Benjamin Boettner

The role of Rho GTPases in disease development

The functionality and efficacy of Rho GTPase signaling is pivotal for a plethora of biological processes. Due to the integral nature of these molecules, the dysregulation of their activities can result in diverse aberrant phenotypes. Dysregulation can, as will be described below, be based on an altered signaling strength on the level of a specific regulator or that of the respective GTPase itself. Alternatively, effector pathways emanating from a specific Rho GTPase may be under- or overactivated. In this review, we address the role of the Rho-type GTPases as a subfamily of the Ras-superfamily of small GTP-binding proteins in the development of various disease phenotypes. The steadily growing list of genetic alterations that specifically impinge on proper Rho GTPase function corresponds to pathological categories such as cancer progression, mental disabilities and a group of quite diverse and unrelated disorders. We will provide an overview of disease-rendering mutations in genes that have been positively correlated with Rho GTPase signaling and will discuss the cellular and molecular mechanisms that may be affected by them.

Rac Regulates Integrin-Mediated Spreading and Increased Adhesion of T Lymphocytes

ABSTRACT Leukocyte adhesion to the extracellular matrix (ECM) is tightly controlled and is vital for the immune response. Circulating lymphocytes leave the bloodstream and adhere to ECM components at sites of inflammation and lymphoid tissues. Mechanisms for regulating T-lymphocyte–ECM adhesion include (i) an alteration in the affinity of cell surface integrin receptors for their extracellular ligands and (ii) an alteration of events following postreceptor occupancy (e.g., cell spreading). Whereas H-Ras and R-Ras were previously shown to affect T-cell adhesion by altering the affinity state of the integrin receptors, no signaling molecule has been identified for the second mechanism. In this study, we demonstrated that expression of an activated mutant of Rac triggered dramatic spreading of T cells and their increased adhesion on immobilized fibronectin in an integrin-dependent manner. This effect was not mimicked by expression of activated mutant forms of Rho, Cdc42, H-Ras, or ARF6, indicating the unique role of Rac in this event. The Rac-induced spreading was accompanied by specific cytoskeletal rearrangements. Also, a clustering of integrins at sites of cell adhesion and at the peripheral edges of spread cells was observed. We demonstrate that expression of RacV12 did not alter the level of expression of cell surface integrins or the affinity state of the integrin receptors. Moreover, our results indicate that Rac plays a role in the regulation of T-cell adhesion by a mechanism involving cell spreading, rather than by altering the level of expression or the affinity of the integrin receptors. Furthermore, we show that the Rac-mediated signaling pathway leading to spreading of T lymphocytes did not require activation of c-Jun kinase, serum response factor, or pp70S6 kinase but appeared to involve a phospholipid kinase.

Control of cell adhesion dynamics by Rap1 signaling

Individual cells in their particular environments adhere to the extracellular matrix (ECM) and their neighbours via integrin-containing and cadherin-containing complexes, respectively. The dynamics of these interactions regulate the formation and maintenance of complex tissues. An expanding body of evidence accentuates the role of the small Rap1 GTPase and its associated signaling network in many of these processes. In this review we will discuss more recently revealed roles of Rap1 signaling by primarily focusing on functions of the Rap1 effectors RIAM, KRIT-1/CCM1 and AF-6/Afadin in junctional regulation of the vascular system and in epithelial cells. Furthermore, we will describe novel findings on the Rap activator PDZ-GEF in the regulation of cell-cell adhesion between epithelial cells and within a stem cell niche.

The Rap GTPase Activator Drosophila PDZ-GEF Regulates Cell Shape in Epithelial Migration and Morphogenesis

ABSTRACT Epithelial morphogenesis is characterized by an exquisite control of cell shape and position. Progression through dorsal closure in Drosophila gastrulation depends on the ability of Rap1 GTPase to signal through the adherens junctional multidomain protein Canoe. Here, we provide genetic evidence that epithelial Rap activation and Canoe effector usage are conferred by the Drosophila PDZ-GEF (dPDZ-GEF) exchange factor. We demonstrate that dPDZ-GEF/Rap/Canoe signaling modulates cell shape and apicolateral cell constriction in embryonic and wing disc epithelia. In dPDZ-GEF mutant embryos with strong dorsal closure defects, cells in the lateral ectoderm fail to properly elongate. Postembryonic dPDZ-GEF mutant cells generated in mosaic tissue display a striking extension of lateral cell perimeters in the proximity of junctional complexes, suggesting a loss of normal cell contractility. Furthermore, our data indicate that dPDZ-GEF signaling is linked to myosin II function. Both dPDZ-GEF and cno show strong genetic interactions with the myosin II-encoding gene, and myosin II distribution is severely perturbed in epithelia of both mutants. These findings provide the first insight into the molecular machinery targeted by Rap signaling to modulate epithelial plasticity. We propose that dPDZ-GEF-dependent signaling functions as a rheostat linking Rap activity to the regulation of cell shape in epithelial morphogenesis at different developmental stages.

Rac and Cdc42 effectors.

Rac and Cdc42, like all members of the Ras superfamily, act as molecular switches, cycling between an inactive GDP-bound state and an active GTPbound state. Until recently, members of the Rho-subfamily were believed to be primarily involved in the regulation of cytoskeletal organization in response to extracellular growth factors. However, studies from numerous groups over the past few years have revealed that the Rho-GTPases play crucial roles in a wide variety of biological processes such as transcriptional regulation, cell growth control, membrane trafficking including endocytosis, exocytosis and phagocytosis, as well as development (Fig. 1). The signal transduction pathways mediating these biological phenomena are complex and intricately interwoven. A major challenge has been the identification of downstream effector molecules by which Rac and Cdc42 mediate these activities. Numerous targets of Rac and Cdc42 have been identified (Fig. 2) and further characterization of some of them has led to a better understanding of the function Rac and Cdc42 display at the molecular level. The different Rac and Cdc42 specific effectors and our current understanding of their physiological roles are the major focus of this chapter.

Ras and Rap1 interaction with AF-6 effector target.

Publisher Summary This chapter describes the Ras and Rap I interaction with AF-6 effector target. The chapter presents a spectrum of investigative approaches that served to demonstrate specific protein interactions among the Ras/Rapl GTPases and their potential effector molecule AF-6 in vivo and in vitro . The Rap types of small GTPases are members of the Ras superfamily and are the molecules that show the most identity with the oncogenic Ras proteins. Whereas the interaction of activated Ras proteins with their downstream effectors Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphatidylinositol 3-kinase (PI3K) leads to a fairly defined picture, the role of Rapl is as yet poorly understood. The two-hybrid analysis and the investigation of the kinetic and thermodynamic properties of Ras-AF-6 and Rapl-AF-6 complexes leads to the same overall outcome—namely, Rap1 appears to form the tightest complex with AF-6-RBD1. The chapter concludes with a discussion on methods to quantitate Ras/Rapl and AF-6 interactions.

Noncanonical Effector Targets Of Oncogenic Ras Proteins

The understanding of the Raf, PI3-kinase and RalGDS mediated pathways that relay physiological signals from oncogenic Ras-proteins has been consistently improved over the recent years. The proliferative, anti-apoptotic and some of the more cell-type and tumor-idiosyncratic effects of Ras GTPases in various scenarios could be ascribed to one of these effectors. However, individual tumor cells undergo drastic changes in their cell fates and differentiation states which likely require the activation of other than Raf-, PI3-kinase- and RalGDS-initiated signaling mechanisms. In addition, Ras GTPases participate in a multitude of developmental processes that entail growth, proliferative, differentiative and migratory programs. Proteins such as AF-6, Nore1, certain protein kinase C (PLC) isoforms, Tiam1, Rin1 and a few others have been identified as candidate Ras-effectors mostly by virtue of their physical interaction properties in various affinity-based protocols but also as a result of genetic and computational approaches. This selection of alternative binding partners for oncogenic Ras-proteins can thus serve as a source for more in depth investigations of particular Ras-related phenomena. The following chapter will scrutinize these molecules with respect to their functions and biochemical properties