Ann Apolloni

Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains

The plasma membrane pits known as caveolae have been implicated both in cholesterol homeostasis and in signal transduction. CavDGV and CavKSY, two dominant-negative amino-terminal truncation mutants of caveolin, the major structural protein of caveolae, significantly inhibited caveola-mediated SV40 infection, and were assayed for effects on Ras function. We find that CavDGV completely blocked Raf activation mediated by H-Ras, but not that mediated by K-Ras. Strikingly, the inhibitory effect of CavDGV on H-Ras signalling was completely reversed by replenishing cell membranes with cholesterol and was mimicked by cyclodextrin treatment, which depletes membrane cholesterol. These results provide a crucial link between the cholesterol-trafficking role of caveolin and its postulated role in signal transduction through cholesterol-rich surface domains. They also provide direct evidence that H-Ras and K-Ras, which are targeted to the plasma membrane by different carboxy-terminal anchors, operate in functionally distinct microdomains of the plasma membrane.

Ras isoforms vary in their ability to activate Raf-1 and phosphoinositide 3-kinase

Ha-, N-, and Ki-Ras are ubiquitously expressed in mammalian cells and can all interact with the same set of effector proteins. We show here, however, that in vivo there are marked quantitative differences in the ability of Ki- and Ha-Ras to activate Raf-1 and phosphoinositide 3-kinase. Thus, Ki-Ras both recruits Raf-1 to the plasma membrane more efficiently than Ha-Ras and is a more potent activator of membrane-recruited Raf-1 than Ha-Ras. In contrast, Ha-Ras is a more potent activator of phosphoinositide 3-kinase than Ki-Ras. Interestingly, the ability of Ha-Ras to recruit Raf-1 to the plasma membrane is significantly increased when the Ha-Ras hypervariable region is shortened so that the spacing of the Ha-Ras GTPase domains from the inner surface of the plasma membrane mimicks that of Ki-Ras. Importantly, these data show for the first time that the activation of different Ras isoforms can have distinct biochemical consequences for the cell. The mutation of specific Ras isoforms in different human tumors can, therefore, also be rationalized.

H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway

ABSTRACT Ras proteins must be localized to the inner surface of the plasma membrane to be biologically active. The motifs that effect Ras plasma membrane targeting consist of a C-terminal CAAX motif plus a second signal comprising palmitoylation of adjacent cysteine residues or the presence of a polybasic domain. In this study, we examined how Ras proteins access the cell surface after processing of the CAAX motif is completed in the endoplasmic reticulum (ER). We show that palmitoylated CAAX proteins, in addition to being localized at the plasma membrane, are found throughout the exocytic pathway and accumulate in the Golgi region when cells are incubated at 15°C. In contrast, polybasic CAAX proteins are found only at the cell surface and not in the exocytic pathway. CAAX proteins which lack a second signal for plasma membrane targeting accumulate in the ER and Golgi. Brefeldin A (BFA) significantly inhibits the plasma membrane accumulation of newly synthesized, palmitoylated CAAX proteins without inhibiting their palmitoylation. BFA has no effect on the trafficking of polybasic CAAX proteins. We conclude that H-ras and K-ras traffic to the cell surface through different routes and that the polybasic domain is a sorting signal diverting K-Ras out of the classical exocytic pathway proximal to the Golgi. Farnesylated Ras proteins that lack a polybasic domain reach the Golgi but require palmitoylation in order to traffic further to the cell surface. These data also indicate that a Ras palmitoyltransferase is present in an early compartment of the exocytic pathway.

Characterization of RasGRP2, a Plasma Membrane-targeted, Dual Specificity Ras/Rap Exchange Factor

Ras proteins operate as molecular switches in signal transduction pathways downstream of tyrosine kinases and G-protein-coupled receptors. Ras is switched from the inactive GDP-bound state to the active GTP-bound state by guanine nucleotide exchange factors (GEFs). We report here the cloning and characterization of RasGRP2, a longer alternatively spliced form of the recently cloned RapGEF, CalDAG-GEFI. A unique feature of RasGRP2 is that it is targeted to the plasma membrane by a combination of N-terminal myristoylation and palmitoylation. In vivo, RasGRP2 selectively catalyzes nucleotide exchange on N- and Ki-Ras, but not Ha-Ras. RasGRP2 also catalyzes nucleotide exchange on Rap1, but this RapGEF activity is less potent than that associated with CalDAG-GEFI. The nucleotide exchange activity of RasGRP2 toward N-Ras is stimulated by diacylglycerol and inhibited by calcium. The effects of diacylglycerol and calcium are additive but are not accompanied by any detectable change in the subcellular localization of RasGRP2. In contrast, CalDAG-GEFI is localized predominantly to the cytosol and lacks Ras exchange activity in vivo. However, prolonged exposure to phorbol esters, or growth in serum, results in localization of CalDAG-GEFI to the cell membrane and restoration of Ras exchange activity. Expression of RasGRP2 or CalDAG-GEFI in NIH3T3 cells transfected with wild type N-Ras results in an accelerated growth rate but not morphologic transformation. Thus, under appropriate growth conditions, CalDAG-GEFI and RasGRP2 are dual specificity Ras and Rap exchange factors.

14-3-3 Facilitates Ras-Dependent Raf-1 Activation In Vitro and In Vivo

ABSTRACT 14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus.

Expression of Epstein-Barr virus nuclear antigens 3, 4, and 6 are altered in cell lines containing B-type virus

A high proportion of HIV-positive sera were found to react with 130- and 180-kDa antigens which were present in the Jijoye cell line. The majority of the HIV-positive sera which detected these antigens also contained antibodies to Epstein-Barr virus (EBV) nuclear antigen 2B (EBNA2B) suggesting a relationship between B-type EBV strains and the expression of the 130K/180K antigens. Cell lines were established by infection of B lymphocytes with different A- and B-type strains of EBV. Incubation of these lines with sera from individuals infected with either A-type or B-type EBV strains demonstrated that the 130K and 180K antigens were only expressed by cell lines containing B-type virus. Sera from individuals infected with A-type EBV did not react with the 180K antigen in any cell lines but could detect EBNAs 3, 4, and 6 antigens in the A-type cell lines. Restriction enzyme analysis of the BamHI E region of the EBV genome revealed marked differences between the A and B types of the virus. These results demonstrate that expression of antigens encoded from the BamHI E region of EBV (EBNAs 3, 4, and 6) are altered in cell lines transformed by B-type strains of EBV.

Arginine Methylation Increases the Stability of Human Immunodeficiency Virus Type 1 Tat

ABSTRACT Arginine methylation of human immunodeficiency virus type 1 (HIV-1) Tat protein downregulates its key function in viral-gene transactivation. The fate of methylated Tat is unknown, so it is unclear whether methylated Tat is degraded or persists in the cell for additional functions. Here we show that the arginine methyltransferase PRMT6 increases Tat protein half-life by 4.7-fold. Tat stabilization depends on the catalytic activity of PRMT6 and requires arginine methylation within the Tat basic domain. In contrast, HIV-1 Rev, which is also methylated by PRMT6, is completely refractory to the stabilizing effect. Proteasome inhibition and silencing experiments demonstrated that Tat can be degraded by a REGγ-independent proteasome, against which PRMT6 appears to act to increase Tat half-life. Our data reveal a proteasome-dependent Tat degradation pathway that is inhibited by arginine methylation. The stabilizing action of PRMT6 could allow Tat to persist within the cell and the extracellular environment and thereby enable functions implicated in AIDS-related cancer, neurodegeneration, and T-cell death.

Protein methylation is required to maintain optimal HIV-1 infectivity

Background:Protein methylation is recognized as a major protein modification pathway regulating diverse cellular events such as protein trafficking, transcription, and signal transduction. More recently, protein arginine methyltransferase activity has been shown to regulate HIV-1 transcription via Tat. In this study, adenosine periodate (AdOx) was used to globally inhibit protein methyltransferase activity so that the effect of protein methylation on HIV-1 infectivity could be assessed.Results:Two cell culture models were used: HIV-1-infected CEM T-cells and HEK293T cells transfected with a proviral DNA plasmid. In both models, AdOx treatment of cells increased the levels of virion in culture supernatant. However, these viruses had increased levels of unprocessed or partially processed Gag-Pol, significantly increased diameter, and displayed reduced infectivity in a MAGI X4 assay. AdOx reduced infectivity equally in both dividing and non-dividing cells. However, infectivity was further reduced if Vpr was deleted suggesting virion proteins, other than Vpr, were affected by protein methylation. Endogenous reverse transcription was not inhibited in AdOx-treated HIV-1, and infectivity could be restored by pseudotyping HIV with VSV-G envelope protein. These experiments suggest that AdOx affects an early event between receptor binding and uncoating, but not reverse transcription.Conclusion:Overall, we have shown for the first time that protein methylation contributes towards maximal virus infectivity. Furthermore, our results also indicate that protein methylation regulates HIV-1 infectivity in a complex manner most likely involving the methylation of multiple viral or cellular proteins and/or multiple steps of replication.

Human immunodeficiency virus type 1 protease regulation of tat activity is essential for efficient reverse transcription and replication.

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Tat protein enhances reverse transcription, but it is not known whether Tat acts directly on the reverse transcription complex or through indirect mechanisms. Since processing of Tat by HIV protease (PR) might mask its presence and, at least in part, explain this lack of data, we asked whether Tat can be cleaved by PR. We used a rabbit reticulocyte lysate (RRL) system to make Tat and PR. HIV-1 PR is expressed as a Gag-Pol fusion protein, and a PR-inactivated Gag-Pol is also expressed as a control. We showed that Tat is specifically cleaved in the presence of PR, producing a protein of approximately 5 kDa. This result suggested that the cleavage site was located in or near the Tat basic domain (amino acids 49 to 57), which we have previously shown to be important in reverse transcription. We created a panel of alanine-scanning mutations from amino acids 45 to 54 in Tat and evaluated functional parameters, including transactivation, reverse transcription, and cleavage by HIV-1 PR. We showed that amino acids 49 to 52 (RKKR) are absolutely required for Tat function in reverse transcription, that mutation of this domain blocks cleavage by HIV-1 PR, and that other pairwise mutations in this region modulate reverse transcription and proteolysis in strikingly similar degrees. Mutation of Tat Y47G48 to AA also down-regulated Tat-stimulated reverse transcription but had little effect on transactivation or proteolysis by HIV PR, suggesting that Y47 is critical for reverse transcription. We altered the tat gene of the laboratory strain NL4-3 to Y47D and Y47N so that overlapping reading frames were not affected and showed that Y47D greatly diminished virus replication and conveyed a reverse transcription defect. We hypothesize that a novel, cleaved form of Tat is present in the virion and that it requires Y47 for its role in support of efficient reverse transcription.