SummarySignaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.

The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] and PtdIns(3,5)P 2 . Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFPtagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stressmediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P 2 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P 2 and PtdIns(4,5)P 2 at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.The actin cytoskeleton plays a fundamental role in regulating cytokinesis and organelle transport. In the budding yeast Saccharomyces cerevisiae genetic and morphological evidence indicates that actin regulates cell growth. In yeast filamentous actin is found in two morphologically identified forms, cables and patches (1, 24). Actin cables are found mainly in the mother cell and extend along the axis of growth, which is asymmetrical to the emerging daughter cell. Cables are involved in regulating organelle inheritance and vesicle targeting. Actin patches are associated with plasma membrane invaginations and are motile structures that move along the plasma membrane in response to osmotic stress (6, 33, 47). It has been speculated that actin patches may be necessary machinery for maintaining secretion or endocytosis. Actin cables and patches may form part of an integrated system, as their distributions often change simultaneously (23). However, the signaling mechanisms regulating the assembly and movement of actin cables and patches in response to osmotic and other stimuli are not well understood.The phosphoinositides are ubiquitous components of eukaryotic membranes and are critic...

Phosphoinositides are ubiquitous membrane components of various intracellular compartments, which regulate many diverse cellular functions including membrane trafficking events, secretion, actin cytoskeletal organization, cellular proliferation, and inhibition of apoptosis (reviewed in Refs. 1-4). Many of these functions are mediated by binding and recruiting signaling proteins which contain specific phosphoinositidebinding domains such as SH2 domains, pleckstrin homology domains, FYVE domains, C2 domains or polybasic domains, thereby localizing these effector proteins to specific membranes (reviewed in Refs. 3 and 4).Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P 2 ) 1 serves as a precursor to second messenger molecules such as inositol (1,4,5)-trisphosphate and phosphatidylinositol (3,4,5)-trisphosphate, but also independent of further modification regulates the actin cytoskeleton and membrane trafficking (1, 5). PtdIns(4,5)P 2 binds to actin-binding proteins such as profilin and gelsolin (6) and displaces capping proteins from actin filaments, allowing polymerization and formation of actin stress fibers (7-9). PtdIns(4,5)P 2 also plays a role in regulating vesicle budding and in the recruitment and activation of proteins involved in the coating of vesicles (2).Cellular levels of PtdIns(4,5)P 2 are regulated by a series of lipid phosphorylation and dephosphorylation reactions mediated by specific lipid kinases and phosphatases. Inositol polyphosphate 5-phosphatases (5-phosphatases) regulate cellular PtdIns(4,5)P 2 levels by hydrolyzing the 5-position phosphate from the inositol ring forming phosphatidylinositol 4-phosphate (PtdIns(4)P) (10, 11). The budding yeast Saccharomyces cerevisiae has four 5-phosphatase genes, INP51, INP52, INP53, and INP54. Inp51p, Inp52p, and Inp53p each comprise an N-terminal SacI domain, a central 5-phosphatase domain, and a C-terminal proline-rich region (12, 13). These enzymes share significant sequence homology with the mammalian homologue synaptojanin, which regulates the recycling of synaptic vesicles in nerve terminals (14). Synaptojanin, Inp52p, and Inp53p contain two catalytic domains, a central 5-phosphatase domain and an N-terminal SacI domain which hydrolyzes PtdIns(3,5)P 2 , PtdIns(4)P, and PtdIns(3)P forming PtdIns (15). Null mutation of any two SacI domain-containing 5-phosphatases results in plasma membrane invaginations and thickened cell walls, defects in polarization of the actin cytoskeleton, and impaired endocytosis (12, 13). However, double SacI domain-containing 5-phosphatase null mutants display normal secretion of invertase suggesting that Inp51p, Inp52p, and Inp53p do not play a role in regulating secretion (16). A triple SacI domain-containing 5-phosphatase null mutant is nonviable suggesting Inp54p cannot function to rescue the loss of these three 5-phosphatases.

Rnd proteins are atypical members of the Rho GTPase family that induce actin cytoskeletal reorganization and cell rounding. Rnd proteins have been reported to bind to the intracellular domain of several plexin receptors, but whether plexins contribute to the Rnd-induced rounding response is not known. Here we show that Rnd3 interacts preferentially with plexin-B2 of the three plexin-B proteins, whereas Rnd2 interacts with all three B-type plexins, and Rnd1 shows only very weak interaction with plexin-B proteins in immunoprecipitations. Plexin-B1 has been reported to act as a GAP for R-Ras and/or Rap1 proteins. We show that all three plexin-B proteins interact with R-Ras and Rap1, but Rnd proteins do not alter this interaction or R-Ras or Rap1 activity. We demonstrate that plexin-B2 promotes Rnd3-induced cell rounding and loss of stress fibres, and enhances the inhibition of HeLa cell invasion by Rnd3. We identify the amino acids in Rnd3 that are required for plexin-B2 interaction, and show that mutation of these amino acids prevents Rnd3-induced morphological changes. These results indicate that plexin-B2 is a downstream target for Rnd3, which contributes to its cellular function.