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Annals of Clinical & Laboratory Science 38:47-56 (2008)
© 2008 Association of Clinical Scientists

Site-Specific Phosphorylation of raf in Cells Containing Oncogenic ras-p21 Is Likely Mediated by jun-N-Terminal Kinase

Victor Adler1,2, Wilbur Bowne3,4, Josef Michl1,5, Kelley A. Sookraj3,4, Kamran Ikram3,4, Sidney Pestka6, Lara Izotova6, Michael Zenilman3, Fred K. Friedman7, Yongxia Qu1,2 and Matthew R. Pincus1,2
1 Department of Pathology, SUNY Downstate Medical Center, and 2 Department of Pathology & Laboratory Medicine, New York Harbor VA Medical Center, Brooklyn, NY; 3 Department of Surgery, SUNY Downstate Medical Center, and 4 Department of Surgery, New York Harbor VA Medical Center, Brooklyn, NY; 5 Departments of Microbiology, Anatomy, and Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY; 6 Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School, Piscataway, NJ; and 7 National Institutes of Health, Bethesda, MD

Address correspondence to Matthew R. Pincus, M.D., Ph.D., New York Harbor VA Medical Center, 800 Poly Place, Brooklyn, NY 11209, USA; tel 718 630 3688; fax 718 630 2960; e-mail: matthew.pincus2{at}med.va.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In a study of interactions between the raf-MEK-MAPK (ERK) and JNK-jun pathways, we found previously that JNK can induce phosphorylation of raf but not vice versa. In this study, we investigate the nature of the JNK-induced phosphorylation of raf. In in vitro experiments in which immunobead-bound raf is phosphorylated by activated JNK, we find strong phosphorylation signals at raf-Ser259 and Ser338. The Ser259 phosphorylation is surprising since it is associated with inhibition of migration of raf to the cell membrane where it can interact with ras-p21. We also find that in oocytes induced to mature with oncogenic ras-p21, which induces high levels of phosphorylated JNK and MAPK, the same pattern of phosphorylation of raf occurs. In contrast, in oocytes induced to mature with insulin, which requires activation of wild-type ras-p21, phosphorylation of raf-Ser338 but not raf-Ser259 occurs. In oncogenic ras-transformed human pancreatic cancer MIA-PaCa-2 cells, phosphorylation of both raf serines occurs. Treatment of these cells with the ras peptide, PNC-2 attached to a penetratin sequence that blocks JNK and MAPK phosphorylation and induces tumor cell necrosis, results in a marked decrease in phosphorylation of raf-Ser259, but not that of raf-Ser338. These results suggest that oncogenic ras-p21 induces phosphorylation of both raf-Ser259 and Ser338 and that raf-Ser 259 phosphorylation may be effected by activated JNK.

Abbreviations: MEK, mitogen (extracellular)-activated kinase, the direct activator of MAPK; MAPK, mitogen-activated protein kinase, also called ERK; TOPK, Lymphokine-Activated Killer T-Cell-Originated Protein Kinase (TOPK); DYRK1A, Dual-Specificity-Tyrosine-Phosphorylation-Regulated Kinase-1A; PNC-2, Ha-ras peptide 96-110; PNC-7, Ha-ras peptide, 35–47; leader peptide or penetratin, trans-membrane-penetrating peptide; PNC-28, peptide containing human p53 sequence 17–26 linked to penetratin sequence; JNK, jun-N-terminal kinase; PKC, protein kinase C.

Keywords: oncogenic ras-p21, raf, phosphorylation, raf-Ser259, raf-Ser338, JNK


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oncogenic and wild-type ras-p21 induce mitogenic signaling through overlapping but distinct pathways [1,2]. Injection of oncogenic (Val 12-containing) ras-p21 into Xenopus laevis oocytes induces oocyte maturation by a pathway that requires its interaction with jun-N-terminal kinase (JNK) and its substrate, jun, the nuclear transcription factor [1–4]. Furthermore, oncogenic ras-p21 requires activation of the raf-MEK-MAP kinase (MAPK) pathway to induce oocyte maturation [5]. These findings contrast with the mode of action of activated wild-type ras-p21 [1,2].

Insulin induces oocyte maturation [6] in a manner that requires activation of endogenous wild-type ras-p21 [7]. Inhibitors of the oncogenic ras-p21-JNK/jun interactions and of JNK-induced activation of jun that block oncogenic ras-p21-induced oocyte maturation have only a minimal effect on insulin-induced maturation [1,2]. Levels of JNK and of MAPK phosphorylation in oocytes induced to mature by injection of oncogenic ras-p21 are much higher than in oocytes that are induced to mature with insulin [5]. These results suggested that activated wild-type ras-p21 does not require activation of JNK and is not completely dependent on the raf-MEK-MAPK pathway in mitogenic signaling [1,2,5]. Since both oncogenic and insulin-activated wild-type ras-p21 require raf on their signal transduction pathways [1,2], and since a major action of raf is activation of the MEK-MAPK phosphorylation cascade, activated wild-type ras-p21 may interact with raf in a manner distinct from that of oncogenic ras-p21 since the two proteins have markedly different effects on MEK and MAPK activations.

In our investigations of the signal transduction pathways activated by oncogenic ras-p21, we found that there are interactions between the raf-MEK-MAPK and JNK-jun pathways [8]. In a recent study, we prepared immuno-beads of raf, MEK, and MAPK, isolated from U-251 human astrocytoma cells and incubated each of these with activated JNK [9]. Conversely, we incubated immuno-beads of JNK with activated forms of raf, MEK, and MAPK. We observed that whereas activated JNK induced phosphorylation of raf, the reverse phosphorylation of JNK by activated raf did not occur [9]. Furthermore, activated MEK induced JNK phosphorylation while activated JNK did not induce MEK phosphorylation [9]. In contrast to these results, no cross-phosphorylations between MAPK and JNK were found to occur. These results suggested that raf, MEK, and JNK are involved in a positive feed-back loop [9].

A surprising finding in the above studies was the direct phosphorylation of raf by activated JNK. Several phosphorylation sites have been identified in raf. In raf-1, phosphorylations of Ser338 and of Tyr340 and Tyr341 appear to be required for activation [10]. Phosphorylation of raf-Ser259 also has been found to be important in regulation of its localization [10]. Phosphorylation of raf-Ser259 enables binding of raf to the scaffolding 14-3-3 protein in the cytosol, which may inhibit its migration to the cell membrane [10].

In this report, we have undertaken to determine if JNK induces phosphorylation of raf at either of the two critical Ser residues, Ser338 and Ser259, to explore whether JNK induces a "signature" phosphorylation of raf that distinguishes it from phosphorylations induced by other kinases both in oocytes and in ras-transformed human tumor cells. In addition, since wild-type and oncogenic ras-p21 appear to interact with raf differently, we have examined the phosphorylation patterns at these two raf sites in oocytes induced to mature with insulin and with microinjected oncogenic ras-p21. Our purpose is to determine if there are differences in these patterns that result from differing interactions of the wild-type and oncogenic proteins with raf.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Proteins.  Val 12-Ha-ras-p21 protein was overexpressed in E. coli using the pGH-L9 expression vector containing the chemically synthesized Ha-ras gene, as previously described [11]. Activated JNK was purchased from Biomol (Plymouth, PA). This JNK has the same activity as that obtained from lysis of anisomycin-treated U-251 cells and eluted from anti-JNK immuno-beads as described [9]. Insulin was purchased from Sigma (St. Louis, MO) and used directly. TPA (12-O-tetradecanoyl-phorbol 13-Acetate) was purchased from Sigma and used directly.

Blocking Peptides.  To demonstrate specificity of antibodies to two different phosphorylated forms of raf, ie, phospho-Ser 259 and phospho-Ser 338, we utilized two phosphopeptides containing the sequences of amino acid residues around each of these two sites. These peptides are: Raf-Ser 259: Pro-Arg-Gly-Ser-Pro-Ser-Pro-Ala-Ser-Val-Ser Raf-ser 338: Gly-Gln-Arg-Asp-Ser-Ser-Tyr-Tyr-Glu-Ile-Ala The phosphorylated Ser residues are highlighted in bold italics for each sequence respectively. These peptides were provided by Santa Cruz Biotechnology (Santa Cruz, CA) (phospho-raf-1 peptide phosphorylated at the Ser residue corresponding to Ser 259 in raf-1) and Cell Signaling Technology (Danvers, MA) (phospho-raf-1 peptide phosphorylated at the Ser residue corresponding to Ser 338 in raf-1).

Antibodies.  Anti-Raf and anti-JNK polyclonal antibodies were purchased from Calbiochem (San Diego, CA). Antibody to phospho-raf-1-Ser259 was purchased from Santa Cruz Biotechnology; antibody to phospho-raf-1-Ser338 was purchased from Cell Signaling Technology.

Oocytes.  Xenopus laevis female frogs (Connecticut Valley Biological [Southhampton, MA]) were obtained as described previously [1,5–9] from collagenase-digested ovarian follicles that were then incubated in Barth’s medium at 19°C for 12–24 hr [1,5–9].

Human Tumor Cell Lines.  MIAPaCa-2 (human pancreatic cancer) cell lines were from American Type Culture Collection (ATCC) (Manassas, VA); U-251 (human astrocytoma) cells were a gift of Dr. D. Weinstin (GliaMed, NY).

Buffers.  Oocyte (Group VI) lysis buffer [5] consisted of 80 mM beta-glycerophosphate, 20 mM EGTA, 20 mM HEPES, pH 7.5, 1 mM PMSF, 2 µg/ml pepstatin, 1 mM leupeptin, 2 µg/ml aprotinin, 1 mM Na3VO4, and 1% Triton X-100. Lysis buffer for cells, used to lyse MIAPaCa-2 and U-251 cells, consisted of 0.35 M LiCl, 50 mM HEPES, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol (DDT), 2 mM MgCl2, 50 mM NPP (nitrophenyl phosphate phosphatase inhibitor), 1 mM sodium vanadate, and an inhibitor "cocktail" consisting of 1 µg/ml each of the protease inhibitors, pepstatin, leupeptin, and aprotinin, and the phosphatase inhibitors, 1 mM sodium orthovanadate, and 5 mM sodium. Kinase buffer, used for phosphorylation reactions, consisted of 50 mM Tris-HCI, pH-7.5; 50 mM LiCl; 10% glycerol; 1 mM-EGTA; 1 mM-DTT; 0.5% NP-40 (non-ionic detergent); and 0.5% Triton X-100.

Oocyte Microinjection and Incubation.  Oocytes were micro-injected with Val 12-p21 (100 µg/ml) and incubated in Barth’s medium (Specialty Media, Lavellette, NJ) or were incubated in Barth’s medium to which insulin was then added so that its final concentration was 10 µg/ml. All incubations were performed at 19°C. Oocyte maturation was determined by observing germinal vesicle breakdown (GVBD) [1,5–9] over a 36 hr period. All experiments were performed in duplicate on at least 100 oocytes.

Cell Cultures.  MIAPaCa-2 and U-251 cells were grown in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with heat inactivated 10% calf or fetal calf serum, 20 mM glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml) and plated on a 6-well plate with density no more then 20000 cells/well in 3 ml of culture medium.

Western Blotting for raf Phosphorylation Sites in MIA-PaCa Cells and in Oocytes.  The same procedure that we previously reported for U-251 cells [9] was applied to MIA-PaCa-2 cells. These cells (1.7 x 109 cells) were washed extensively with washing buffer (20mM Tris/1mM EGTA/0.15M NaCl, pH 7.5). Cells were harvested with a rubber policeman and were resuspended in 9 vol of buffer containing 20mM HEPES pH 7.4, 100 mM LiCl, 1 mM EGTA, 0.5% NP-40, 0.5% Triton X-100e, 1 mM DTT, 50 mM nitro-phenyl-phosphate, and 1% protease inhibitors mixture (Sigma). After homogenization (Dounce, 50 strokes), the cell suspension was incubated 10 min in ice and centrifuged at 12000 x g for 5 min. The proteins (40 µg) were electrophoresed in 12.5% PAAG gel (Bio-Rad), transferred on PVDF membrane, and subjected to immunobloting with primary anti-raf and anti-raf phospho-specific (Ser259 or Ser338) polyclonal anti-rabbit antibody, each at a 1:1000 dilution. The membrane was then incubated for 1 hr at room temperature with 50 ml of goat anti-rabbit secondary antibody (Pierce Supersignal West Pico Chemiluminescent kit) that was diluted 1:25,000 in TBS-T buffer. The membrane was again washed 4 times for 10 min each with 50 ml of TBS-T, and the signals were developed with enhancer (per manufacturer’s instructions) on x-ray film.

For oocytes, we followed the procedure described in a prior study [5]. Briefly, between 100–200 oocytes matured with Val 12-ras-p21 or with insulin were lysed in Group VI oocyte lysis buffer described above, following a procedure we described previously [5]. Samples of lysates containing constant amounts of protein (either 50 or 75 µg) were subjected to SDS PAGE on a 12% resolving gel and the proteins then electrophoretically transferred onto nitrocellulose membranes overnight at 4°C; the membranes were then blocked with non-fat dry milk in Tris-buffered saline with 1% Tween-20 (TBS-T) (pH 7.6) and then incubated with the appropriate anti-raf, anti-phospho-raf-Ser259 or anti-phos-pho-raf-Ser338 antibody (1:1000 dilution for each antibody). All incubations were performed for 12 hr at 4°C, after which the membranes were washed 3 times with TBS-T and incubated with anti-rabbit secondary antibody (Amersham, Piscataway, NJ) at 1:4000 dilution. Detection was performed with the ECL chemiluminescence detection kit (Amersham).

Testing Antibody Specificity.  The above experiments were repeated in the presence of one or the other of the two blocking peptides containing the amino acid sequences at the phosphorylation sites for raf Ser259 and raf Ser338. In these experiments, MIA-PaCa-2 cells were incubated overnight with 12.5 µg/ml TPA to induce high levels of raf phosphorylation especially at Ser 259 [12]. The cells were then lysed as described in the preceding section. However, each of the two primary phospho-specific antibodies was diluted 1:1000 with each peptide (1.0 ug/ml). The blotting procedure was as described in the preceding section.

Immunobeads Containing Unactivated raf from U-251 Cells.  Immunoaffinity beads containing bound raf were prepared as described in our previous paper [9]. Briefly, confluent U-251 cells (1 x 106) were lysed in cell lysis buffer (see above) and the lysate was then incubated with primary anti-raf antibody (0.1 µg of antibody for every 500 µg of total protein), followed by addition of 1 µl of 1 µg/ul biotinylated anti-IgG (Pierce, Rockford, IL). To this mixture, 25 µl of streptavidin beads (Pierce) was added, and the mixture was incubated overnight at 4°C, washed, and used in the kinase assay with JNK.

JNK-Induced Phosphorylation of Bead-Bound raf was achieved as described in our prior paper [9]. Immunoaffinity bead-bound raf was incubated with activated JNK. The beads were then centrifuged and washed with kinase buffer, subjected to SDS PAGE, and transferred to nitrocellulose membranes which were then blotted with anti-raf or anti-raf-phospho-serine-259 or anti-raf-phospho-serine-338 (diluted 1:2000 in TBS-T, containing 0.25% BSA) using a protocol identical to that used previously to detect phospho-raf [9]. The membrane was incubated with goat anti-rabbit secondary antibody (Pierce) and developed as described previously [9].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antibody Specificity.  To confirm the specificity of each of the two primary antibodies to phosphorylated raf-Ser259 and raf-Ser338, we utilized the original phosphopeptides used to develop each antibody as blocking peptides. For example, we incubated the anti-Ser259 antibody with the raf-Ser259 phosphopeptide and then blotted whole MIA-PaCa-2 cell lysate with this antibody; in addition, we incubated the cell lysate with the anti-raf-Ser259 antibody in the presence of the raf-Ser338 phosphopeptide as a control. The results are shown in Fig. 1Go. Lane 1 shows the blot for cell lysate from untreated cells with antibody in the absence of peptide. Lane 2 shows the same blot except for cells treated with TPA, a phorbol ester known to induce activation of PKC that, in turn, induces phosphorylation of raf Ser259. As can be seen in this figure, the level of raf-phospho-Ser259 is higher, as expected. Lane 3, the result of the negative control, shows that the presence of raf-phospho-Ser338 peptide does not affect the level of immune complex with the anti-raf-phospho-Ser259 antibody (compare lanes 2 and 3). On the other hand, if the anti-raf-phospho-Ser259 antibody is incubated with the raf-phospho-Ser259 peptide, the blot of the cell lysate, shown in lane 4, shows that there is marked diminution of the immune complex. These results suggest that the anti-raf-phospho-Ser259 antibody is specific and does not cross-react with raf-phospho-Ser338. We obtained similar results (not shown) in which we found that raf-phospho-Ser259 peptide had no effect on the amount of immune complex formed when the anti-raf-phospho-Ser338 antibody was used to blot whole cell lysate. On the other hand, the raf-phospho-Ser338 peptide caused a marked diminution of the immune complex when the anti-phospho-raf-Ser338 antibody was used to blot the cell lysate. These results together confirm that the antibodies are specific and do not cross-react.


Figure 1
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  		Fig. 1. Effects of the two raf phosphopeptides on the level of immune complex formed in blots of MIA-PaCa-2 cell lysates with anti-raf-phospho-Ser 259 antibody. Lane 1, blot of cell lysate from cells untreated with TPA with antibody alone; lane 2, same as lane 1 except cells were treated with TPA; lane 3, same as lane 2 except antibody incubated with raf-phospho-Ser 338 peptide; lane 4, same as lane 3, except antibody incubated with raf-phospho-Ser 259 peptide.

 
Phosphorylation of Immuno-bead-Bound raf by Activated JNK.  In our prior paper [9], we showed that JNK induces high levels of phosphorylation of raf. We blotted raf beads that had been incubated with activated JNK and ATP with phospho-specific antibodies against phospho-raf-Ser259 and phos-pho-raf-Ser338. As shown in Fig. 2Go, activated JNK induces elevated levels of phosphorylation of both Ser338 and Ser259. This finding suggests that JNK can induce phosphorylation at both sites of raf intracellularly. To determine if this occurs and if this is a unique feature of JNK-induced phosphorylation, we turned to the oocyte system.


Figure 2
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  		Fig. 2. Cell-free phosphorylation of immuno-bead-bound raf by activated JNK. Lane 1 shows results for untreated immunobeads of raf blotted for phospho-raf-Ser259 (A), phospho-raf-Ser338 (B), and total raf (C). Lane 2 shows results for immunobeads of raf incubated with JNK + ATP [9] for phospho-raf-Ser259 (A), phospho-raf-Ser338 (B), and total raf (C).

 
raf Phosphorylation Pattern in Oocytes.  In oocytes, oncogenic ras-p21 induces high levels of JNK while insulin induces only low levels of JNK phosphorylation [5]. Therefore, we compared the phosphorylation pattern of raf in oocytes that were induced to mature with oncogenic ras-p21 with that in oocytes induced to mature with insulin. Fig. 3Go shows that in oocytes induced to mature with oncogenic ras-p21 (Lane 3, B and C), both Ser259 and Ser338 are phosphorylated. In contrast, in oocytes induced to mature with insulin, there is a strong raf-Ser338 phosphorylation signal and significantly less intense raf-Ser259 phosphorylation signal (lane 2, B and C). This finding suggests that oncogenic and activated wild-type ras-p21 proteins induce different phosphorylation patterns on raf, a critical target for both proteins [8]. Since oncogenic ras-p21, but not insulin, induces high levels of JNK phosphorylation, our finding a strong phosphorylation signal on raf-Ser259 only in the oocytes matured with oncogenic ras-p21 is consistent with JNK-induced phosphorylation of this serine in raf.


Figure 3
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  		Fig. 3. Blots for phospho-raf-Ser259 and phospho-raf-Ser338 in oocytes induced to mature with oncogenic Val 12-ras-p21 or insulin. In the figure, lane 1 is for blots of untreated oocytes; lane 2, for oocytes induced to mature with insulin; and lane 3, for oocytes induced to mature with microinjected Val 12-ras-p21. (A) blots for total raf; (B) blots for phospho-raf-Ser259; (C) blots for phospho-raf-Ser338. Total raf is relatively constant in all three lanes and serves as an internal loading control.

 
raf Phosphorylation Pattern in a ras-Transformed Human Pancreatic Cancer MIA-PaCa-2 Cell Line.  To determine if oncogenic ras induces the same pattern of raf phosphorylation in ras-transformed human cancer cells, we blotted lysates of MIA-PaCa-2 cells with anti-raf-Ser259 and raf-Ser338 antibodies. As shown in Fig. 4Go, the pattern of phosphorylation of raf in these cells parallels the in vitro results in Fig. 2Go. As can be seen in Fig. 4Go, there are significant phosphorylation signals at raf-Ser259 and raf-Ser338 (lane 1, B and D, respectively) in untreated MIA-PaCa-2 cells. (The blot for phospho-raf-Ser338 yields a double band, the lower band being a degradation product.) This pattern is consistent with our finding in oocytes, ie, oncogenic ras induces strong phosphorylation of both serines in raf. The raf phosphorylation pattern in this cell line is consistent with JNK-induced phosphorylation of raf.


Figure 4
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  		Fig. 4. Blots for phospho-raf forms of lysates of ras-transformed MIA-PaCa-2 human pancreatic cancer cells. In each data set, labeled A–E, lane 1 is for untreated MIA-PaCa-2 cells; lane 2 is for MIAPaCa-2 cells treated with a control peptide, called PNC-29, that does not affect cell growth (see [13] and [14]); lane 3 is for MIAPaCa-2 cells treated for 48 hr with PNC 2-leader anti-ras peptide. (A) Blots for total raf; (B) blots for phospho-raf-Ser259; (C) blots for total JNK; (D) blots for phospho-raf-Ser338; and (E) blots for phospho-JNK. The data shown in (C) serve as an internal loading control showing that total JNK remains constant for all 3 conditions, ie, lanes 1–3.

 
As discussed elsewhere [1,2,13,14], there are two ras peptides, corresponding to ras-p21 residues 35-47 (PNC-7) and 96-110 (PNC-2), both of which block oncogenic ras-p21-, but not insulin-, induced oocyte maturation. We have found that PNC-2 specifically blocks the interaction of oncogenic ras-p21 with JNK [3,4]. We have found that each peptide blocks JNK phosphorylation in oocytes. We have attached both peptides to a cell membrane-traversing penetratin sequence from Antennapedia [14], called "leader," and have incubated these peptides with MIA-PaCa-2 cells [13]. We find that both peptides induce tumor cell necrosis of these cells [13; Adler et al, Cancer Chemother Pharmacol, in press].

We have therefore blotted lysates of these cells, after treatment of these cells with PNC-2-leader, with the anti-phospho-raf antibodies. As shown in Fig. 4Go, lanes 1 in frames B and D show significant expression of phospho-raf-Ser259 and phospho-raf-Ser338, respectively, in untreated MIA-PaCa-2 cells. Lanes 3 in frames B and D show that phosphorylation of raf-Ser259 (frame B) is significantly reduced by treatment of the cells with PNC-2-leader peptide, while there appears to be no effect on phosphorylation of raf-Ser338 (frame D). As shown in lanes 2 of frames B and D, treatment of the cells with the negative control PNC-29 peptide [13,14] results in no change in either phosphorylation signal, supporting the specificity of the effect of the PNC-2-leader peptide.

Treatment of these cells with PNC-2-leader peptide likewise diminishes the level of phospho-JNK [13]. As shown in Fig. 4Go, lane 1 of frame E shows significant levels of phospho-JNK in untreated cells. However, as shown in lane 3 of frame E, PNC-2-leader peptide strongly diminishes the level of phospho-JNK. This effect is specific to PNC-2-leader peptide since the negative control PNC-29 peptide has no effect on the phospho-JNK level (lane 2, frame E). As can be seen in frame C, blots for total JNK, the levels are similar for all three lanes and are unaffected by treatment of the cells with either peptide.

The result that JNK phosphorylation is strongly reduced with a concomittant and selective reduction in raf-Ser259 phosphorylation is compatible with the in vitro result, shown in Fig. 1Go, that JNK induces phosphorylation of raf-Ser259.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Oncogenic ras-p21 Appears To Induce raf Phosphorylation at Ser259.  Our results with the oocyte system and ras-transformed MIA-PaCa-2 cells suggest that oncogenic ras-p21 induces phosphorylation of ras at both Ser259 and Ser338. In contrast to this pattern, in the oocyte system, insulin-activated wild-type ras-p21 induces phosphorylation of raf Ser338 but not Ser259 (Fig. 3Go). This result suggests that oncogenic ras-p21 activates raf in a manner that is distinct from that of the wild-type protein.

Raf as a Branchpoint on the Signal Transduction Pathways of Oncogenic and Activated Wild-Type ras-p21.  In previous studies in oocytes, we found that raf is an essential target for both oncogenic and activated wild-type ras-p21 [8]. Since raf is known to activate both MEK and MAPK, we expected elevated levels of these proteins in oocytes induced to mature with both oncogenic and wild-type ras-p21 proteins. However, surprisingly, activation of MEK and MAPK was much higher in oocytes induced to mature with oncogenic ras-p21 than with insulin-activated wild-type ras-p21 [5,9]. This finding suggested that activated wild-type ras-p21 could interact with raf in a manner so as to activate alternate downstream pathways. This conclusion was corroborated by several previous findings, namely that: (A) MAP kinase phosphatase completely blocks oncogenic ras-p21-induced maturation while it only partially blocks insulin-induced maturation [8], implying alternate downstream pathway activation; and that (B) the anti-ras peptide, PNC-7 [1,2,14], that interacts with the amino terminal regulatory domain of raf [15], blocks oncogenic ras-p21- but not insulin-induced oocyte maturation. This peptide strongly blocks MAPK phosphorylation [1,2,5,13], suggesting that the raf-activated MEK-MAPK pathway is essential to oncogenic, but not activated wild-type, ras-p21.

Taken together, these results imply that oncogenic and activated wild-type ras-p21 interact with raf in different manners such that each protein activates different downstream pathways. When oncogenic ras-p21 interacts with raf, there is a strong activation of the MEK-MAPK pathway. When wild-type ras-p21 interacts with raf, there is much weaker activation of this pathway. Recently, we have identified several dual specificity kinases [16,17], ie, Lymphokine-Activated Killer T-Cell-Originated Protein Kinase (TOPK), a direct raf target [18], and dual-specificity tyrosine-phosphorylated and regulated kinase 1A (DYRK1A) that lie downstream of raf and that appear to be essential to signal transduction by insulin-activated wild-type ras-p21 in oocytes [16,17].

Possible Pathways Activated by Oncogenic and Wild-Type ras-p21 Downstream of raf.  Since the different interactions of each ras-p21 protein with raf give rise to different raf phosphorylation patterns and result in the activation of different downstream pathways, we propose the scheme shown in Fig. 5Go to account for the above observations. In this scheme, oncogenic ras-p21 is shown to interact with raf giving rise to phosphorylations at Ser259 and Ser338. This combination results in activated raf that stimulates the MEK-MAPK pathway. In contrast, insulin-activated wild-type ras-p21 interacts with raf so as to induce phosphorylation at Ser 338 resulting in some activation of the MEK-MAPK pathway but also resulting predominantly in activation of an alternate pathway involving TOPK and DYRK1A and possibly hitherto unidentified pathway elements.


Figure 5
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  		Fig. 5. Scheme summarizing the overlapping but distinct signal transduction pathways induced by oncogenic (left, ras-p21*) and activated wild-type (right) ras-p21 protein. The figure shows that both proteins interact with raf but give rise to different phosphorylation patterns, the oncogenic protein inducing phosphorylation of both raf-Ser259 and Ser338 and the activated wild-type protein inducing phosphorylation of raf-Ser338 but not Ser259. The oncogenic ras pathway shown in this figure is based on the results discussed in the Discussion section and in [9]. Plus signs for the oncogenic ras-p21 signal transduction pathway in the figure represent positive stimulation on a circular feedback pathway involving raf, MEK, and JNK [9]. The signal transduction elements shown for wild-type ras-p21 are based on the results in [21] and [22]. For wild-type ras-p21, the thick arrow represents the major pathway that is postulated to be activated by raf while the thinner arrow represents a less major pathway (MEK-MAPK) that is also activated by raf.

 
Also shown in Fig. 5Go is a second unique feature of the oncogenic ras-p21-induced signal transduction pathway. Based on previous results in which we found that activated JNK induces raf phosphorylation and that activated MEK induces JNK phosphorylation [9]. On the basis of these findings, we proposed that there is a cyclic positive feedback loop involving JNK, raf, and MEK [9]. A major implication of this scheme is that it may be possible to inhibit oncogenic ras-p21 interactions with raf and/or activation of the MEK-MAPK pathway without interfering with the normal functioning of wild-type ras-p21. This conclusion is corroborated by our findings that PNC-7 peptide, that blocks oncogenic ras-p21-raf interactions and diminishes MEK and MAPK activation, has only a minimal effect on insulin-activated wild-type ras-p21. This suggests an approach for the design of selective anti-cancer agents.

Possible Role of JNK in Oncogenic ras-Induced raf Ser259 Phosphorylation.  We have found previously in oocytes that oncogenic, but not insulin-activated wild-type ras-p21, interacts with JNK. In this study, we now find that activated JNK induces phosphorylation of immuno-bead-bound raf Ser259 in the cell-free system obtained from U-251 cells (Fig. 2Go). Since oncogenic ras-p21 induces JNK activation, we surmise that the oncogenic ras-activated JNK phosphorylates raf at Ser259. This conclusion is supported in this study with oocytes induced to mature with oncogenic ras-p21. Oncogenic ras induces high levels of phosphorylated JNK in these oocytes and results comcomittantly in high levels of raf-phospho-Ser259 (Fig. 3Go). In ras-transformed MIA-PaCa-2 pancreatic cancer cells, which express high levels of phospho-JNK (Fig. 4Go), we have found high levels of phospho-raf-Ser259 (Fig. 4Go). These levels decrease markedly in the presence of the peptide, PNC2-leader, that blocks the oncogenic ras-p21-JNK interaction and JNK phosphorylation [1,2], while the level of phospho-raf-Ser338 remains constant (Fig. 4Go). The fact that PNC-2-leader blocks raf Ser259 but not Ser338 phosphorylation suggests that the kinase activity of JNK (and/or some associated kinases) on raf is specific for raf-Ser 259.

On the other hand, a number of other kinases, including protein kinase C (PKC) and Akt, have been implicated in inducing phosphorylation of raf Ser259. Additionally, phorbol ester that induces PKC activation likewise is known to induce phosphorylation of raf-Ser259 [12]. We previously found that PKC is an important downstream target of oncogenic but not insulin-activated wild-type ras-p21 [19]. Therefore, kinases other than JNK may be responsible for inducing phosphorylation of raf Ser259 either uniquely or in conjunction with JNK. However, our findings discussed above, especially that purified activated JNK induces strong phosphorylation of raf Ser259 in a cell-free system, point to JNK as a major participant in induction of this phosphorylation.

Role of raf Phosphorylated at Ser259.  Our results showing that oncogenic ras-p21 and JNK (at least in the cell-free system) induce raf Ser259 phosphorylation are surprising in view of several studies that suggest that phosphorylation of raf-Ser259 results in diminished stimulation of the MEK-MAPK pathway [10,20–22]. These studies suggest that replacement of Ser259 by Ala results in enhanced activation of the MEK-MAPK pathway. Phosphorylation of Ser259 is thought to result in the binding of raf to the 14-3-3 cytosolic scaffolding protein that may inhibit its ability to migrate to the cell membrane where it can interact with ras-p21 [10,20–22].

On the other hand, raf-1 and PKC-alpha have been shown to cooperate synergistically in the transformation of NIH3T3 cells [23]. A critical step appears to be phosphorylation of raf-1 at Ser259 with PKC-alpha, suggesting that phosphorylation of Ser259 can lead to different, possibly opposing, effects intracellularly. In a breast cancer cell line, activated Akt phosphorylates raf at Ser259, resulting in downregulation of the MEK-MAPK pathway but at the same time promoting cell proliferation [24].

Since raf contains numerous sites for phosphorylation that include Ser43, Ser259, Ser338, Tyr340, Tyr341, Ser445, Thr598, and Ser601 [10], it is possible that specific combinations of phosphorylations including that of raf-Ser259 result in raf retardation of cell proliferation and/or inhibition of the MEK-MAPK pathway, while other combinations stimulate either or both of these events. Our results in this study suggest that in a human pancreatic cancer cell line there is a high level of phosphorylation of raf-Ser259 that is downregulated by a peptide (PNC-2-leader) known to inhibit oncogenic ras-JNK interaction and to induce tumor cell necrosis [1–4,13; V Adler et al, manuscript submitted]. In these cells it is possible that some of the raf-Ser259 species induce cell proliferation while others result in auto-down-regulation of the raf-MEK-MAPK pathway. We are now investigating the patterns of phosphorylation of raf that contain phosphorylated raf-Ser259.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pincus MR. Development of new anti-cancer peptides from conformational energy analysis of the oncogenic ras-p21 protein and its complexes with target proteins. Front Biosci 2004;9:3486–3509.[Medline]
  2. Pincus MR, Brandt-Rauf PW, Michl J, Friedman FK. ras-p21-Induced cell transformation: unique signal transduction pathways and implications for the design of new chemotherapeutic agents. Cancer Invest 2000; 18:39–50.[Medline]
  3. Adler V, Pincus MR, Brandt-Rauf PW, Ronai Z. Complexes of ras-p21 with jun-n-kinase and c-jun proteins. PNAS USA 1995;92:10585–10589.[Abstract/Free Full Text]
  4. Adler V, Pincus MR, Polatskaya A, Montano X, Friedman FK, Ronai Z. Activation of c-jun nh2 kinase by uv irradiation is dependent on p21ras. J Biol Chem 1996;271,23304–23309.[Abstract/Free Full Text]
  5. Ranginwale M, Smith S, Flom J, Chie L, Kanovsky M, Chung D, Friedman FK, Robinson RC, Brandt-Rauf PW, Yamaizumi Z, Michl J, Pincus MR. Differences in patterns of activation of map kinases induced by oncogenic ras-p21 and insulin in oocytes. Exp Cell Res 2001; 269:162–169.[Medline]
  6. Birchmeier C, Broek D, Wigler M. ras Proteins can induce meiosis in Xenopus oocytes. Cell 1985;43:615–621.[Medline]
  7. Deshpande AK, Kung H-F. Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins. Mol Cell Biol 1987;7:1285–1288.[Abstract/Free Full Text]
  8. Chie L, Amar S, Kung H-F, Lin MCM, Chen H, Chung D, Adler V, Ronai Z, Friedman FK, Robinson RC, Kovac C, Brandt-Rauf PW, Yamaizumi Z, Michl J, Pincus MR. Induction of oocyte maturation by jun-n-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-mek signal transduction pathway. Cancer Chemother Pharmacol 2000;45:441–449.[Medline]
  9. Adler V, Qu Y, Smith SJ, Izotova L, Pestka S, Kung H-F, Lin M, Friedman FK, Chie L, Chung DL, Boutjdir M, Pincus MR. Functional interactions of raf and mek with jun-n-terminal kinase (JNK) result in a positive feedback pathway loop on the oncogenic ras signaling pathway. Biochemistry 2005;44:10784–10795.[Medline]
  10. Mercer KE, Pritchard CA. Raf proteins and cancer: b-raf is identified as a mutational target. Biochim Biophys Acta 2003;1653:25–40.[Medline]
  11. Miura K, Inouye Y, Nakamor H, Iwai S, Ohtsuka E, Ikehara M, Noguchi S, Nishimura S. Synthesis and expression of a synthetic gene for the activated human c-Ha-ras protein. Jpn J Cancer Res 1986;77:45–51.
  12. Sendoh H, Hu C-D, Wu D, Song C, Yamawaki-Kataoka Y, Kotani J, Okada T, Shima F, Kariya K-I, Kataoka T. Role of raf-1 conserved region 2 in regulation of ras-dependent raf-1 activation. Biochem Biophys Res Commun 2000;271:596–602.[Medline]
  13. Pincus MR, Michl J, Bowne W, Zenilman M. Anti-cancer peptides from the ras-p21 and p53 proteins. In: Research Advances in Cancer (RM Mohan, Ed) Global Research Network Pub, Kerala, India, 2007; in press.
  14. Kanovsky M, Michl J, Botzolaki G, Morin J, Kovac C, Chunh D, Friedman FK, Pincus MR. Peptides, designed from molecular modeling studies of the ras-p21 protein, induce phenotypic reversion of a pancreatic carcinoma cell line but have no effect on normal pancreatic acinar cell growth. Cancer Chemother Pharmacol. 2003;52:202–208.[Medline]
  15. Chie L, Friedman, FK, Kung H-F, Lim MCM, Chung DL, Pincus MR. Identification of the site of inhibition of mitogenic signaling by oncogenic ras-p21 by a ras effector peptide. J Protein Chem 2002;21:367–369.[Medline]
  16. Qu Y, Adler V, Chu T, Platica O, Michl J, Pestka S, Izotova L, Boutjdir M, Pincus MR. Two dual specificity kinases are preferentially induced by wild-type rather than by oncogenic ras-p21 in Xenopus oocytes. Front Biosci 2006;11:2420–2427.[Medline]
  17. Qu Y, Adler V, Izotova L, Pestka S, Bowne W, Michl J, Boutjdir M, Friedman FK, Pincus MR. Dual-specificity kinases, TOPK and DYRK1A, are critical for oocyte maturation induced by wild-type- but not by oncogenic-ras-p21 protein. Front Biosci 2007;12,5089–5097.[Medline]
  18. Yuryev A, Wennogle LP Novel raf kinase protein-protein interactions found by an exhaustive yeast two-hybrid analysis. Genomics 2003;81;112–125.[Medline]
  19. Chung DL, Brandt-Rauf PW, Weinstein IB, Nishimura S, Yamaizumi Z, Murphy RB, Pincus MR. Evidence that the ras-oncogene-encoded 21 protein induces oocyte maturation via activation of protein kinase C. PNAS USA 1992;89,1993–1996.[Abstract/Free Full Text]
  20. Dhillon AS, Pollock C, Steen H, Mischak H, Kolch W. Cyclic AMP-dependent kinase regulates raf-1 kinase mainly by phosphorylation of serine 259. Mol Cell Biol 2002;22:3237–3246.[Abstract/Free Full Text]
  21. Yip-Schneider MT, Miao W, Lin A, Barnard S, Tzivion G, Marshall MS. Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association Biochem J 2000;351:151–159.[Medline]
  22. Abraham D, Podar K, Pacher M, Kubicek M, Welzel N, Hemmings BA, Dilworth, SM, Mischak H, Kolch W, Baccarini M. Raf-1-associated protein phosphatase 2a as a positive regulator of kinase activation. J Biol Chem 2000;275;22300–22304.[Abstract/Free Full Text]
  23. Kolch W, Heidecker G, Kochs G, Hummel R, Vahidi H, Mischak H, Finkenzeller G, Marme D, Rapp UR. Protein kinase C-alpha activates RAF-1 by direct phosphorylation. Nature 1993;364:249–252.[Medline]
  24. Zimmerman S, Moelling, K. Phosphorylation and regulation of RAF by Akt (protein kinase B) Science 1999;26:1741–1744.




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