Kent L. Rossman

GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors

Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure to do so can have significant consequences and is reflected in the aberrant function of Dbl-family GEFs in some human diseases.

Increasing complexity of Ras signaling

The initial discovery that ras genes endowed retroviruses with potent carcinogenic properties and the subsequent determination that mutated ras genes were present in a wide variety of human cancers, prompted a strong suspicion that the growth-promoting actions of mutated Ras proteins contribute to their aberrant regulation of growth stimulatory signaling pathways. In 1993, a remarkable convergence of experimental observations from genetic analyses of Drosophila, S. cerevisiae and C. elegans as well as biochemical and biological studies in mammalian cells came together to define a clear role for Ras in signal transduction. What emerged was an elegant linear signaling pathway where Ras functions as a relay switch that is positioned downstream of cell surface receptor tyrosine kinases and upstream of a cytoplasmic cascade of kinases that included the mitogen-activated protein kinases (MAPKs). Activated MAPKs in turn regulated the activities of nuclear transcription factors. Thus, a signaling cascade where every component between the cell surface and the nucleus was defined and conserved in worms, flies and man. This was a remarkable achievement in our efforts to appreciate how the aberrant function of Ras proteins may contribute to the malignant growth properties of the cancer cell. However, the identification of this pathway has proven to be just the beginning, rather than the culmination, of our understanding of Ras in signal transduction. Instead, we now appreciate that this simple linear pathway represents but a minor component of a very complex signaling circuitry. Ras signaling has emerged to involve a complex array of signaling pathways, where cross-talk, feedback loops, branch points and multi-component signaling complexes are recurring themes. The simplest concept of a signaling cascade, where each component simply relays the same message to the next, is clearly not the case. In this review, we summarize our current understanding of Ras signal transduction with an emphasis on new complexities associated with the recognition and/or activation of cellular effectors, and the diverse array of signaling pathways mediated by interaction between Ras and Ras-subfamily proteins with multiple effectors.

The Ras superfamily at a glance

The Ras superfamily of small guanosine triphosphatases (GTPases) comprise over 150 human members (Table S1 in [supplementary material][1]), with evolutionarily conserved orthologs found in Drosophila, C. elegans, S. cerevisiae, S. pombe, Dictyostelium and plants ([Colicelli, 2004][2]). The Ras

Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy?

There is now considerable and increasing evidence for a causal role for aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases function as GDP–GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase-activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. In this Review, we assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.

Rho family proteins and Ras transformation: the RHOad less traveled gets congested

The Rho family of small GTPases has attracted considerable research interest over the past 5 years. During this time, we have witnessed a remarkable increase in our knowledge of the biochemistry and biology of these Ras-related proteins. Thus, Rho family proteins have begun to rival, if not overshadow, interest in their more celebrated cousins, the Ras oncogene proteins. The fascination in Rho family proteins is fueled primarily by two major observations. First, like Ras, Rho family proteins serve as guanine nucleotide-regulated binary switches that control signaling pathways that in turn regulate diverse cellular processes. Rho family proteins are key components in cellular processes that control the organization of the actin cytoskeleton, activate kinase cascades, regulate gene expression, regulate membrane trafficking, promote growth transformation and induce apoptosis. Second, at least five Rho family proteins have been implicated as critical regulators of oncogenic Ras transformation. Thus, it is suspected that Rho family proteins contribute significantly to the aberrant growth properties of Ras-transformed cells. Rho family proteins are also critical mediators of the transforming actions of other transforming proteins and include Dbl family oncogene proteins, G protein-coupled receptors and G protein α subunits. Thus, Rho family proteins may be key components for the transforming actions of diverse oncogene proteins. Major aims of Rho family protein studies are to define the molecular mechanism by which Rho family proteins regulate such a diverse spectrum of cellular behavior. These efforts may reveal novel targets for the development of anti-Ras and anti-cancer drugs.

Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.

The principal guanine nucleotide exchange factors for Rho family G proteins contain tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains that catalyse nucleotide exchange and the activation of G proteins. Here we have determined the crystal structure of the DH and PH domains of the T-lymphoma invasion and metastasis factor 1 (Tiam1) protein in complex with its cognate Rho family G protein, Rac1. The two switch regions of Rac1 are stabilized in conformations that disrupt both magnesium binding and guanine nucleotide interaction. The resulting cleft in Rac1 is devoid of nucleotide and highly exposed to solvent. The PH domain of Tiam1 does not contact Rac1, and the position and orientation of the PH domain is markedly altered relative to the structure of the uncomplexed, GTPase-free DH/PH element from Sos1. The Tiam1/Rac1 structure highlights the interactions that catalyse nucleotide exchange on Rho family G proteins, and illustrates structural determinants dictating specificity between individual Rho family members and their associated Dbl-related guanine nucleotide exchange factors.

Signaling Interplay in Ras Superfamily Function

Ras proteins function as signaling hubs that are activated by convergent signaling pathways initiated by extracellular stimuli. Activated Ras in turn regulates a diversity of downstream cytoplasmic signaling cascades. Ras proteins are founding members of a large superfamily of small GTPases that have significant sequence and biochemical similarities. Recent observations have established a complex signaling interplay between Ras and other members of the family. A key biochemical mechanism facilitating this crosstalk involves guanine nucleotide exchange factors (GEFs), which serve as regulators and effectors, as well as signaling integrators, of Ras signaling.

A crystallographic view of interactions between Dbs and Cdc42: PH domain‐assisted guanine nucleotide exchange

Dbl‐related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl‐family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1·Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl‐family proteins will utilize their PH domains similarly to Dbs.

Increasing complexity of ras signal transduction: Involvement of rho family proteins

Publisher Summary The chapter illustrates current understanding of Ras signal transduction, with emphasis on the involvement of Rho family proteins and on how Rho family proteins contribute to Ras function. A remarkable convergence of experimental observations from genetic analyses of Drosophila melanogaster, Saccharomyces cerevisiae, and Caenorhabditis eleguns, as well as biochemical and biological studies in mammalian cells, together define a clear role for Ras in signal transduction. This remarkable achievement appreciates how the aberrant function of Ras proteins contribute to the malignant growth properties of the cancer cell. Ras signaling involves a complex array of signaling pathways, in which cross-talk, feedback loops, branch points, and multicomponent signaling complexes are recurring themes. The chapter summarizes two major emerging themes that refocus the perceptions of how Ras functions in signal transduction. First, it has become clear that the Raf-1 serine/threonine kinase is not the sole downstream effector of Ras. Second, the functions of certain Ras-related proteins that constitute part of the Rho family proteins are shown to be important for Ras transformation. This, together with evidence implicating these small guanosine triphosphates (GTPases) downstream of Ras, has prompted considerable effort in understanding how the function of Rho family proteins contribute to Ras signaling and transformation.

Structural basis for the selective activation of Rho GTPases by Dbl exchange factors.

Activation of Rho-family GTPases involves the removal of bound GDP and the subsequent loading of GTP, all catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl-family. Despite high sequence conservation among Rho GTPases, Dbl proteins possess a wide spectrum of discriminatory potentials for Rho-family members. To rationalize this specificity, we have determined crystal structures of the conserved, catalytic fragments (Dbl and pleckstrin homology domains) of the exchange factors intersectin and Dbs in complex with their cognate GTPases, Cdc42 and RhoA, respectively. Structure-based mutagenesis of intersectin and Dbs reveals the key determinants responsible for promoting exchange activity in Cdc42, Rac1 and RhoA. These findings provide critical insight into the structural features necessary for the proper pairing of Dbl-exchange factors with Rho GTPases and now allow for the detailed manipulation of signaling pathways mediated by these oncoproteins in vivo.