The Ras-related GTPases Rac and Cdc42 contribute to tumorigenesis and metastasis through the activation of diverse downstream effector proteins but how cells regulate activation of distinct subsets of effectors in different contexts is unknown. We have found that the number of Rac and Cdc42 molecules in cells is greatly exceeded by the total number of effector molecules and propose that competition between effectors must limit effector activation. This hypothesis is critically important because competition between effectors implies that loss or overexpression of an individual effector, such as can occur in cancer or Wiskott-Aldrich syndrome, would alter signaling by other pathways. This concept of an interconnected effector network, as opposed to distinct pathways that operate independently, has profound implications for the molecular basis of these diseases. Thus, understanding the interrelations of effector signaling pathways is crucial for predicting biological responses to pharmacological strategies targeting individual effector pathways. The goal of this proposal is to explicate the regulatory mechanisms controlling the specificity of effector activation by Rac and Cdc42. We have developed an experimental system using cytoplasmic extracts of Xenopus laevis eggs in which endogenous Rac and Cdc42 can be activated in a tunable manner and the activation of endogenous downstream effectors N-WASP and the p21-activated kinases (Paks) can be measured quantitatively. Our central hypothesis, based on preliminary studies, is that superimposed on a background of simple competition between effectors, two regulatory mechanisms bias effector activation;scaffolding proteins and pathway crosstalk. In our first aim we will determine the importance of competition on Rac/Cdc42 effector activation by modestly modulating protein levels of individual effectors and measuring the activation of others. In the second aim we will elucidate the role of a scaffolding protein, Toca-1, in biasing Rac/Cdc42 effector selection and will determine if guanine nucleotide exchange factors can serve as scaffolding factors to bias effector pathway activation. In the third aim we will illuminate the role of crosstalk in regulating Rac/Cdc42 effector selection by determining if phosphorylation of N- WASP by Paks regulates N-WASP activation by Rac/Cdc42. These studies will reveal fundamental mechanisms of signaling specificity that likely apply to other small GTPases, such as Ras, that activate multiple downstream pathways of clinical importance in cancer.

Public Health Relevance

The Rho family GTPases Rac and Cdc42 regulate many other proteins that can contribute to the disease process in cancer, mental retardation, and immune disorders. Although many of the partners of Rac and Cdc42 are known, we do not know how the regulation of the various partners is coordinated. This proposal will illuminate how this coordination could be corrupted in disease and will provide crucial information for the selection of optimal drug targets for new therapeutic approaches.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Flicker, Paula F
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Research Institute of Fox Chase Cancer Center
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