The kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase, resulting in phosphorylation of ternary complex factor and Jun by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKC alpha-mediated Raf-1 activation in NIH3T3 fibroblasts. PKC alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKC alpha induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKC alpha. Consistent with such a direct interaction is the observation that Raf-1 and PKC alpha cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.

Raf-1 serine-threonine protein kinase has the hallmarks of a critical switch that connects growth factor receptor activation at the cell membrane with transcriptional events in the nucleus. We show by use of Raf-1 dominant-negative mutants that Raf-1 is required for serum-, TPA-, and Ras-induced expression from the oncogene-responsive element in the polyomavirus enhancer. The minimal region of Raf-1 that displays this dominant-negative phenotype (Raf-C4] contains a cysteine finger motif. Raf-C4 appears to function by titrating out a Raf-1-activating factor that is induced by Ras following serum or TPA treatment of NIH-3T3 cells. In addition, we show that Raf-1 and Ras cooperate in trans-activation through the oncogene-responsive element and that the cysteine-rich region is necessary for this effect.

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.

Many growth factors regulate the cytoplasmic Raf-1 protein kinase, consistent with its having a central role in transduction of growth signals. The kinase is ubiquitously expressed and can promote proliferation, presumably in a manner dependent on growth-factor receptors and membrane-associated oncogenes. We have now examined the dependence of serum- and TPA (12-O-tetradecanoylphorbol-13-acetate)-regulated NIH/3T3 cell growth on RAF-1 kinase to determine whether Raf-1 is essential for receptor signalling. We inhibited Raf-1 function by expressing c-raf-1 antisense RNA or kinase-defective c-raf-1 mutants. Antisense RNA for c-raf-1 interferes with proliferation of normal NIH/3T3 cells and reverts raf-transformed cells. In revertant cells, DNA replication induced by serum or TPA was eliminated or reduced proportionately to the reduction in Raf protein levels. Expression of a kinase-defective Raf-1 mutant (craf301) or a regulatory domain fragment (HCR) inhibited serum-induced NIH/3T3-cell proliferation and raf transformation even more efficiently. Inhibition by antisense RNA or craf301 blocked proliferation and transformation by Ki- and Ha-ras oncogenes. We conclude that raf functions as an essential signal transducer downstream of serum growth factor receptors, protein kinase C and ras.

In resting cells, c-Jun is phosphorylated on five sites. Three of these sites reside next to its DNA binding domain and negatively regulate DNA binding. In response to expression of oncogenic Ha-Ras, phosphorylation of these sites decreases, while phosphorylation of two other sites within c-Jun's activation domain is greatly enhanced. Phosphorylation of these residues, serines 63 and 73, stimulates the transactivation function of c-Jun and is required for oncogenic cooperation with Ha-Ras. We now show that the same changes in c-Jun phosphorylation are elicited by a variety of transforming oncoproteins with distinct biochemical activities. These oncoproteins, v-Sis, v-Src, Ha-Ras, and Raf-1, participate in a signal transduction pathway that leads to increased phosphorylation of serines 63 and 73 on c-Jun. While oncogenic Ha-Ras is a constitutive stimulator of c-Jun activity and phosphorylation, the normal c-Ha-Ras protein is a serum-dependent modulator of c-Jun's activity. c-Jun is therefore a downstream target for a phosphorylation cascade involved in cell proliferation and transformation.

We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.

Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV.

A series of wild-type and mutant rafgenes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine-and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.The raffamily of proto-oncogenes consists of three active members: A-raf-1, B-raf, and c-raf-1 (2,4,5,20,22,23). The three proteins show the greatest stretch of homology in the carboxy-terminal half, which, in the case of c-raf-1, has been shown to have serine-and threonine-specific protein kinase activity (33, 48). Physiologically, Raf protein kinases function as information shuttles that communicate between the cell surface and nucleus. From Ki-ras revertant and anti-ras antibody microinjection studies, it is known that c-raf-1 acts independently of and perhaps downstream from Ras in signal transduction (2,22,36,41,42,52). In addition, we recently showed that Raf is hyperphosphorylated and enzymatically activated in cells which are transformed by src, fms, or ras, and in cells which have been treated with platelet-derived growth factor or 12-0-tetradecanoylphorbol-13-acetate (TPA) (35). In response to platelet-derived growth factor and TPA, Raf protein is also translocated from the cytoplasm to the perinuclear space, as is evident from immune fluorescence and cell fractionation studies (44). Ultimately, rafis thought to exert its effect by modulation of transcription factor activity via phosphorylation (42). Evidence for this comes from experiments in which cells transfected with activated raf genes showed increased transcriptional activity from an APl-dependent promoter in transient transfection assays, suggesting that Raf is involved in the regulation of transcription factors of the jun-AP1 gene family (56). Raf function is essential for normal rates of cell proliferation, as demonstrated by the lethal effect of mutations in the Drosophila melanogaster D-raf-1 locus (37).c-raf-1, the most-studied member of the family, encodes a * Corresponding auth...