin, spectrin and p190B RhoGAP.

Keywords

Kazrin, ARVCF, Cadherin, Catenin, spectrin, p190B