Rac controls PIP5K localisation and PtdIns(4,5)P2 synthesis, which modulates vinculin localisation and neurite dynamics.

In N1E-115 cells, neurite retraction induced by neurite remodelling factors such as lysophosphatidic acid, sphingosine 1-phosphate and semaphorin 3A require the activity of phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks). PIP5Ks synthesise the phosphoinositide lipid second messenger phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2], and overexpression of active PIP5K is sufficient to induce neurite retraction in both N1E-115 cells and cerebellar granule neurones. However, how PIP5Ks are regulated or how they induce neurite retraction is not well defined. Here, we show that neurite retraction induced by PIP5Kß is dependent on its interaction with the low molecular weight G protein Rac. We identified the interaction site between PIP5Kß and Rac1 and generated a point mutant of PIP5Kß that no longer interacts with endogenous Rac. Using this mutant, we show that Rac controls the plasma membrane localisation of PIP5Kß and thereby the localised synthesis of PtdIns(4,5)P2 required to induce neurite retraction. Mutation of this residue in other PIP5K isoforms also attenuates their ability to induce neurite retraction and to localise at the membrane. To clarify how increased levels of PtdIns(4,5)P2 induce neurite retraction, we show that mutants of vinculin that are unable to interact with PtdIns(4,5)P2, attenuate PIP5K- and LPA-induced neurite retraction. Our findings support a role for PtdIns(4,5)P2 synthesis in the regulation of vinculin localisation at focal complexes and ultimately in the regulation of neurite dynamics.

Rac3 inhibits adhesion and differentiation of neuronal cells by modifying GIT1 downstream signaling.

Rac1 and Rac3 are highly homologous regulatory proteins that belong to the small GTPases of the Rho family. Previously, we showed that Rac3 induces cell rounding and prevents neuronal differentiation, in contrast to its close relative Rac1, which stimulates cell spreading and neuritogenesis. To explain these opposing effects, we investigated whether Rac1 and Rac3 interact with different proteins. Here, we show that both Rac1 and Rac3 interact with GIT1, a multifunctional Arf-GAP protein, which regulates cell-matrix adhesion, cell spreading and endocytosis. However, in contrast to Rac1, the Rac3-GIT1 interaction is not mediated by betaPix. Interestingly, Rac3 expression severely attenuates the interaction between GIT1 and paxillin, accompanied by defective paxillin distribution, focal adhesion formation and disturbed cell spreading. Moreover, in Rac3-expressing cells, Arf6 activity is strongly reduced and the Arf6-GAP activity of GIT1 is required for Rac3 downstream signaling. Indeed, expression of wild-type Arf6 or the Arf6-GEF ARNO induced cell spreading in the otherwise rounded Rac3-expressing cells. Our data suggest that Rac3 and Rac1 oppose each other's function by differently modulating GIT1 signaling. Rac1 induces adhesion and differentiation by activating PAK1 and stimulating the GIT1-paxillin interaction, whereas Rac3 blocks this interaction and inactivates Arf6 by stimulating the GAP function of GIT1, thereby preventing cell spreading and differentiation.

Rac1 and Rac3 have opposing functions in cell adhesion and differentiation of neuronal cells.

Rac1 and Rac3 are highly homologous members of the Rho small GTPase family. Rac1 is ubiquitously expressed and regulates cell adhesion, migration and differentiation in various cell types. Rac3 is primarily expressed in brain and may therefore have a specific function in neuronal cells. We found that depletion of Rac1 by short interference RNA leads to decreased cell-matrix adhesions and cell rounding in neuronal N1E-115 cells. By contrast, depletion of Rac3 induces stronger cell adhesions and dramatically increases the outgrowth of neurite-like protrusions, suggesting opposite functions for Rac1 and Rac3 in neuronal cells. Consistent with this, overexpression of Rac1 induces cell spreading, whereas overexpression of Rac3 results in a contractile round morphology. Rac1 is mainly found at the plasma membrane, whereas Rac3 is predominantly localized in the perinuclear region. Residues 185-187, present in the variable polybasic rich region at the carboxyl terminus are responsible for the difference in phenotype induced by Rac1 and Rac3 as well as for their different intracellular localization. The Rac1-opposing function of Rac3 is not mediated by or dependent on components of the RhoA signaling pathway. It rather seems that Rac3 exerts its function through negatively affecting integrin-mediated cell-matrix adhesions. Together, our data reveal that Rac3 opposes Rac1 in the regulation of cell adhesion and differentiation of neuronal cells.

A plasmid-borne Rap-Phr system of Bacillus subtilis can mediate cell-density controlled production of extracellular proteases.

Bacillus subtilis uses two-component signal transduction systems to sense intra- and extracellular stimuli to adapt to fluctuating environmental situations. Regulator aspartate phosphatases (Raps) have important roles in these processes, as they can dephosphorylate certain response-regulators, and are themselves subject to cell-density-controlled inhibition by secreted Phr (phosphate regulator) peptides. Eleven chromosomal genes encode this family of phosphatases, but in addition, certain strains contain endogenous plasmids with genes for homologous Rap-Phr systems. Plasmid pTA1060 encodes Rap60 and its antagonistic signalling molecule Phr60. Strikingly, expression of Rap60 in B. subtilis 168 strongly repressed the production of proteolytic enzymes. In fact, the transcription of the aprE gene, encoding a major extracellular protease, was shown to be decreased upon Rap60 expression, whereas this effect could be antagonized by the extracellular addition of synthetic Phr60 pentapeptide. Finally, transcription studies suggest that Rap60 dephosphorylates a component of the phosphorelay and is coupled to aprE transcription by the transition-state regulator AbrB. In conclusion, these data show that endogenous plasmids contain functional Rap-Phr systems and for the first time, that Rap-Phr systems can mediate cell-density controlled production of secreted proteases. This quorum-sensing mechanism might enable B. subtilis to suppress protease production under conditions of low cell densities when nutrients are still available in sufficient amounts.

Rap1 regulates E-cadherin-mediated cell-cell adhesion.

The small GTPase Rap is best characterized as a critical regulator of integrin-mediated cell adhesion, although its mechanism of action is not understood. Rap also influences the properties of other cell-surface receptors and biological processes, although whether these are a consequence of effects on integrins is not clear. We show here that Rap also plays an important role in the regulation of cadherin-mediated cell-cell adhesion in epithelial cells. Expression of constitutively active Rap1A restored cadherin-mediated cell-cell contacts in mesenchymal Ras-transformed Madin-Darby canine kidney cells, resulting in reversion to an epithelial phenotype. Activation of endogenous Rap via the Rap exchange factor Epac1 also antagonized hepatocyte growth factor-induced disruption of adherens junctions. Inhibition of Rap signaling resulted in disruption of epithelial cell-cell contacts. Rap activity was required for adhesion of cells to recombinant E-cadherin extracellular domains, i.e. in the absence of integrin-mediated adhesion. These findings suggest that Rap signaling positively contributes to cadherin-mediated adhesion and that this occurs independently of effects on integrin-mediated adhesion. Our results imply that Rap may function in a broader manner to regulate the function of cell-surface adhesion receptors.