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 de®ne 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 de®ned and conserved in worms,¯ies and man. This was a remarkable achievement in our e orts to appreciate how the aberrant function of Ras proteins may contribute to the malignant growth properties of the cancer cell. However, the identi®cation 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 e ectors, and the diverse array of signaling pathways mediated by interaction between Ras and Ras-subfamily proteins with multiple e ectors.

The Fas cell surface receptor induces apoptosis upon receptor oligomerization. We have identified a novel signaling protein, termed Daxx, that binds specifically to the Fas death domain. Overexpression of Daxx enhances Fas-mediated apoptosis and activates the Jun N-terminal kinase (JNK) pathway. A C-terminal portion of Daxx interacts with the Fas death domain, while a different region activates both JNK and apoptosis. The Fas-binding domain of Daxx is a dominant-negative inhibitor of both Fas-induced apoptosis and JNK activation, while the FADD death domain partially inhibits death but not JNK activation. The Daxx apoptotic pathway is sensitive to both Bcl-2 and dominant-negative JNK pathway components and acts cooperatively with the FADD pathway. Thus, Daxx and FADD define two distinct apoptotic pathways downstream of Fas.

Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era.

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.

Abstract. We have examined the role of ms-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rabla and rablb (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rablbl:u prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rabla, rablb, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.