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M McMahon

Publications and source records attributed to M McMahon.

At least 73 records · Page 4Linked to original sources
PubMed1998

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors.

Adenovirus E1A Proteins↗
PubMed1998

Comprehensive nutrition plan improves cardiovascular risk factors in essential hypertension.

Increased arterial pressure is known to be influenced by a variety of nutrients. Compliance with dietary recommendations for risk reduction is often limited by the complexity of their implementation. In addition, how improvements in total diet, rather than single nutrients, influence concomitant cardiovascular risk factors has not been thoroughly explored. We assessed the effects of a nutritionally complete prepared meal program, the Campbell's Center for Nutrition and Wellness plan (CCNW), compared with dietary therapy in which participants received a structured nutritional assessment and prescription and selected their own foods, in 101 women and men with mild-to-moderate hypertension. Outcome measures included blood pressure (BP), lipids and lipoproteins, glucose, glycosylated hemoglobin (HbA1c), insulin, homocysteine, nutrient intake, compliance, and quality of life. Both dietary interventions significantly lowered BP (P < .0001), while simultaneously improving the overall cardiovascular risk profile. Significantly greater benefits were observed with the CCNW plan as compared with the participant selected diet in cholesterol and LDL levels (both P < .0001), LDL:HDL (P < .001), HbA1c (P < .05), homocysteine (P < .001), total nutrient intake (P < .0001), compliance (P < .0001), and quality of life (P < .001). This study demonstrates that improving the total diet to include the full array of recommended dietary guidelines, rather than focusing on single nutrients, has significant benefits for the cardiovascular risk profile of hypertensive persons beyond BP control. Compared with typical dietary therapy, the comprehensive CCNW meal plan has significantly greater effects on multiple cardiovascular risk factors while yielding greater compliance and improved quality of life.

Adult↗
PubMed1998

Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells.

Raf is a key serine-threonine protein kinase which participates in the transmission of growth, anti-apoptotic and differentiation messages. These signals can be initiated after receptor ligation and are transmitted to members of the MAP kinase cascade that subsequently activate transcription factors controlling gene expression. Raf is a member of a multigene family which includes: Raf-1, A-Raf and B-Raf. The roles that individual Raf kinases play in the regulation of normal and malignant hematopoietic cell growth are not clear. The following studies show that all three Raf kinases are functionally present in certain human hematopoietic cells, and their aberrant expression can result in abrogation of cytokine dependency. Cytokine-dependent TF-1 cells were infected with retroviruses encoding amino-terminal deleted (delta) A-Raf, B-Raf and Raf-1 proteins. These Raf proteins were conditionally inducible as they were fused to the hormone-binding domain of the estrogen receptor (ER). A hierarchy in the abilities of Raf-containing retroviruses to abrogate cytokine dependency was observed as deltaA-Raf:ER was 20- to 200-fold more efficient than either deltaRaf-1:ER or deltaB-Raf:ER, respectively. This result was unexpected as A-Raf is an intrinsically weaker kinase than either Raf-1 or B-Raf. The activated Raf proteins induced downstream MEK and MAP (ERK1 and ERK2) kinase activities in the cells which proliferated in response to Raf activation. Furthermore, a functional MEK signaling pathway was necessary as treatment of the cells with a MEK1-inhibitor suppressed Raf-mediated proliferation. To determine whether the regulatory phosphorylation residues contained in the modified Raf oncoproteins were necessary for transformation, they were altered by site-directed mutagenesis. Substitution of the regulatory phosphorylation tyrosine residues with phenylalanine in either A-Raf or Raf-1 reduced the capacity of these oncoproteins to abrogate cytokine dependency. In contrast, changing the critical aspartic acid residues of B-Raf to either tyrosine or phenylalanine increased the frequency of estradiol-responsive cells. Thus, the amino acids present in the regulatory residues modulated the capability of Raf proteins to abrogate the cytokine dependency of TF-1 cells. Differences in the levels of Raf and downstream kinase activities were observed between cytokine-dependent and estradiol-responsive deltaRaf:ER-infected cells as estradiol-responsive cells usually expressed more Raf and MEK activity than GM-CSF-dependent, deltaRaf:ER-infected cells. Abrogation of cytokine dependency by the activated deltaRaf:ER proteins was associated with autocrine growth factor synthesis which was sufficient to promote the growth of uninfected TF-1 cells. In summary, these observations indicate that the aberrant expression of certain activated deltaRaf:ER oncoproteins can alter the cytokine dependency of human hematopoietic TF-1 cells. These cells will be useful in evaluating the roles of the individual Raf oncoproteins in signal transduction, cell cycle progression, autocrine transformation, regulation of apoptosis and differentiation. Moreover, these Raf-infected cells may be important in evaluating the efficacy of novel anticancer drugs designed to inhibit Raf and downstream signal transduction molecules.

Apoptosis↗
PubMed1998

Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation.

STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.

Animals↗
PubMed1998

Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating factor 1/c-fms signaling.

An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.

3T3 Cells↗
PubMed1997

Oncogenic raf-1 induces the expression of non-histone chromosomal architectural protein HMGI-C via a p44/p42 mitogen-activated protein kinase-dependent pathway in salivary epithelial cells.

The enzyme activity of mitogen-activated protein kinase (MAP kinase) increases in response to agents acting on a variety of cell surface receptors, including receptors linked to heterotrimeric G proteins. In this report, we demonstrated that Raf-1 protein kinase activity in the mouse parotid glands was induced by chronic isoproterenol administration in whole animals. To investigate the molecular nature underlying cellular responses to Raf-1 activation, we have stably transfected rat salivary epithelial Pa-4 cells with human Raf-1-estrogen receptor fusion gene (DeltaRaf-1:ER) and used mRNA differential display in search of messages induced by DeltaRaf-1:ER activation. Through this approach, the gene encoding non-histone chromosomal protein HMGI-C was identified as one of the target genes activated by oncogenic Raf-1 kinase. Activation of Raf-1 kinase resulted in a delayed and sustained increase of HMGI-C expression in the Pa-4 cells. The induction of HMGI-C mRNA level is sensitive to both the protein synthesis inhibitor cycloheximide and transcription inhibitor actinomycin D. The role of the extracellular signal-related kinase (ERK) signaling pathway in the HMGI-C induction was highlighted by the result that the MAP kinase kinase (MEK) inhibitor, PD 98059, blocked DeltaRaf-1:ER- and 12-O-tetradecanoylphorbol-13-acetate-stimulated HMGI-C induction. Altogether, these findings support the notion that the Raf/MEK/ERK signaling module, at least in part, regulates transcriptional activation of the chromosomal architectural protein HMGI-C.

Amino Acid Sequence↗
PubMed1997

Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1.

The catalytic domains of the Raf family of protein kinases (deltaRaf) differ in their ability to activate MEK in vitro and in vivo and in their ability to oncogenically transform mammalian cells. The kinase domain of B-Raf is more active than the equivalent portion of Raf-1 which in turn is more active than A-Raf. In Raf-1 the phosphorylation or mutation to aspartic acid of two key tyrosine residues upstream of the ATP binding site has been demonstrated to significantly potentiate catalytic activity. In A-Raf the analogous amino acids are also tyrosine whereas in B-Raf they are aspartic acid. To determine if these differences in amino acid sequence influence the relative catalytic activity of the Raf kinase domains we constructed forms of deltaA-Raf, deltaB-Raf and deltaRaf-1 that encode either aspartic acid [DD], phenylalanine [FF] or tyrosine [YY] at these positions. These proteins were expressed both in mammalian cells as fusions with the hormone binding domain of the estrogen receptor and as epitope-tagged proteins in Sf9 insect cells to test their oncogenic and catalytic potentials. When expressed in Rat1 or 3T3 cells in the presence of hormone all of the deltaRaf-1:ER and deltaA-Raf:ER proteins were transforming with the exception of the [FF] form of deltaA-Raf. In general the [DD] forms of the deltaRaf-1:ER and deltaA-Raf:ER proteins were the most potently oncogenic which correlated with their ability to elicit activation of the MAP kinase pathway. Consistent with the transformation data, the catalytic activity of the [DD] forms of deltaA-Raf:ER and deltaRaf-1:ER was about ten times greater than the cognate [FF] and [YY] forms of the proteins. By contrast all of the deltaB-Raf:ER proteins were highly transforming and deltaB-Raf catalytic activity was largely unaffected by mutation of the aforementioned aspartic acids to either tyrosine or phenylalanine. Similar results were obtained with epitope-tagged forms of deltaA-Raf, deltaB-Raf and deltaRaf-1 expressed in Sf9 cells. These data provide support for the model that key tyrosine residues in the protein kinase domains of A-Raf and Raf-1 are important in the regulation of catalytic activity. In addition they demonstrate that the higher intrinsic activity of B-Raf cannot be explained simply by the presence of aspartic acids at the analogous positions.

Amino Acids↗
PubMed1997

Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca2+-dependent signal pathways in Rat-1 cells.

Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate had little effect on the early peak of ERK1 activity but potentiated the sustained phase. Cycloheximide also potentiated ERK1 activation in Rat-1 cells expressing DeltaRaf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-1. Since cycloheximide did not potentiate MEK activity but abrogated the expression of mitogen-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, we speculated that the level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells. Inhibition of LPA-stimulated MEK and ERK activation with PD98059 and pertussis toxin, a selective inhibitor of Gi-protein-coupled signaling pathways, reduced LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1 expression. Specific activation of the MEK/ERK pathway by DeltaRaf-1:ER had little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells. Activation of PKC played little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression was blocked by buffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity. In Rat-1DeltaRaf-1:ER cells, we observed a strong synergy of MKP-1 expression when cells were stimulated with estradiol in the presence of ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under conditions where these agents did not synergize for ERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERK-dependent and -independent signals may regulate MKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression.

Animals↗
PubMed1997

Cooperating oncogenes converge to regulate cyclin/cdk complexes.

The cooperation of oncogenes in the transformation of primary rat Schwann cells is a strikingly synergistic process. We have explored the molecular mechanisms involved. Activation of an inducible Raf kinase results in morphologically transformed cells that are arrested in G1 via the induction of p21(CiP1) and subsequent inhibition of cyclin/cdk activity. In contrast, coexpression of SV40 large T (LT) or a dominant-negative mutant of p53 abolishes p21(CiP1) induction and alleviates the growth arrest. Moreover in this scenario, Raf activation results in an increase in the specific activity of cyclin/cdk complexes with Raf and LT cooperating to superinduce cyclin A/cdk2 activity and stimulate proliferation in the absence of mitogens. Thus, signaling by Raf and its cooperating partners converges at the regulation of cyclin/cdk complexes, with the cellular responses to Raf modulated by p53.

Animals↗
PubMed1997

Nutritional management of cardiovascular risk factors. A randomized clinical trial.

BACKGROUND: Adherence to dietary recommendations for disease management is often hindered by the complexity of incorporating them into the daily diet. Nutrition and cardiovascular scientists and food technologists collaborated to develop a prepared meal plan that meets national dietary guidelines for cardiovascular risk reduction. OBJECTIVE: To assess the clinical effects of this plan, which incorporates all National Academy of Sciences National Research Council recommended dietary allowances for vitamins, minerals, and macronutrients, compared with a patient-selected American Heart Association Step I and Step II diet plan. METHODS: This multicenter, randomized, parallel-intervention trial was conducted at 10 medical centers in the United States and Canada and involved 560 men and women with hypertension, dyslipidemia, or diabetes. Following calculation of prescriptions to meet individual nutritional requirements based on the Harris-Benedict equation, participants were randomized to the Campbell's Center for Nutrition and Wellness (CCNW) plan, which is composed of prepackaged breakfast, lunch, and dinner meals provided to participants, or a nutritionist-guided American Heart Association Step I and Step II diet, in which participants self-selected foods to meet their nutrition prescription for 10 weeks. MAIN OUTCOME MEASURES: Blood pressure (BP); lipid, glucose, glycosylated hemoglobin (HbA1c), and insulin levels; body weight; dietary intake; and quality of life. RESULTS: Patients' BP, lipid levels, carbohydrate metabolism, weight, and quality of life (P < or = .001 for all findings except low-density lipoprotein-high-density lipoprotein ratio, P = .25) all improved on both nutrition plans. Mean differences (+/-SD) between baseline and treatment clinical values for the CCNW and the self-selected diet groups (between-group P values), respectively, were as follows: systolic BP, -6.4 +/- 9.2 mm Hg and -4.6 +/- 9.0 mm Hg (P = .02); diastolic BP, -4.2 +/- 5.7 mm Hg and -3.0 +/- 5.1 mm Hg (P = .006); cholesterol, -0.32 +/- 0.58 mmol/L and -0.27 +/- 0.56 mmol/L (-12.4 +/- 22.5 mg/dL and -10.4 +/- 21.9 mg/dL) (P = .30); glucose, -0.65 +/- 1.88 mmol/L and -0.75 +/- 2.03 mmol/L (-11.7 +/- 34.0 mg/dL and -13.5 +/- 36.6 mg/dL) (P = .10); and HbA1c, -0.4% +/- 0.8% and -0.3% +/- 0.7% (P = .66). Weight loss with the CCNW and self-selected plans, respectively, was as follows: men, -4.5 +/- 3.6 kg and -3.5 +/- 3.3 kg; and women, -4.8 +/- 3.0 kg and -2.8 +/- 2.8 kg. Quality of life was significantly improved for daily and work activities (P < .05) and nutritional health perceptions (P < .05) with the CCNW plan relative to the self-selected group. Overall nutrient intake and compliance were both significantly (P < .001) better with the CCNW plan. CONCLUSIONS: Nutritionally balanced meals that meet the recommendations of national health organizations improved multiple risk factors for patients with cardiovascular disease. The CCNW plan resulted in greater clinical benefits, nutritional completeness, and compliance than the self-selected diet. The CCNW is a comprehensive nutrition plan, convenient for both prescription and practice, and appears viable for effecting favorable dietary changes in patients at high risk for cardiovascular disease.

Adult↗
PubMed1997

Dietary compliance and cardiovascular risk reduction with a prepared meal plan compared with a self-selected diet.

Noncompliance with therapeutic diets remains a major obstacle to achieving improvements in cardiovascular disease (CVD) morbidity and mortality. This study compared dietary compliance and CVD risk factor response to two dietary interventions designed to treat hypertension, dyslipidemia, and diabetes mellitus. In a multicenter trial, 560 adults were randomly assigned to either a self-selected, mixed-food plan (n = 277), or a nutrient-fortified prepared meal plan (n = 283); each was designed to provide 15-20% of energy from fat, 55-60% from carbohydrate, and 15-20% from protein. Nutrient intake was estimated from 3-d food records collected biweekly throughout the 10-wk intervention. Compliance was determined by evaluating the participants' ability to meet specific criteria for energy intake [+/-420 kJ (100 kcal) from the midpoint of the prescribed energy range], fat intake (< 20%, < 25%, or < 30% of energy from total fat), and the National Cholesterol Education Program/American Heart Association Step 1 and 2 diet recommendations. Compliance with energy, fat, and Step 1 and 2 criteria was better in participants who followed the prepared meal plan than in those who followed the self-selected diet (P < 0.0001). Compliant participants in both groups achieved greater reductions in body weight, systolic and diastolic blood pressure, and total and low-density-lipoprotein cholesterol than noncompliant participants (P < 0.05). In general, better endpoint responses were observed with lower fat intakes regardless of group assignment. The prepared meal plan is a simple and effective strategy for meeting the many nutrient recommendations for CVD risk reduction and improving dietary compliance and CVD endpoints.

Adult↗
PubMed1997

Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1.

Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Ras and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of deltaRaf-1:ER, a conditionally active form of oncogenic human Raf-1. Promoter activation by deltaRaf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp -974 and -988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following deltaRaf-1:ER activation. Cotransfection of Ets-2 and p44 mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to deltaRaf-1:ER. Potentiated activation required both p44 MAP kinase catalytic activity and threonine 72 in the Pointed domain of Ets-2. Biochemical assays demonstrated the ability of the p42 and p44 MAP kinases to phosphorylate Ets-2 on threonine 72. Importantly, in intact cells, the kinetics of phosphorylation of Ets-2 on this residue closely mirror the activation of the p42 and p44 MAP kinases and the observed onset of HB-EGF gene transcription following deltaRaf-1:ER activation. These data firmly establish Ets-2 as a direct target of the Raf-MEK-MAP kinase signaling pathway and strongly implicate Ets-2 in the regulation of HB-EGF gene expression.

3T3 Cells↗
PubMed1997

Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.

The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.

3T3 Cells↗
PubMed1996

Role of the Raf/mitogen-activated protein kinase pathway in p21ras desensitization.

Desensitization of p21(ras) after stimulation of cells by growth factors and phorbol 12-myristate 13-acetate (PMA) correlates with hyperphosphorylation of the guanine nucleotide exchange factor Son-of-sevenless (Sos) and its dissociation from the adaptor protein Grb2 (Cherniack, A., Klarlund, J. K., Conway, B. R., and Czech, M. P. (1995) J. Biol. Chem. 270, 1485-1488). To test the role of the Raf/mitogen-activated protein (MAP) kinase pathway, we utilized cells expressing a chimera composed of the catalytic domain of p74Raf-1 and the hormone binding domain of the estradiol receptor (DeltaRaf-1:ER). Estradiol markedly stimulated DeltaRaf-1:ER and the downstream MEK and MAP kinases in these cells as well as Sos phosphorylation. However, the dissociation of Grb2 from Sos observed in response to PMA was not apparent upon DeltaRaf-1:ER activation. Furthermore, stimulation of DeltaRaf-1:ER did not impair GTP loading of p21(ras) in response to platelet-derived growth factor or epidermal growth factor. We conclude that activation of the Raf/MAP kinase pathway alone in these cells is insufficient to cause disassembly of Sos from Grb2 or to interrupt the ability of Sos to catalyze activation of p21(ras).

3T3 Cells↗
PubMed1996

Oncogenic Raf-1 activates p70 S6 kinase via a mitogen-activated protein kinase-independent pathway.

Cell proliferation requires the co-ordinate triggering of several protein kinases of Ser/Thr specificity such as p70 S6 kinase (S6K), which phosphorylates the ribosomal S6 protein and thus increases translation of mRNAs with polypyrimidine tracts. The multiplicity of signaling pathways leading to p70 S6K activation are not fully elucidated. However, several reports have indicated that the activation of p70 S6K is independent of mitogen-activated protein kinase (MAPK) activation. Interestingly, we and others have shown that constitutive activation of the MAPK pathway promotes cell proliferation, suggesting that this cascade is able to activate p70 S6K, a key step to trigger cell cycle entry. In this report we demonstrate that transfection of constitutively active mitogen-activated protein kinase kinase 1 in CCL 39 cells leads to activation of p70 S6K. Furthermore, we have established a cell line that stably expresses DeltaRaf-1:ER, an estradiol-regulated form of oncogenic Raf-1. The addition of estradiol to these cells was sufficient to elicit rapid activation of mitogen-activated protein kinase kinase 1, MAPK, and p70 S6K. Surprisingly, the activation of p70 S6K is not mediated by MAPK because blocking MAPK activation by expression of the phosphatase MKP-1 did not prevent p70 S6K activation by DeltaRaf-1:ER. In conclusion, we have demonstrated that activation of p70 S6K by DeltaRaf-1:ER is mediated by a new MAPK-independent pathway. This pathway is resistant to low nanomolar concentrations of wortmannin, indicating that it does not involve membrane-bound phosphatidylinositol-trisphosphate kinase activation.

Animals↗