Lea Guo

Rho3 of Saccharomyces cerevisiae, Which Regulates the Actin Cytoskeleton and Exocytosis, Is a GTPase Which Interacts with Myo2 and Exo70

ABSTRACT The Rho3 protein plays a critical role in the budding yeastSaccharomyces cerevisiae by directing proper cell growth. Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S. cerevisiae cDNA library to identify proteins interacting with Rho3. Two proteins, Exo70 and Myo2, were identified in this screen. Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain. In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins. In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2. Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1. The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins. The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5′-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5′-O-(3-thiodiphosphate). Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence. In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele ofRHO3, RHO3E129,A131 , which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.

Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.

Drosophila Rheb GTPase is required for cell cycle progression and cell growth.

Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. In a screen to identify genes affecting hindgut morphogenesis in Drosophila, we identified a P-element insertion in dRheb, a novel, highly conserved member of the Ras superfamily of G-proteins. Overexpression of dRheb in the developing fly (using the GAL4:UAS system) causes dramatic overgrowth of multiple tissues: in the wing, this is due to an increase in cell size; in cultured cells, dRheb overexpression results in accumulation of cells in S phase and an increase in cell size. Using a loss-of-function mutation we show that dRheb is required in the whole organism for viability (growth) and for the growth of individual cells. Inhibition of dRheb activity in cultured cells results in their arrest in G1 and a reduction in size. These data demonstrate that dRheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that dRheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, we find that flies with reduced dRheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator TOR. In cultured cells, the effect of overexpressing dRheb was blocked by the addition of rapamycin. These results imply that dRheb is involved in TOR signaling.

Single amino-acid changes that confer constitutive activation of mTOR are discovered in human cancer.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a variety of cellular functions such as growth, proliferation and autophagy. In a variety of cancer cells, overactivation of mTOR has been reported. In addition, mTOR inhibitors, such as rapamycin and its derivatives, are being evaluated in clinical trials as anticancer drugs. However, no active mutants of mTOR have been identified in human cancer. Here, we report that two different point mutations, S2215Y and R2505P, identified in human cancer genome database confer constitutive activation of mTOR signaling even under nutrient starvation conditions. S2215Y was identified in large intestine adenocarcinoma whereas R2505P was identified in renal cell carcinoma. mTOR complex 1 prepared from cells expressing the mutant mTOR after nutrient starvation still retains the activity to phosphorylate 4E-BP1 in vitro. The cells expressing the mTOR mutant show increased percentage of S-phase cells and exhibit resistance to cell size decrease by amino-acid starvation. The activated mutants are still sensitive to rapamycin. However, they show increased resistance to 1-butanol. Our study points to the idea that mTOR activating mutations can be identified in a wide range of human cancer.

Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells

The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties. We used a subtractive forward two-hybrid approach to identify small molecule compounds that block the interaction of Ras with Raf. These compounds (MCP1 and its derivatives, 53 and 110) reduced serum-induced transcriptional activation of serum response element as well as Ras-induced transcription by way of the AP-1 site. They also inhibited Ras-induced Raf-1 activation in human embryonic kidney 293 cells, Raf-1 and mitogen-activated protein kinase kinase 1 activities in HT1080 fibrosarcoma cells, and epidermal growth factor-induced Raf-1 activation in A549 lung carcinoma cells. The MCP compounds caused reversion of ras-transformed phenotypes including morphology, in vitro invasiveness, and anchorage-independent growth of HT1080 cells. Decreased level of matrix metalloproteinases was also observed. Further characterization showed that MCP compounds restore actin stress fibers and cause flat reversion in NIH 3T3 cells transformed with H-Ras (V12) but not in NIH 3T3 cells transformed with constitutively active Raf-1 (RafΔN). Finally, we show that MCP compounds inhibit anchorage-independent growth of A549 and PANC-1 cells harboring K-ras mutation. Furthermore, MCP110 caused G1 enrichment of A549 cells with the decrease of cyclin D level. These results highlight potent and specific effects of MCP compounds on cancer cells with intrinsic Ras activation.

Cdk inhibitors, roscovitine and olomoucine, synergize with farnesyltransferase inhibitor (FTI) to induce efficient apoptosis of human cancer cell lines.

Farnesyltransferase inhibitor (FTI) induces apoptosis of transformed cells. This involves changes in mitochondria, including decrease of mitochondrial membrane potential and the release of cytochrome c. The released cytochrome c then induces events leading to the activation of caspase-3. In this study, we report that purine derivative cyclin-dependent kinase (Cdk) inhibitors, roscovitine and olomoucine, dramatically enhance this FTI-induced apoptosis of human cancer cell lines. We noticed the synergy between Cdk inhibitors and FTI through our screen to identify compounds that enhance FTI-induced apoptosis of promyelocytic leukemic cell line HL-60. The Cdk inhibitors by themselves do not induce apoptosis at the concentrations used. Roscovitine synergizes with FTI to release cytochrome c from mitochondria. In addition, we detected synergistic effects of FTI and roscovitine to inhibit hyperphosphorylation of retinoblastoma protein. Enhancement of FTI-induced apoptosis by roscovitine is not unique to HL-60 cells, since similar synergy was observed with a leukemic cell line CEM and a prostate cancer cell line LNCaP. In LNCaP cells, in addition to roscovitine and olomoucine, phophatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, was effective in enhancing FTI-induced apoptosis. However, the effects of roscovitine appear to be distinct from those of LY294002, since roscovitine did not affect Akt activity while LY294002 significantly decreased the activity of Akt. Our finding of the synergy between FTI and Cdk inhibitor is significant for understanding the mechanism of action of FTI as well as for clinical use of FTI.

Identification of Dominant Negative Mutants of Rheb GTPase and Their Use to Implicate the Involvement of Human Rheb in the Activation of p70S6K

Rheb GTPases represent a unique family of the Ras superfamily of G-proteins. Studies on Rheb in Schizosaccharomyces pombe and Drosophila have shown that this small GTPase is essential and is involved in cell growth and cell cycle progression. The Drosophila studies also raised the possibility that Rheb is involved in the TOR/S6K signaling pathway. In this paper, we first report identification of dominant negative mutants of S. pombe Rheb (SpRheb). Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. We show that transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants blocked nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells.

Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast.

Rheb GTPase is a key player in the control of growth, cell cycle and nutrient uptake that is conserved from yeast to humans. To further our understanding of the Rheb pathway, we sought to identify hyperactivating mutations in the Schizosaccharomyces pombe Rheb, Rhb1. Hyperactive forms of Rhb1 were found to result from single amino acid changes at valine‐17, serine‐21, lysine‐120 or asparagine‐153. Expression of these mutants confers resistance to canavanine and thialysine, phenotypes which are similar to phenotypes exhibited by cells lacking the Tsc1/Tsc2 complex that negatively regulates Rhb1. The thialysine‐resistant phenotype of the hyperactive Rhb1 mutants is suppressed by a second mutation in the effector domain. Purified mutant proteins exhibit dramatically decreased binding of GDP, while their GTP binding is not drastically affected. In addition, some of the mutant proteins show significantly decreased GTPase activities. Thus the hyperactivating mutations are expected to result in an increase in the GTP‐bound/GDP‐bound ratio of Rhb1. By using the hyperactive mutant, Rhb1K120R, we have been able to demonstrate that Rhb1 interacts with Tor2, one of the two S. pombe TOR (Target of Rapamycin) proteins. These fission yeast results provide the first evidence for a GTP‐dependent association of Rheb with Tor.

Inhibitors of Protein Geranylgeranyltransferase I and Rab Geranylgeranyltransferase Identified from a Library of Allenoate-derived Compounds

Protein geranylgeranylation is critical for the function of a number of proteins such as RhoA, Rac, and Rab. Protein geranylgeranyltransferase I (GGTase-I) and Rab geranylgeranyltransferase (RabGGTase) catalyze these modifications. In this work, we first describe the identification and characterization of small molecule inhibitors of GGTase-I (GGTI) with two novel scaffolds from a library consisting of allenoate-derived compounds. These compounds exhibit specific inhibition of GGTase-I and act by competing with a substrate protein. Derivatization of a carboxylic acid emanating from the core ring of one of the GGTI compounds dramatically improves their cellular activity. The improved GGTI compounds inhibit proliferation of a variety of human cancer cell lines and cause G1 cell cycle arrest and induction of p21CIP1/WAF1. We also report the identification of novel small molecule inhibitors of RabGGTase. These compounds were identified first by screening our GGTI compounds for those that also exhibited RabGGTase inhibition. This led to the discovery of a common structural feature for RabGGTase inhibitors: the presence of a characteristic six-atom aliphatic tail attached to the penta-substituted pyrrolidine core. Further screening led to the identification of compounds with preferential inhibition of RabGGTase. These compounds inhibit RabGGTase activity by competing with the substrate protein. These novel compounds may provide valuable reagents to study protein geranylgeranylation.

A novel approach to tag and identify geranylgeranylated proteins.

A recently developed proteomic strategy, the “GG‐azide”‐labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido‐geranylgeranyl analog and chemoselective derivatization of azido‐geranylgeranyl‐modified proteins by the “click” chemistry, using a tetramethylrhodamine‐alkyne. The resulting conjugated proteins can be separated by 1‐D or 2‐D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC‐MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The “GG‐azide”‐labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post‐translational modifications.