Cdc42p and Rho1p are sequentially activated and mechanistically linked to vacuole membrane fusion

Small monomeric GTPases act as molecular switches, regulating many biological functions via activation of membrane localized signaling cascades. Activation of their switch function is controlled by GTP binding and hydrolysis. Two Rho GTPases, Cdc42p and Rho1p, are localized to the yeast vacuole where they regulate membrane fusion. Here, we define a method to directly examine vacuole membrane Cdc42p and Rho1p activation based on their affinity to probes derived from effectors. Cdc42p and Rho1p showed unique temporal activation which aligned with distinct subreactions of in vitro vacuole fusion. Cdc42p was rapidly activated in an ATP-independent manner while Rho1p activation was kinetically slower and required ATP. Inhibitors that are known to block vacuole membrane fusion were examined for their effect on Cdc42p and Rho1p activation. Rdi1p, which inhibits the dissociation of GDP from Rho proteins, blocked both Cdc42p and Rho1p activation. Ligands of PI(4,5)P₂ specifically inhibited Rho1p activation while pre-incubation with U73122, which targets Plc1p function, increased Rho1p activation. These results define unique activation mechanisms for Cdc42p and Rho1p, which may be linked to the vacuole membrane fusion mechanism.     

Small GTPases in vesicle trafficking

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.     

Mechanics of the cellular actin cortex: From signalling to shape change

The actin cortex is a thin layer of actin, myosin and actin-binding proteins that underlies the membrane of most animal cells. It is highly dynamic and can undergo remodelling on timescales of tens of seconds, thanks to protein turnover and myosin-mediated contractions. The cortex enables cells to resist external mechanical stresses, controls cell shape and allows cells to exert forces on their neighbours. Thus, its mechanical properties are the key to its physiological function. Here, we give an overview of how cortex composition, structure and dynamics control cortex mechanics and cell shape. We use mitosis as an example to illustrate how global and local regulation of cortex mechanics gives rise to a complex series of cell shape changes.     

Molecular mechanism of co-translational protein targeting by the signal recognition particle

The signal recognition particle (SRP) is a key component of the cellular machinery that couples the ongoing synthesis of proteins to their proper localization, and has often served as a paradigm for understanding the molecular basis of protein localization within the cell. The SRP pathway exemplifies several key molecular events required for protein targeting to cellular membranes: the specific recognition of signal sequences on cargo proteins, the efficient delivery of cargo to the target membrane, the productive unloading of cargo to the translocation machinery and the precise spatial and temporal coordination of these molecular events. Here we highlight recent advances in our understanding of the molecular mechanisms underlying this pathway, and discuss new questions raised by these findings.     

Protein kinase D regulates RhoA activity via rhotekin phosphorylation

The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin.     

Functional implications of caspase-mediated RhoGDI2 processing during apoptosis of HL60 and K562 leukemia cells

RhoGDI2, a cytosolic regulator of Rho GTPase, is cleaved during apoptosis in a caspase-3 dependent fashion. By using 2D-gel electrophoresis, mass spectrometry and Western blotting we investigate in this paper the functional consequences of RhoGDI2 processing. We can show that loss of the N-terminal 19 amino acids results in a shift of the isoelectric point of the truncated RhoGDI2 (NΔ19) to a more basic value due to the removal of 9 acidic amino acids from the N-terminus, which may be responsible for enhanced retention of the N-terminally truncated protein within the nuclear compartment. Fusion of the p53 nuclear export signaling sequence MFRELNEALELK to NΔ19 (NΔ19NES) abolished its apoptosis promoting properties, while overexpression of NΔ19 significantly increased the susceptibility to apoptosis induction by the proteasome inhibitor PSI and by staurosporine. These results suggest that cleavage of RhoGDI2 by caspase-3 is not a functionally irrelevant bystander effect of caspase activation during apoptosis, but rather expedites progression of the apoptotic process.     

PAR1-mediated RhoA activation facilitates CCL2-induced chemotaxis in PC-3 cells

Patients with advanced prostate cancer often exhibit increased activation of the coagulation system. The key activator of the coagulation cascade is the serine protease thrombin which is capable of eliciting numerous cellular responses. We previously reported that the thrombin receptor PAR1 is overexpressed in prostate cancer. To investigate further the role of PAR1 in prostate cancer metastasis, we examined the effects of thrombin activation on cell adhesion and motility in PC-3 prostate cancer cells. Activation of PAR1-induced dynamic cytoskeletal reorganization and reduced PC-3 binding to collagen I, collagen IV, and laminin (P < 0.01) but not fibronectin. Expression of the cell surface integrin receptors did not change as assessed by flow cytometry. Immunofluorescence microscopy revealed that PAR1 stimulation caused reorganization of the focal adhesions, suggesting that PAR1 activation in PC-3 cells may be modulating cell adhesion through integrin function but not expression. Furthermore, RhoA was activated upon stimulation with thrombin with subsequent cell contraction, decreased cell adhesion, and induced migration towards monocyte chemoattractant protein 1 (MCP-1; CCL2). Thus, it appears that thrombin stimulation plays a role in prostate cancer metastasis by decreasing cell adhesion to the extracellular matrix and positioning the cell in a "ready state" for migration in response to a chemotactic signal. Further exploration is needed to determine whether PAR1 activation affects other signaling pathways involved in prostate cancer.     

Guanine Nucleotide Exchange Factor OSG-1 Confers Functional Aging via Dysregulated Rho Signaling in Caenorhabditis elegans Neurons

Genetic studies usually focus on quantifying and understanding the existence of genetic control on expected phenotypic outcomes. However, there is compelling evidence suggesting the existence of genetic control at the level of environmental variability, with some genotypes exhibiting more stable and others more volatile performance. Understanding the mechanisms responsible for environmental variability not only informs medical questions but is relevant in evolution and in agricultural science. In this work fully sequenced inbred lines of Drosophila melanogaster were analyzed to study the nature of genetic control of environmental variance for two quantitative traits: starvation resistance (SR) and startle response (SL). The evidence for genetic control of environmental variance is compelling for both traits. Sequence information is incorporated in random regression models to study the underlying genetic signals, which are shown to be different in the two traits. Genomic variance in sexual dimorphism was found for SR but not for SL. Indeed, the proportion of variance captured by sequence information and the contribution to this variance from four chromosome segments differ between sexes in SR but not in SL. The number of studies of environmental variation, particularly in humans, is limited. The availability of full sequence information and modern computationally intensive statistical methods provides opportunities for rigorous analyses of environmental variability.     

Discovery and optimization of indole and 7-azaindoles as Rho kinase (ROCK) inhibitors (part-II)

Therapeutic interventions with Rho kinase (ROCK) inhibitors may effectively treat several disorders such as hypertension, stroke, cancer, and glaucoma. Herein we disclose the optimization and biological evaluation of potent novel ROCK inhibitors based on substituted indole and 7-azaindole core scaffolds. Substitutions on the indole C3 position and on the indole NH and/or amide NH positions all yielded potent and selective ROCK inhibitors (25, 42, and 50). Improvement of aqueous solubility and tailoring of in vitro and in vivo DMPK properties could be achieved through these substitutions.     

Modulation of a GEF switch: autoinhibition of the intrinsic guanine nucleotide exchange activity of p115-RhoGEF

p115-RhoGEF (p115) belongs to the family of RGS-containing guanine nucleotide exchange factors for Rho GTPases (RGS-RhoGEFs) that are activated by G12 class heterotrimeric G protein α subunits. All RGS-RhoGEFs possess tandemly linked Dbl-homology (DH) and plekstrin-homology (PH) domains, which bind and catalyze the exchange of GDP for GTP on RhoA. We have identified that the linker region connecting the N-terminal RGS-homology (RH) domain and the DH domain inhibits the intrinsic guanine nucleotide exchange (GEF) activity of p115, and determined the crystal structures of the DH/PH domains in the presence or absence of the inhibitory linker region. An N-terminal extension of the canonical DH domain (the GEF switch), which is critical to GEF activity, is well folded in the crystal structure of DH/PH alone, but becomes disordered in the presence of the linker region. The linker region is completely disordered in the crystal structure and partially disordered in the molecular envelope calculated from measurements of small angle x-ray scattering (SAXS). It is possible that Gα subunits activate p115 in part by relieving autoinhibition imposed by the linker region.