The p150-Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization.

The Jun N-terminal kinase (JNK) is a downstream effector of Rac and Cdc42 GTPases involved in actin reorganization [1-3]. A role of the Drosophila JNK homologue, Basket (DJNK/Bsk), in the regulation of cell shape changes and actin reorganization arises from its function in the process of dorsal closure [4-6]. One potential mechanism for induction of cytoskeletal changes by JNK is via transcriptional activation of the decapentaplegic gene (dpp, a member of the TGFbeta superfamily) [6]. A direct link between JNK signalling and actin organization has not yet been found, however. We have identified a novel DJNK-interacting protein, p150-Spir, that belongs to the Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2) family of proteins involved in actin reorganization [7] [8]. It is a multidomain protein with a cluster of four WH2 domains, a modified FYVE zinc-finger motif [9], and a DEJL motif, a docking site for JNK [10], at its carboxy-terminal end. In mouse fibroblasts, p150-Spir colocalized with F-actin and its overexpression induced clustering of filamentous actin around the nucleus. When coexpressed with p150-Spir in NIH 3T3 cells, JNK translocated to and colocalizes with p150-Spir at discrete spots around the nucleus. Carboxy-terminal sequences of p150-Spir were phosphorylated by JNK both in vitro and in vivo. We conclude that p150-Spir is a downstream target of JNK function and provides a direct link between JNK and actin organization.

The Spir actin organizers are involved in vesicle transport processes.

The p150-Spir protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spir proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spir encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spir family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as "Spir-box." The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of p150-Spir and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spir protein colocalized specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spir localization pattern was dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibited the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein was arrested in membrane structures, largely colocalizing with the TGN marker TGN46. Our findings that the Spir actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport.

Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1.

The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (deltaRaf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion mutant-estrogen receptor fusion protein (c-Raf-1-BxB-ER) (N-BxB-ER cells). The transformed morphology of these cells is dependent on the presence of the estrogen antagonist 4-hydroxytamoxifen. Addition of 4-hydroxytamoxifen to N-BxB-ER cells arrested by density or serum starvation causes reentry of these cells into cell proliferation. Increases in the cell number are obvious by 24 h after activation of the oncogenic c-Raf-1 protein in confluent cells. The onset of proliferation in serum-starved cells is further delayed and takes about 48 h. In both cases, the proliferative response of the oncogenic c-Raf-1-induced cell proliferation is weaker than the one mediated by serum and does not lead to exponential growth. This is reflected in a markedly lower expression of the late-S- and G2/M-phase-specific cyclin B protein and a slightly lower expression of the cyclin A protein being induced at the G1/S transition. Oncogenic activation of c-Raf-1 induces the expression of the heparin binding epidermal growth factor. The Jnk1 kinase is putatively activated by the action of the autocrine growth factor. The kinetics of Jnk1 kinase activity is delayed and occurs by a time when we also detect DNA synthesis and the expression of the S-phase-specific cyclin A protein. This finding indicates that oncogenic activation of the c-Raf-1 protein can trigger the entry into the cell cycle without the action of the autocrine growth factor loop. The activation of the c-Raf-1-BxB-ER protein leads to an accumulation of high levels of cyclin D1 protein and a repression of the p27Kip1 cyclin-dependent kinase inhibitor under all culture conditions tested.

High-intensity Raf signals convert mitotic cell cycling into cellular growth.

The selection of NIH 3T3 cells expressing a hydroxytamoxifen-inducible c-Raf-1-estrogen receptor fusion protein (c-Raf-1-BxB-ER) in the absence or presence of the inducer results in dramatic differences in the expression levels of the fusion protein. Hydroxytamoxifen-mediated constitutive activation of the Raf signal favors the selection of cells expressing low levels of c-Raf-1-BxB-ER. Cells selected in the absence of hydroxytamoxifen express up to 20 times higher levels of the inducible Raf kinase. Activation of the oncogenic Raf kinase in cells expressing low levels leads to a weak activation of the Raf/Mek/Erk cascade and the induction of S phase in confluent cells. The activation of cells expressing high levels of the kinase leads to a strong persistent signal and inhibits DNA synthesis and mitosis in proliferating cells. The inhibition of DNA synthesis and cell division is presumably due to the elevated expression of the cyclin-dependent kinase inhibitor p21cip1, similar to cells exposed to ionizing radiation. Despite the inhibition of DNA synthesis and mitosis, the constitutive activity of the Raf signaling pathway is still able to initiate cell growth. Activation of the high-intensity Raf signal in arrested serum-starved cells induces cell growth up to a size corresponding to that of M-phase cells in the absence of DNA synthesis. High-intensity Raf signals in proliferating cells consistently lead to an accumulation of cells with the size of M-phase cells and the DNA content of G1 cells or G2-M-phase cells. Therefore, the activation of Raf kinase is sufficient to drive cell growth, even in the presence of high levels of the cyclin-dependent kinase inhibitor p21cip1.

Myc protein structure: localization of DNA-binding and protein dimerization domains.

Wildtype and mutant v-Myc proteins were overexpressed in Escherichia coli using the T7 RNA polymerase system, and the in vitro DNA-binding activities of partially or highly purified proteins were analysed by native DNA-cellulose chromatography. For the construction of the expression plasmids, cloned proviral DNA from wildtype MC29 or from its spontaneous deletion mutant Q10C was used, the latter lacking internal v-myc sequences. Both the wildtype (p59) and the mutant (p42) recombinant protein contain at their amino termini 12 amino acids encoded by the vector, followed by 11 gag amino acids and 9 amino acids encoded by v-myc sequences derived from noncoding c-myc sequences. In addition, p59 contains 416 amino acids encoded by v-myc sequences derived from the complete chicken c-myc coding region, whereas p42 lacks 120 amino acids from the central region of the Myc protein including the highly acidic domain. Two additional proteins were engineered which contain the first 309 (p53) or the last 107 (p16) amino acids, respectively, of the Myc protein sequence in addition to vector-encoded amino acids. The p16 protein represents the carboxyl terminus of the Myc protein sequence containing both a muscle determination gene (MyoD1) homology region, including a basic motif and an amphipathic helix-loop-helix motif, and a leucine heptad repeat. All proteins, except p53 which lacks the carboxyl-terminal Myc protein sequences, bound to native DNA-cellulose and were eluted with 200-500 mM NaCl. Based on the DNA-binding activities of recombinant or spontaneous mutant v-Myc proteins extracted from bacterial or from transformed avian cells, we conclude that the DNA-binding domain of avian Myc proteins is confined within the last 86 carboxyl-terminal amino acids. The same region is also shown to be necessary and sufficient for Myc protein dimerization. This 86-amino acid region essentially encompasses a putative basic DNA contact surface and a tandem array of two presumed protein dimerization motifs, helix-loop-helix and leucine repeat.

Cell cycle targets of Ras/Raf signalling.

The regulation of cell proliferation in multicellular organisms is a complex process, which is primarily regulated by external growth factors provided by surrounding cells. Once induced to proliferate, the passage through the mitotic cell cycle is directed by the components of the so called cell cycle machinery. Over the last years research on growth factor signal-transduction and the components of the cell cycle system has lead to a detailed knowledge of the mechanisms by which growth factors transmit their signals and of the relationship between the components of the cell cycle machinery. The remaining question how the growth factor mediated signal-transduction cascades couple with the cell cycle regulators has recently been a focus of interest.

Regulation of c-myc expression by Ras/Raf signalling.

The c-myc gene is induced upon growth factor stimulation of arrested cells. The interaction of a mitogen with a transmembrane receptor triggers a variety of parallel signal transduction cascades. In order to analyse the role of the Ras/Raf cascade in the regulation of c-myc expression we have established fibroblast cell lines harboring conditional systems activating or inhibiting this pathway. Fusion of the c-Raf-1 kinase domain with the hormone binding domain of the estrogen receptor (c-Raf-1-BxB-ER) provides a 4-hydroxytamoxifen regulated form of the oncogenic c-Raf-1 kinase. We have generated NIH3T3 cells stably expressing the chimeric Raf protein (N-BxB-ER). 4-hydroxytamoxifen mediated activation of the fusion protein in serum starved N-BxB-ER induces the expression of the c-myc gene within 2-6 h. Deletion of the c-Raf-1 kinase domain generates a mutant c-Raf-1 protein (c-Raf-1-C4B), which can directly interact with the effector domain of the Ras protein and thereby block Ras mediated signalling. We have established a NIH3T3 based cell line expressing the c-Raf-1-C4B protein under the control of a tetracycline responsive promoter (N-C4B-tet). Serum starved cells expressing the c-Raf-1-C4B protein exhibit a significantly reduced induction of c-myc expression following serum stimulation compared to the same cells not expressing the Ras inhibitor. The induction of c-myc mRNA following the activation of the isolated Raf/Mek/Erk cascade in addition to the partial inhibition of serum mediated induction of c-myc expression in the presence of the Ras inactivating c-Raf-1-C4B mutant strongly indicates an involvement of the Ras/Raf pathway in the regulation of c-myc expression.

Second cancer risk following Hodgkin's disease: a 20-year follow-up study.

PURPOSE: To determine risk factors for the development of second primary cancers during long-term follow-up of patients with Hodgkin's disease (HD). PATIENTS AND METHODS: We assessed the risk of second cancers (SCs) in 1939 HD patients, who were admitted to the Netherlands Cancer Institute (NKI; Amsterdam) or the Dr Daniel den Hoed Cancer Center (DDHK; Rotterdam) between 1966 and 1986. For 97% of the cohort, we obtained a medical status up to at least January 1989. The median follow-up duration of the patients was 9.2 years; for 17% of the patients, follow-up was longer than 15 years. For more than 98% of all second tumors, the diagnosis was confirmed through a pathology report. RESULTS: In all, 146 patients developed a SC, compared with 41.9 cases expected on the basis of incidence rates in the general population (relative risk [RR], 3.5; 95% confidence interval [CI], 2.9 to 4.1). The mean 20-year actuarial risk of all SCs was 20% (95% CI, 17% to 24%). Significantly increased RRs were observed for leukemia (RR, 34.7; 95% CI, 23.6 to 49.3), non-Hodgkin's lymphoma (NHL) (RR, 20.6; 95% CI, 13.1 to 30.9), lung cancer (RR, 3.7; 95% CI, 2.5 to 5.3), all gastrointestinal cancers combined (RR, 2.0; 95% CI, 1.2 to 3.1), all urogenital cancers combined (RR, 2.4; 95% CI, 1.4 to 3.7), melanoma (RR, 4.9; 95% CI, 1.6 to 11.3), and soft tissue sarcoma (RR, 8.8; 95% CI, 1.8 to 25.8). As compared with the general population, the cohort experienced an excess of 63 cancer cases per 10,000 person-years. Cox-model analysis indicated the following as significant risk factors for developing leukemia: first-year treatment with chemotherapy (CT), follow-up treatment with CT, age at diagnosis of HD greater than 40 years, splenectomy, and advanced stage. Patients treated with CT in the 1980s had a substantially lower risk of leukemia than patients treated in the 1970s (10-year actuarial risks of 2.1% and 6.4%, respectively; P = .07). Significant risk factors for NHL were older age, male sex, and combined modality treatment as compared with either modality alone. Risk of lung cancer was strongly related to radiotherapy (RT), while an additional role of CT could not be demonstrated. After more than 15 years of follow-up, women treated with mantle-field irradiation before age 20 years had a greater than forty-fold increased risk of breast cancer (P < .001). CONCLUSION: While the long-term consequences of HD treatment as administered in the 1960s and 1970s are still evolving, it is promising that patients who received the new treatment regimens introduced in the 1980s have a much lower leukemia risk than patients treated in earlier years. Beginning 10 years after initial RT, the follow-up program of women who received mantle-field irradiation before age 30 years should routinely include breast palpation and yearly mammography.

Second cancer risk following testicular cancer: a follow-up study of 1,909 patients.

PURPOSE: Improved survival in testicular cancer has been accompanied by concern about long-term side effects of therapy. We assessed the evolution of second cancer (SC) risk over a prolonged follow-up period, which has been rarely studied in large patient series. PATIENTS AND METHODS: We estimated the risk of SCs in 1,909 patients with testicular cancer diagnosed in the Netherlands from 1971 to 1985. Complete medical information was obtained up to at least January 1988 for 92% of patients. Median follow-up was 7.7 years. For 89% of second tumors the diagnosis was confirmed through review of histologic slides; for an additional 8%, the diagnosis was verified by pathology reports only. RESULTS: Seventy-eight patients developed a SC 1 year or more after start of treatment, as compared with 47.6 expected on the basis of incidence rates in the general population (relative risk [RR], 1.6; 95% confidence interval [CI], 1.3 to 2.1). The mean 15-year actuarial risk of all SCs was 9.8% (95% CI, 7.5% to 12.8%). Significantly increased RRs were observed for all gastrointestinal cancers combined (RR, 2.6; 95% CI, 1.7 to 3.9), stomach cancer (RR, 3.7; 95% CI, 1.8 to 6.8), contralateral testicular cancer (CLTC) (RR, 35.7; 95% CI, 21.8 to 55.2), and leukemia (RR, 5.1; 95% CI, 1.4 to 13.0). Patients who had received irradiation to the paraaortic lymph nodes and who survived testicular cancer for more than 5 years were at particularly high risk of developing stomach cancer (RR, 6.9; 95% CI, 3.3 to 12.7). The median interval between the diagnosis of testicular cancer and stomach cancer was 12.4 years. Patients treated with chemotherapy (CT) did not experience an increase in SCs in general. Indeed, CT-treated patients, as compared with those who received radiotherapy (RT), or surgery alone, had significantly reduced risk of CLTC. This finding might be attributed to an eradicating effect of CT on carcinoma in situ or subclinical CLTC. The excess risk of leukemia was not found to be clearly related to CT. CONCLUSION: Testicular cancer patients who receive RT experience elevated risk of gastrointestinal tumors. CT does not seem to increase SC risk and may even decrease the risk of a CLTC. Following testicular cancer, the 15-year actuarial risk of all SCs is only about half the risk experienced by patients with Hodgkin's disease.

Leukemia risk following Hodgkin's disease: relation to cumulative dose of alkylating agents, treatment with teniposide combinations, number of episodes of chemotherapy, and bone marrow damage.

PURPOSE: The development of leukemia is one of the most serious long-term complications of modern treatment for Hodgkin's disease (HD). This study was undertaken to examine the relation between risk of leukemia and various treatment factors (including cumulative dose of cytostatic drugs and interaction with radiotherapy [RT]), while also assessing the effect of treatment-induced bone marrow damage. PATIENTS AND METHODS: We conducted a case-control study in a cohort of 1,939 patients treated for HD between 1966 and 1986 in the Netherlands. Detailed information from the medical records was obtained for 44 cases of leukemia and 124 matched controls, in whom leukemia had not developed. RESULTS: The cumulative dose of mechlorethamine was the most important factor in determining leukemia risk. As compared with patients who received RT alone, patients treated with six or fewer cycles of combinations including nitrogen mustard (mechlorethamine) and procarbazine had an eightfold increased risk of developing leukemia (P = .08), while patients who received more than six of such cycles had a greater than 40-fold excess risk (P < .001). Treatment with lomustine or a combination of teniposide and cyclophosphamide also significantly increased the risk of leukemia. Patients who had received chemotherapy (CT) during two or more time periods had a nearly 40-fold increased risk of leukemia as compared with patients treated only once. The extent of RT did not further increase leukemia risk among patients who also received CT. A significantly increased risk of leukemia was found among patients with low platelet counts, both in response to initial therapy and during follow-up. Patients who experienced 2 or more half-year periods with platelet counts less than 75 x 10(6)/mL had an approximately fivefold risk of developing leukemia, and a similar risk increase was found for patients who responded to initial treatment with a > or = 70% decrease of platelet counts (as compared with patients who had a < or = 50% decrease). CONCLUSION: In addition to mechlorethamine, lomustine and teniposide combinations were also linked to an elevated risk of developing leukemia. Since the number of CT episodes was found to be a strong determinant of leukemia risk, it is important that new therapies for HD continue to yield high initial cure rates. Further studies are warranted to investigate whether patients at high risk for developing leukemia may be identified from the response of their platelets to initial therapy for HD.

Cyclin D2 and Ha-Ras transformed rat embryo fibroblasts exhibit a novel deregulation of cell size control and early S phase arrest in low serum.

The D-type cyclins are growth factor-regulated delayed early functions which peak at the G1/S transition, are thought to regulate entry into S phase and have been implicated in tumorigenesis. Here, we show that cyclin D2 can co-operate with Ha-Ras to impose a novel transformed state on rat embryo fibroblasts (REF). While clonal cyclin D2/Ha-Ras REF transformants exhibit a characteristic transformed phenotype in high serum, in low serum they arrest cell proliferation and display profound morphological and cytological changes indicating loss of control of cell mass and deregulation of the G1/S transition. Notably, in low serum, despite re-establishment of actin cables and arrest of proliferation, cell mass continues to increase, creating giant cells up to 10 x normal size. Also, during low-serum culture the cells make a very gradual but progressive entry into S phase, reaching a 2.4N DNA content after 6 days. PCNA is expressed and 2N and 4N cells are largely absent, and thus the cells undergo a novel S phase arrest. While transfer to low serum induced the retinoblastoma protein to enter its dephosphorylated state, and cyclin A, cyclin B and cdc2 levels to decrease, all as normal, cyclin E, cdk4, cdk2 and the exogenous cyclin D2 persisted at high levels. These results indicate that cyclin D2 and Ha-Ras can transform cells when mitogenic signals from growth factors are provided. However, in low serum, co-operation of cyclin D2 and Ha-Ras provides only a subset of the progression signals and these are sufficient for G1-related cell mass increase and S phase entry, but are insufficient for full cell cycling.

Deregulated messenger RNA expression during T cell apoptosis.

The IL-2 dependent murine cytotoxic T cell line CTLL-2 undergoes programmed cell death when deprived of its specific cytokine. We analyzed the expression of cell cycle related genes after IL-2 deprivation. Here we show that a generalized decrease and re elevation of the levels of mRNA takes place as part of the apoptotic program. The levels of several mRNAs encoding cell cycle functions, including cyclin D2, cyclin D3, cyclin B1, c-myc and max all declined at 1.5-3 h following IL-2 deprivation. Notably, the maxmRNA, which was shown to be expressed in proliferating, growth arrested and differentiated cells, is down regulated with the same kinetics as the other mRNAs. Surprisingly, the mRNAs whose levels declined at 1.5-3 h rose again at 10-14 h, a time which closely followed the time of the first detection of apoptotic DNA degradation, at 8 h, but which precedes actual loss of viability, at 14 h, as measured by trypan blue exclusion. Of all analyzed genes only the expression of the S-phase specific histone H4 gene resists the initial decrease and declines gradually over the course of cell death. Measurement of c-Myc protein synthesis at a late stage of the apoptotic program revealed that the accumulated reinduced mRNA is not translated into protein. Because transcriptional regulation has been shown to be dependent on the chromatin structure, the reinduction may be triggered by relaxation of the chromatin caused by alterations in the chromatin structure of apoptotic cells.

Sequence-specific DNA binding by Myc proteins.

Myc proteins have a tripartite carboxyl-terminal domain containing specific amino acid sequence motifs: a basic motif, a helix-loop-helix motif, and a leucine heptad repeat. Similar sequence motifs have been identified in several eukaryotic transcription factors and were shown to facilitate protein-DNA and protein-protein interactions. By using recombinant v-Myc proteins obtained by bacterial expression of full-length or partially deleted avian v-myc alleles, the functional relevance of these sequence motifs for Myc protein oligomerization and for DNA binding was investigated. All recombinant v-Myc proteins that have retained the carboxyl-terminal domain dimerize and specifically bind to double-stranded DNA containing the palindromic core sequence CACGTG. This and a closely related DNA sequence element have been defined previously as part of the binding sites for human transcription factors USF and TFE3, which specifically bind to the adenovirus major late promoter or the muE3 motif within the immunoglobulin heavy-chain enhancer, respectively. It is shown that a 61-amino-acid peptide sequence containing only the bipartite basic motif/helix-loop-helix domain of Myc is necessary and sufficient for dimerization and sequence-specific DNA binding of v-Myc recombinant proteins.

Lung-targeted expression of the c-Raf-1 kinase in transgenic mice exposes a novel oncogenic character of the wild-type protein.

The c-Raf-1 kinase is a downstream effector of Ras signaling. Both proteins are highly oncogenic when they are mutationally activated, but only the Ras GTPase is frequently mutated in naturally occurring tumors. Although the c-Raf-1 protein was found to be amplified in different lung cancer cell lines, overexpression of the wild-type c-Raf-1 protein was shown to be insufficient to transform cultured cells. Here we have addressed the question of whether overexpression of the wild-type c-Raf-1 kinase can induce lung cancer in mice. We show that lung-targeted expression of oncogenically activated or wild-type c-Raf-1 proteins induces morphologically indistinguishable lung adenomas in transgenic mice. Compared with mice transgenic for the activated c-Raf-1-BxB, tumor development is delayed and occurs at a lower incidence in wild-type c-Raf-1 transgenic mice. Our studies show that the c-Raf-1 expression level is a critical parameter in tumor development and should be analyzed in more detail to evaluate its potential in the induction of cancer.

Influence of age and comorbidity on treatment choice and survival in elderly patients with breast cancer.

To determine the effect of age and comorbid diseases on treatment choice and survival, the medical records of 300 breast cancer patients of 55 years and older were reviewed. All patients were admitted to the Netherlands Cancer Institute (NKI) for first treatment between 1980 and 1987. Patients were classified according to severity level of comorbid diseases. Physicians were found to treat women of 75 years and older less often with adjuvant radiotherapy after a mastectomy, and more often to employ only primary endocrine treatment for local stage disease, as compared with younger patients. According to the treatment guidelines of the institute, the study sample was divided into patients who received standard vs. non-standard treatment. The treatment of 38 women (13.1%) did not correspond with the guidelines. Of these, 84% were 75 years and older and 50% had a severe comorbidity status. Logistic regression analysis indicated that advanced age, per se, was a better indicator of the risk of not being treated according to protocol than the comorbidity status. Cox multivariate analyses demonstrated that neither the severity of the comorbidity status nor the differences in treatment between younger and older patients had a significant effect on the risk of dying from breast cancer or on the risk of developing recurrences. In this analysis, age 75 years or more proved to be a significant and independent predictor of a worse overall and disease-specific survival as compared to age between 65-74 years.

Cloning of the breakpoints of a deletion associated with choroidermia.

In order to characterize a previously described submicroscopic deletion encompassing (part of) the choroideremia (tapetochoroidal dystrophy: TCD) gene, we have cloned a 10.5-kb EcoRI fragment from the patient's DNA; this fragment carries the junction between both deletion endpoints ("junction fragment"). The distal portion of this fragment defines a new marker within, or just distal to, the TCD gene. This marker has been employed to confirm the diagnosis in several affected family members, and to rule out carriership in a female at risk with conspicuous clinical signs.

Ral and Rho-dependent activation of phospholipase D in v-Raf-transformed cells.

Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals. We reported previously that although the transformed phenotype induced by v-Src was dependent upon Raf-1, the PLD activity induced by v-Src was independent of Raf-1. This observation suggested to us that Raf would not likely be an activator of PLD. However, upon examination of PLD activity in v-Raf-transformed cells, surprisingly, we found that PLD activity is elevated to levels that were even higher than that observed in v-Src-transformed cells. To characterize the mechanism of v-Raf-induced PLD activity, we examined the dependence of v-Raf-induced PLD activity upon protein kinase C (PKC) the small GTPases Ral and Rho, which have all been implicated in the activation of PLD. The v-Raf-induced PLD activity was inhibited by dominant negative mutants for both Ral and Rho. The dependence upon Ral was particularly surprising since Ral is a downstream target of Ras, which is an upstream activator of Raf. Depleting cells of PKC by long term phorbol ester treatment actually increased PLD activity in v-Raf-transformed cells, indicating that v-Raf-induced PLD activity is not dependent on PKC. These data describe a novel mechanism for PLD activation by v-Raf that is independent of PKC, but dependent upon both Ral and Rho GTPases.