Pierre McCrea - US grants

Cadherin cell-cell adhesion proteins play central roles in both the establishment (morphogenesis), and maintenance (tumor suppression), of a cell's differentiated state. Cadherins are present in all vertebrate solid tissues, and may be common to invertebrate tissues as well. The highly conserved cytoplasmic domains of cadherins associate with three (thus-far) identified polypeptides named alpha-catenin, beta-catenin, and plakoglobin (gamma-catenin), which are essential to cadherin-mediated cell-cell adhesion. alpha-Catenin and beta-catenin have also recently been found associated with the intracellular tumor-suppresser gene product APC, thus increasing the complexity of the catenins' known interactions and potential functions. beta-Catenin and plakoglobin are distinct proteins found within the same cells, but are highly homologous to each other in structure and amino acid sequence. Their specific versus shared structure-functions have not yet been determined, but the catenins collectively are thought to mediate the cadherins' (and APC's? interaction with the actin cytoskeleton - and likely with other intracellular proteins that we wish to identify. We have also recently obtained evidence suggesting that beta-catenin participates in the Wnt developmental signaling pathway. Our research is guided by three Specific Aims: A) Define the structure-function of beta-catenin. B) Define the structure-function of beta-catenin. C) Discover and characterize additional cytoplasmic proteins that interact with cadherins or catenins. In meeting these objectives, the experimental systems employed will include Xenopus eggs and embryos, cultures of canine MDCK and Xenopus A6 cell lines, and the yeast "two-hybrid" system. Epitope-tagged mutant constructs of beta-catenin and plakoglobin, and beta-catenin-plakoglobin chimeric constructs, will be transfected into MDCK and A6 tissue culture cells, and the resulting phenotypes evaluated using phase-contrast and immunofluorescence microscopy. Mutant plakoglobin constructs will also be tested in Xenopus embryos, to examine its structure-function (relative to that of beta-catenin) in a developmental context. Immunoprecipitation of the epitope-tagged beta-catenin and plakoglobin constructs will permit identification of intracellular interactions leading to the observed phenotypes. Novel proteins interacting with the cadherin-catenin complex will be identified by using chemical cross-linker and yeast two-hybrid approaches.

The catenin proteins are of high biological interest given that they bind the cytoplasmic domains of cadherin cell-cell adhesion proteins, forming a complex essential to the morphogenic sorting of tissues and the subsequent maintenance of tissue identities. Of additional intrigue, catenins bind receptor tyrosine kinases, various actin-associated proteins, the APC tumor suppressor (best characterized in the progression of human colon cancer), and components of signaling pathways that require their direct association with nuclear transcription factors. We have collaboratively revealed that the cadherin-catenin complex contains the p120 catenin, which bears structural homology with both beta-catenin and plakoglobin (gamma-catenin), and is a preferred substrate of src kinase and growth factor receptors. Our recent studies have examined the role of p120 in vertebrate development, and indicate that p120 function is critical for proper cell migration, likely resulting from its modulation of cadherin adhesive function. We have further shown that in contrast to beta-catenin, p120 does not engage in Wnt/ beta-catenin-directed nuclear signaling. However, since p120 was recently revealed to bind a novel transcription factor, Kaiso, of the POZ/zinc-finger family, p120 is likely to modulate distinct (non-Wnt/beta-catenin) transcriptional events in the nucleus. This proposal aims to discern the functions of Xenopus p120 (Xp120) and Xenopus Kaiso (XKaiso) in early vertebrate embryogenesis. The experimental system to be employed is the amphibious frog Xenopus, which will offer experimental advantages including the capacity to functionally evaluate Xp120 and XKaiso from both cellular and developmental perspectives. Aims Addressed: 1. What are the developmental functions of Xp120 in association with Cadherins? 2. What are the developmental functions of XKaiso, and to what extent are they modulated via Xp120?

DESCRIPTION (provided by applicant): The catenin proteins contribute to the biology of all animal cells and tissues, displaying essential embryonic and adult functions within a variety of sub-cellular compartments. In addition to binding cadherin at the plasma membrane, B- catenin is well known to associate in the nucleus with transcription factors of the TCF/LEF family in transducing developmental Wnt signals crucial to development, for example, dorsal axis formation, limb patterning and neurogenesis. More recently, the ARVCF and p120 catenins have gained notice in the context of cadherin function at the plasma membrane and other potential roles within the nucleus. Perhaps the most prominent finding was that p120 modulates Rho-family GTPases crucial to cell motility and adhesion. As will be made evident, we have likewise accumulated evidence that ARVCF is functionally coupled to RhoA, and outline in this Proposal our plans to address the developmental role of ARVCF (and p120) in modulating Rho-family GTPases. To gain further insight of ARVCF function, we have conducted yeast two-hybrid screens for interacting protein partners. We recently identified three novel protein associations that were authenticated using in vitro and in vivo co precipitation approaches. We term these three proteins CAPs (Catenin Associated Protein), and expect that the physical association of each CAP reflects a distinct functional interaction which we propose to examine in the context of this Proposal. For our studies, we will employ the Xenopus embryo system that allows for the facile introduction (injection) of exogenous constructs/reagents and the rapid development of this vertebrate within an external environment. We will conduct experiments largely based upon loss-of-function (morpholino), exogenous expression and rescue approaches. Aims Addressed 1. What functional relationship exists between ARVCF (and Xp120) and each CAP? 2. What functional relationship exists between ARVCF (and p120) and Rho-family GTPases? Hypotheses Tested 1. CAPs modulate ARVCF (and p120) developmental functions 2. ARVCF (and p120) modulate the developmental functions of Rho-family GTPases