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Mechanisms of Cellular Signaling and Transformation by Ras and Rac Proteins

J.H. Jackson, J.K. Voice, D. Warnock, V. Hill, X. Gao, Q. Chen

MECHANISMS OF RAS SIGNALING

Ras proteins lie at the heart of signal transduction pathways that link cell-surface receptors to the nucleus. Diverse extracellular ligands activate their respective receptors, and the activated receptors induce a guanine nucleotide exchange factor (GEF) to convert inactive GDP-bound Ras to active GTP-bound Ras. Activated Ras subsequently binds 1 or more effector proteins, and the activated effector initiates a signaling cascade that ultimately induces normal cell growth or differentiation. Ras proteins can be made constitutively active by point mutations, and these mutations contribute to the pathogenesis of many human cancers.

Although many of the components involved in Ras-mediated signal transduction have been elucidated, critical questions remain. For instance, human cells contain 4 homologous Ras proteins, H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B, but it is unknown whether these 4 homologs have different biological functions or participate in distinct signal transduction pathways. Recently, we showed that the Ras GEF, Ras-GRF, can selectively activate a single Ras homolog in vivo. In addition, we found that the 4 Ras homologs have substantially different abilities to activate downstream effector proteins, such as Raf-1, in vivo and to induce cellular transformation. Furthermore, in collaboration with R. Klemke, Department of Immunology, we found that the Ras homologs vary in their abilities to induce organization of the actin cytoskeleton and to stimulate cell motility. Our combined results support the notion that each Ras homolog may participate in distinct signaling cascades and have different biological functions.

We are further assessing the biological properties and signaling pathways of each of the 4 Ras homologs. In addition, because the 4 Ras proteins are essentially identical except for their hypervariable C-terminal domains, we are evaluating whether residues within the hypervariable domains account for the distinct biological properties or signaling pathways. Previously, we showed that a polybasic domain within the hypervariable region of K-Ras 4B is crucial for the transforming activity of K-Ras 4B, and recent results indicate that this domain also dictates GEF specificity and is required for activation of Raf-1 effectors.

Our studies should greatly improve our understanding of Ras-mediated signal transduction and could form the theoretical basis for the development of Ras homolog--specific inhibitors that could be useful in the treatment of cancer and inflammatory and immunologic diseases.

MECHANISMS OF RAC SIGNALING

Rac 1 is a member of the Ras superfamily of GTP-binding proteins. Rac 1 regulates the activity of the superoxide anion--generating NADPH oxidase system of phagocytes, plays a central role in organization of the actin cytoskeleton, and is essential for Ras-induced transformation. In addition, mutant, constitutively active Rac 1 can induce cellular transformation, invasion, and metastasis. Similar to Ras proteins, Rac 1 is activated by upstream GEFs and binds effector proteins that signal downstream. Human cells contain 3 homologous Rac proteins, Rac 1, Rac 2, and Rac 3, that are essentially identical except for the hypervariable C-terminal domains. Rac 1, but not Rac 2 or Rac 3, contains a polybasic domain within its hypervariable region that is virtually identical to the polybasic domain of K-Ras 4B.

Because of our studies indicating the crucial importance of the polybasic domain of K-Ras 4B, we investigated the biological significance of the polybasic domain of Rac 1. In collaboration with U. Knaus, Department of Immunology, we found that Rac 1 binds to and activates the effector protein PAK 1 far more efficiently than Rac 2 does, and we showed that the polybasic domain directly accounts for the enhanced ability of Rac 1 to bind to and activate PAK 1. In addition, in collaboration with P. Heyworth, Department of Molecular and Experimental Medicine, and M. Schwartz, Department of Vascular Biology, we found that the polybasic domain is also crucial for Rac 1--mediated activation of NADPH oxidase and membrane ruffling but is not required for Rac 1--mediated cell transformation or binding of Rac 1 to the effector protein POR 1.

Our studies suggest that the polybasic domain may be a novel effector domain that enables Rac, as well as Ras, homologs to activate different downstream effector proteins. We are now assessing whether the 3 Rac homologs have different biological functions, selectively activate other effector proteins, or are activated by different GEFs in vivo.

PUBLICATIONS

Knaus, U.G., Wang, Y., Reilly, A.M., Warnock, D., Jackson, J.H. Structural requirements for PAK activation by Rac GTPases. J. Biol. Chem. 273:21512, 1998.

Jones, M.K., Jackson, J.H. Ras-GRF activates H-Ras, but not N-Ras or K-Ras 4B, protein in vivo. J. Biol. Chem. 273:1782, 1998.