1. The role of Rho family proteins in leukocyte transendothelial migration.

We have ongoing projects on the role of Rho family proteins in leukocyte transendothelial migration. Most of our work so far has focused on the function of RhoA in leukocytes (e.g. primary human monocytes), but we wish to explore the role of these proteins also in endothelial cells. For leukocytes to migrate across an endothelial monolayer, the leukocytes have to migrate through endothelial cell junctions. Our hypothesis is that leukocyte interactions with endothelial cells signal via endothelial adhesion molecules, such as selectins, PECAM, integrins, etc., to the Rho family of proteins, which in turn regulate the adhesive strength of the endothelial cell-cell junctions. One project will be to explore this hypothesis and to analyze the signaling pathways downstream from leukocyte engagement of endothelial cell adhesion molecules and how these pathways may affect the integrity of endothelial cell-cell adhesions.

2. Tyrosine phosphatases and integrin-mediated adhesion.

In response to integrin-mediated adhesion, there is an elevation in the tyrosine phosphorylation of FAK, paxillin, p130Cas, and other proteins. Relatively little is known about the role of tyrosine phosphatases in this process. Several tyrosine phosphatases have been indentified that interact with tyrosine phosphorylated proteins downstream from integrins and yet we know very little about how these phosphatases are regulated and which ones are critical in the signaling pathways initiated by integrin engagement. We have an ongoing project on the tyrosine phosphatase, PTP-PEST, and have tools to examine several other phosphatases that are candidates for participating in the signaling pathways that emanate from integrin-mediated adhesion.

3. Identification of novel focal adhesion components.

During the past 20 years, many structural and signaling proteins have been discovered in focal adhesions, both by us and other labs. Most of the identification of new focal adhesion components has been a random process. New proteins continue to be discovered at these sites, suggesting that the list of components is far from complete. We propose to analyze the composition of focal adhesions more systematically by isolating these structures, resolving their proteins on SDS gels and using Mass Spectrometry and proteomics to identify all the proteins present in focal adhesions.

4. Signaling downstream from cell-cell junctions.

We have recently demonstrated that the formation of cadherin-mediated cell-cell junctions stimulates the activities of Rac1 and Cdc42, but causes a depression in the activity of RhoA. Many epithelial cells require the development of cell-cell adhesions, as well as appropriate adhesion to extracellular matrix, in order to differentiate. We wish to explore whether signals, such as the elevation of Rac1 and Cdc42 activity, downstream from cell junction assembly, contribute to promoting the differentiated phenotype of epithelial cells.

5. The role of diaphanous in stress fiber assembly downstream from RhoA.

RhoA stimulates the assembly of stress fibers and focal adhesions. Our lab demonstrated that this is due in part to the stimulation of contractility. RhoA elevates myosin light chain phosphorylation by stimulating the protein kinase, Rho-kinase. Besides Rho-kinase, the diaphanous proteins are also downstream effectors of active RhoA and contribute to the assembly of stress fibers. In particular, diaphanous proteins appear to regulate the diameter of stress fibers and the size of focal adhesions. We have constitutively active and dominant negative forms of diaphanous and wish to explore how these regulate the organization of actin filaments within stress fibers.

6. The characterization of novel Rho family guanine nucleotide exchange factors.

The activity of Rho family proteins is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Many GEFs have been identified in the human genome but have not been characterized with respect to their Rho family specificity, mode of regulation or tissue/cell distribution. We have isolated the cDNAs for several uncharacterized GEFs and one project in the lab aims to determine the specificity of these GEFs and to explore the pathways that lead to their activation.