Abstract
The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.
Highlights
The small Rho GTPases are monomeric GTP-binding proteins that play a role in a variety of cellular processes that depend on the actin cytoskeleton, such as morphogenesis, endocytosis, phagocytosis, cytokinesis and migration
Activation of Rho GTPases is regulated by three types of proteins: The guanine nucleotide dissociation inhibitors (GDIs), the guanine nucleotide exchange factors (GEFs), and GTPase activating proteins (GAPs) [6]
epidermal growth factor (EGF)-induced filopodia formation is illustrated in Fig. 1A, showing staining of the actin cytoskeleton and the localization of the vasodilator-stimulated phosphoprotein (VASP)
Summary
The small Rho GTPases are monomeric GTP-binding proteins that play a role in a variety of cellular processes that depend on the actin cytoskeleton, such as morphogenesis, endocytosis, phagocytosis, cytokinesis and migration. They act as nucleotide-dependent switches cycling between an active, GTP-bound state and an inactive, GDP-bound state. The Rac1-associated protein (Sra-1), a subunit of the pentameric WAVE complex, does not harbor a CRIB-motif and can directly bind to Rac but not to Cdc42 [4] Each of these effectors contributes to the cytoskeletal reorganization downstream of Rac and Cdc, driving the formation of a diverse array of actin structures, e. The GAPs stimulate the intrinsic GTPase activity and convert the GTP-bound form of Rho GTPases to the inactive, GDP-bound form [9]
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