Anne B. Vojtek

Mammalian Ras interacts directly with the serine/threonine kinase raf

We have identified proteins that interact with H-Ras using a two hybrid system screen of a mouse cDNA library. Approximately 50% of the clones identified encoded portions of the c-Raf and A-Raf serine/threonine kinases. Overlaps among these clones define a conserved 81 residue region of the N-terminus of Raf as the Ras interaction region. We show that Raf interacts with wild-type and activated Ras, but not with an effector domain mutant of Ras or with a dominant-interfering Ras mutant. Using purified bacterially expressed fusion proteins, we show, furthermore, that Ras and the N-terminal region of Raf associate directly in vitro and that this interaction is dependent on GTP bound to Ras.

Increasing Complexity of the Ras Signaling Pathway

Ras is a key regulator of cell growth in all eukaryotic cells. Genetic, biochemical, and molecular studies in Caenorhabditis elegans, Drosophila, and mammalian cells have positioned Ras centrally in signal transduction pathways that respond to diverse extracellular stimuli, including peptide growth factors, cytokines, and hormones. The biological activity of Ras is controlled by a regulated GDP/GTP cycle. Guanine nucleotide exchange factors (GEFs; RasGRF1/2 and Sos1/2) promote the formation of the active, GTP-bound form of Ras (1). GTPaseactivating proteins (GAPs; p120 GAP and NF1) accelerate the intrinsic GTP hydrolytic activity of Ras to promote formation of the inactive, GDP-bound form of Ras (1). Mutations in Ras at amino acids 12, 13, or 61 make Ras insensitive to GAP action and, hence, constitutively active in transforming mammalian cells (2, 3). These activating mutations in Ras are prevalent in a wide spectrum of human cancers. It has been estimated that 30% of all human tumors contain an activating mutation in Ras. The frequency of Ras mutations varies depending on tumor type, with the highest frequencies seen in lung, colon, thyroid, and pancreatic carcinomas (3). The frequency of Ras mutations is likely to be an underestimation of the contribution of aberrant signaling through the Ras pathway to human malignancies because chronic up-regulation of the Ras pathway can occur in the absence of mutations in Ras itself (4–6).

Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem–loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.

Rho family members: Activators of MAP kinase cascades

Low molecularweight GTP-binding proteins are molecular switches that control a diverse array of biological processes (Bourne et al., 1991). When bound to GTP, these proteins transduce signals to effector proteins. When bound to GDP, these proteins are in an inactive state. The cycling between the active, GTP-bound state and the inactive, GDP-bound state is controlled by positive and negative modulators that directly contact the GTP-binding protein. Positive modulators, guanine nucleotide dissociation stimulators (GDSs), catalyze the dissociation of GDP. Subsequently, GTP binds, and the protein is active. Negative modulators, GTPase-activating proteins (GAPS), stimulate the intrinsic GTP hydrolytic activity of the GTPbinding proteins and, hence, the formation of the inactive, GDP-bound state. Approximately 50 low molecular weight GTPases have been described (Chardin, 1991). The family members can be subdivided, on the basis of sequence homology, into five classes: Ras, Rab, Arf, Ran, and Rho. Ras family members (H-Ras, K-Ras, N-Ras, R-Ras, TC21, RaplAl Rap1 B, and RapPAIRapPB) play salient roles in cell growth and development. The members of the Rab/Arf subfamilies monitor and direct the movements of vesicles within the cell. Ran is required for nuclear protein import. And, finally, the Rho family members (Cdc42/G25K, Racl, Rac2, RhoA, RhoB, and RhoC) play dynamic roles in the regulation of the actin cytoskeleton. Regulated changes in the actin cytoskeleton are required for cytokinesis, for cell motility, for vesicle trafficking, for pinocytosis, and, in the yeast Saccharomyces cerevisiae, for bud site selection and for polarized cell growth. Each of the small GTPases of the Rho subfamily participates in distinct patterns of actin reorganization. Rat is required for membrane ruffling, whereas Rho is required for the formation of stress fibers (Ridley and Hall, 1992; Ridley et al., 1992). Cdc42 was first identified in the yeast S. cerevisiae, where it is required for polarized cell growth (Adamset al., 1990); subsequently, a homolog of this yeast protein, Cdc42Hs (G25K), was identified from mammalian cells, where it regulates the formation of actin-containing microspikes, called filopodia (Kozma et al., 1995; Nobes and Hall, 1995). The observation that chronic activation of Cdc42, Rat, and Rho by deregulated exchange factors induces malignant transformation as well as morphological changes (Khosravifar et al., 1994; Michiels et al., 1995) has suggested that these GTPases may regulate nuclear as well as cytoplasmic effects. Three recent papers in Cell (Coso et al., 1995; Minden et al., 1995; Hill et al., 1995) describe a link from these GTP-binding proteins to the nucleus and, for Cdc42 and Rat, to a mitogen-activating protein kinase (MAPK) cascade. Coso et al. (1995) and Minden et al. (1995) made extensive use of transient transfection assays with an epitopetagged cJun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) cDNA clone as reporter. Protein kinase assays of the recovered tagged JNK were used to monitor activity of the MAPK pathway that leads to the JNKs (in order, MAP or extracellular-regulated kinase [ERK] kinase 1 [MEKKl], SAP or ERK kinase [SEKIIMAPK kinase 4 [MKK4], and JNK; Figure 1). By cotransfecting various mutants of Cdc42, Racl, and RhoA that lock the small GTPase into a constitutively active or inactive state, Coso et al. and Minden et al. were able to show that JNK activation is dependent on Cdc42 and Racl , but not RhoA. In addition, transfection of a RhoGAP, which inactivates all Rho family members, reduces activation of JNK by cytokines, and transfection of Dbl or Ost, GDSs for Cdc42 and also RhoA, efficiently activates JNK. The Dbl-induced activation of JNK was abolished upon cotransfection of JNK with an N-terminal region of the serinelthreonine kinase PAK85 (Manser et al., 1994; Martin et al., 1995) which includes a Cdc42IRacbinding domain and may be expected to block signaling from Dbl through Cdc42 by titrating the GTPase. This result indicates that the Dbl activation of JNK is mediated through its activation of Cdc42, not RhoA. Minden et al. also detected activation of Jun (a JNK substrate) in cells expressing activated Rat and showed that Rat activation of JNK is blocked by kinasedefective mutants of either SEWMKK4 or MEKKl, which are expected to sequester their respective activators. These results suggest that Cdc42 and Racl can activate

Ras-Raf Interaction: Two-Hybrid Analysis

The identification of proteins mediating Ras effects, such as the serine/threonine kinases of the Raf family, has advanced our understanding of how signals are transmitted from the extracellular milieu to the nucleus. The modified two-hybrid system has proved to be a powerful tool for identifying specific protein interactions, such as those between Ras and Raf. We hope that the insight gained from the Ras screen, as well as insights from other two hybrid screens, will prove valuable in the application of this system to other enigmatic questions in biology.

p110δ, a Novel Phosphatidylinositol 3-Kinase Catalytic Subunit That Associates with p85 and Is Expressed Predominantly in Leukocytes

We have identified a novel p110 isoform of phosphatidylinositol 3-kinase from human leukocytes that we have termed p110δ. In addition, we have independently isolated p110δ from a mouse embryo library on the basis of its ability to interact with Ha-RasV12 in the yeast two-hybrid system. This unique isoform contains all of the conserved structural features characteristic of the p110 family. Recombinant p110δ phosphorylates phosphatidylinositol and coimmunoprecipitates with p85. However, in contrast to previously described p110 subunits, p110δ is expressed in a tissue-restricted fashion; it is expressed at high levels in lymphocytes and lymphoid tissues and may therefore play a role in phosphatidylinositol 3-kinase-mediated signaling in the immune system.

Simultaneous inhibition of GSK3α and GSK3β using hairpin siRNA expression vectors

Abstract Short interfering RNAs (siRNAs) can mediate sequence-specific inhibition of gene expression in mammalian cells. We and others have recently developed expression vector-based systems for synthesizing siRNAs or hairpin siRNAs in mammalian cells. Expression vector-based RNA interference (RNAi) effectively suppresses expression of target genes and is likely to be a powerful tool for analysis of gene function. Here we compare inhibition by vectors expressing hairpin siRNA designs either with different loop sequences connecting the two siRNA strands, or with duplex regions of different lengths. Our results suggest that lengthening the 19-nucleotide duplex region of a relatively ineffective hairpin siRNA can increase inhibition, but increasing the length of an effective 19-nt hairpin siRNA does not increase inhibition. We also demonstrate that hairpin siRNA vectors can be used to inhibit two target genes simultaneously. We have targeted glycogen synthase kinase-3α (GSK-3α) and GSK-3β, two related kinases involved in the regulation of a variety of cellular processes and also implicated in the pathogenesis of several human diseases. Inhibition of either GSK-3α or GSK-3β by transfection of hairpin siRNA vectors leads to elevated expression of the GSK-3 target β-catenin, whereas inhibition of both kinases further increases β-catenin expression. Our results suggest that vector-based siRNA inhibition may be useful for dissecting the functional roles of GSK-3α and GSK-3β in somatic cells. The ability to inhibit two or more genes simultaneously with hairpin siRNA expression vectors should facilitate studies of gene function in mammalian cells.

Akt Regulates Basic Helix-Loop-Helix Transcription Factor-Coactivator Complex Formation and Activity during Neuronal Differentiation

ABSTRACT Neural basic helix-loop-helix (bHLH) transcription factors regulate neurogenesis in vertebrates. Signaling by peptide growth factors also plays critical roles in regulating neuronal differentiation and survival. Many peptide growth factors activate phosphatidylinositol 3-kinase (PI3K) and subsequently the Akt kinases, raising the possibility that Akt may impact bHLH protein function during neurogenesis. Here we demonstrate that reducing expression of endogenous Akt1 and Akt2 by RNA interference (RNAi) reduces neuron generation in P19 cells transfected with a neural bHLH expression vector. The reduction in neuron generation from decreased Akt expression is not solely due to decreased cell survival, since addition of the caspase inhibitor z-VAD-FMK rescues cell death associated with loss of Akt function but does not restore neuron formation. This result indicates that Akt1 and Akt2 have additional functions during neuronal differentiation that are separable from neuronal survival. We show that activated Akt1 enhances complex formation between bHLH proteins and the transcriptional coactivator p300. Activated Akt1 also significantly augments the transcriptional activity of the bHLH protein neurogenin 3 in complex with the coactivators p300 or CBP. In addition, inhibition of endogenous Akt activity by the PI3K/Akt inhibitor LY294002 abolishes transcriptional cooperativity between the bHLH proteins and p300. We propose that Akt regulates the assembly and activity of bHLH-coactivator complexes to promote neuronal differentiation.

Akt2 Negatively Regulates Assembly of the POSH-MLK-JNK Signaling Complex

We demonstrate that POSH, a scaffold for the JNK signaling pathway, binds to Akt2. A POSH mutant that is unable to bind Akt2 (POSH W489A) exhibits enhanced-binding to MLK3, and this increase in binding is accompanied by increased activation of the JNK signaling pathway. In addition, we show that the association of MLK3 with POSH is increased upon inhibition of the endogenous phosphatidylinositol 3-kinase/Akt signaling pathway. Thus, the assembly of an active JNK signaling complex by POSH is negatively regulated by Akt2. Further, the level of Akt-phosphorylated MLK3 is reduced in cells expressing the Akt2 binding domain of POSH, which acts as a dominant interfering protein. Taken together, our results support a model in which Akt2 binds to a POSH-MLK-MKK-JNK complex and phosphorylates MLK3; phosphorylation of MLK3 by Akt2 results in the disassembly of the JNK complex bound to POSH and down-regulation of the JNK signaling pathway.

TC21 Causes Transformation by Raf-Independent Signaling Pathways

Although the Ras-related protein TC21/R-Ras2 has only 55% amino acid identity with Ras proteins, mutated forms of TC21 exhibit the same potent transforming activity as constitutively activated forms of Ras. Therefore, like Ras, TC21 may activate signaling pathways that control normal cell growth and differentiation. To address this possibility, we determined if regulators and effectors of Ras are also important for controlling TC21 activity. First, we determined that Ras guanine nucleotide exchange factors (SOS1 and RasGRF/CDC25) synergistically enhanced wild-type TC21 activity in vivo and that Ras GTPase-activating proteins (GAPs; p120-GAP and NF1-GAP) stimulated wild-type TC21 GTP hydrolysis in vitro. Thus, extracellular signals that activate Ras via SOS1 activation may cause coordinate activation of Ras and TC21. Second, we determined if Raf kinases were effectors for TC21 transformation. Unexpectedly, yeast two-hybrid binding analyses showed that although both Ras and TC21 could interact with the isolated Ras-binding domain of Raf-1, only Ras interacted with full-length Raf-1, A-Raf, or B-Raf. Consistent with this observation, we found that Ras- but not TC21-transformed NIH 3T3 cells possessed constitutively elevated Raf-1 and B-Raf kinase activity. Thus, Raf kinases are effectors for Ras, but not TC21, signaling and transformation. We conclude that common upstream signals cause activation of Ras and TC21, but activated TC21 controls cell growth via distinct Raf-independent downstream signaling pathways.