RESUMEN
In previous studies, involving molecular modeling of wild-type and oncogenic forms of the ras-p21 protein bound to GTPase activating protein GAP and the ras-specific guanine nucleotide exchange-promoting protein, SOS, we identified specific domains of GAP and SOS proteins that differ in conformation when the computed average structures of the corresponding wild-type and oncogenic complexes are superimposed. Additionally, in these previous studies, we have synthesized peptides corresponding to these domains and found that all of them inhibit either or both oncogenic (Val 12-containing) p21- and insulin-activated wild-type p21-induced oocyte maturation. To document further the specificity of the inhibition of these peptides for the ras signal transduction pathway, we have now tested their effects on progesterone-induced maturation that occurs by a ras-independent pathway. None of these peptides, including a peptide corresponding to residues 980-989 of SOS that completely blocks oncogenic p21-induced maturation and also causes extensive inhibition of insulin-induced maturation, affects progesterone-induced maturation, suggesting that all of these peptides are specific for the ras pathway. Since our approach to the design of peptides that can inhibit oncogenic ras-p21 selectively is based on identifying domains that differ in conformation between oncogenic and wild-type complexes, we have now further synthesized peptides that correspond to domains of GAP (residues 903-910) and SOS (residues 792-804) that do not differ in conformation when the average structures are superimposed. These peptides do not inhibit either oncogenic p21- or insulin-induced oocyte maturation, supporting the overall strategy of using peptides from domains that change conformation as the ones most likely to inhibit oncogenic and/or wild-type ras-p21. These results further support the specificity of inhibition of the GAP and SOS peptides from the conformationally distinct domains of both proteins.
Asunto(s)
Proteínas Activadoras de GTPasa/farmacología , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Son Of Sevenless/farmacología , Animales , Femenino , Proteína Oncogénica p21(ras)/fisiología , Oocitos/efectos de los fármacos , Progesterona/antagonistas & inhibidores , Estructura Terciaria de Proteína , Xenopus laevisRESUMEN
In previous studies we have found that oncogenic (Val 12)-ras-p21 induces Xenopus laevis oocyte maturation that is selectively blocked by two ras-p21 peptides, 35-47, also called PNC-7, that blocks its interaction with raf, and 96-110, also called PNC-2, that blocks its interaction with jun-N-terminal kinase (JNK). Each peptide blocks activation of both JNK and MAP kinase (MAPK or ERK) suggesting interaction between the raf-MEK-ERK and JNK-jun pathways. We further found that dominant negative raf blocks JNK induction of oocyte maturation, again suggesting cross-talk between pathways. In this study, we have undertaken to determine where these points of cross-talk occur. First, we have immunoprecipitated injected Val 12-Ha-ras-p21 from oocytes and found that a complex forms between ras-p21 raf, MEK, MAPK, and JNK. Co-injection of either peptide, but not a control peptide, causes diminished binding of ras-p21, raf, and JNK. Thus, one site of interaction is cooperative binding of Val 12-ras-p21 to raf and JNK. Second, we have injected JNK, c-raf, and MEK into oocytes alone and in the presence of raf and MEK inhibitors and found that JNK activation is independent of the raf-MEK-MAPK pathway but that activated JNK activates raf, allowing for activation of ERK. Furthermore, we have found that constitutively activated MEK activates JNK. We have corroborated these findings in studies with isolated protein components from a human astrocyte (U-251) cell line; that is, JNK phosphorylates raf but not the reverse; MEK phosphorylates JNK but not the reverse. We further have found that JNK does not phosphorylate MAPK and that MAPK does not phosphorylate JNK. The stress-inducing agent, anisomycin, causes activation of JNK, raf, MEK, and ERK in this cell line; activation of JNK is not inhibitable by the MEK inhibitor, U0126, while activation of raf, MEK, and ERK are blocked by this agent. These results suggest that activated JNK can, in turn, activate not only jun but also raf that, in turn, activates MEK that can then cross-activate JNK in a positive feedback loop.
Asunto(s)
Retroalimentación Fisiológica , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptor Cross-Talk , Animales , Butadienos/farmacología , Línea Celular Tumoral , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Complejos Multiproteicos , Nitrilos/farmacología , Oocitos , Fosforilación , Transducción de Señal , Xenopus laevisRESUMEN
ras-p21 protein binds to the son-of-sevenless (SOS) guanine nucleotide-exchange promoter that allows it to exchange GDP for GTP. Previously, we performed molecular dynamics calculations on oncogenic (Val 12-) and wild-type ras-p21 bound to SOS. By superimposing the average structures of these two complexes, we identified four domains (residues 631-641, 676-691, 718-729, and 994-1004) in SOS that change conformation and were candidates for being effector domains. These calculations were performed in the absence of three crystallographically undefined loops (i.e., residues 591-596, 654-675, and 742-751). We have now modeled these loops into the SOS structure and have re-performed the dynamics calculations. We find that all three loop domains undergo large changes in conformation that involve mostly changes in their positioning and not their individual conformations. We have also identified another potential effector domain (i.e., residues 980-989). Overall, our current results suggest that SOS interactions with oncogenic ras-p21 may enhance ras-p21 mitogenic signaling through prolonging its activation by maintaining its binding to GTP and by allowing its effector domains to interact with intracellular targets.
Asunto(s)
Proteína Oncogénica p21(ras)/química , Proteínas Son Of Sevenless/química , Animales , Sitios de Unión , Humanos , Proteína Oncogénica p21(ras)/metabolismo , Estructura Terciaria de Proteína/fisiología , Transducción de Señal/fisiología , Proteínas Son Of Sevenless/metabolismoRESUMEN
Oncogenic ras-p21 directly activates jun-N-terminal kinase (JNK) and its substrate, jun as a unique step on its mitogenic signal transduction pathway. This activation is blocked by the specific JNK-jun inhibitor, glutathione-S-transferase-pi (GST-pi). Four domains of GST-pi have been implicated in this regulatory function: 34-50, 99-121, 165-182, and 194-201. The 34-50 domain is unique in that it does not affect GST-pi binding to JNK-jun but blocks jun phosphorylation by JNK. We now find that it completely blocks oncogenic (Val 12-) ras-p21-induced oocyte maturation but has no effect on insulin-induced oocyte maturation. Because the latter process requires activation of wild-type ras-p21, this peptide appears to be specific for inhibiting only the oncogenic form of ras-p21, suggesting its use in treating ras-induced tumors.
Asunto(s)
Glutatión Transferasa/metabolismo , Isoenzimas/metabolismo , Mitosis/fisiología , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Péptidos/metabolismo , Transducción de Señal/fisiología , Animales , Femenino , Gutatión-S-Transferasa pi , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias/metabolismo , Oocitos/metabolismo , XenopusRESUMEN
In the accompanying paper, we found, using molecular dynamics calculations, four domains of the ras-specific SOS guanine nucleotide exchange protein (residues 589-601, 654-675, 746-761, and 980-989) that differ markedly in conformation when SOS is complexed with either oncogenic (Val 12-) ras-p21 or wild-type ras-p21. Three of these domains contain three crystallographically undefined loops that we modeled in these calculations, and one is a newly identified non-loop domain containing SOS residues 980-989. We have now synthesized peptides corresponding to these four domains and find that all of them block Val 12-ras-p21-induced oocyte maturation. All of them also block insulin-induced oocyte maturation, but two of these peptides, corresponding to SOS residues 589-601 and 980-989, block oncogenic ras to a significantly greater extent. These results suggest that SOS contains domains, including the three loop domains, that are important for ras signaling and that several of these domains can activate different pathways specific to oncogenic or wild-type ras-p21.
Asunto(s)
Biología Computacional , Fragmentos de Péptidos/metabolismo , Transducción de Señal/fisiología , Proteínas Son Of Sevenless/metabolismo , Proteínas ras/metabolismo , Animales , Femenino , Oocitos/metabolismo , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Proteínas Son Of Sevenless/química , Xenopus , Proteínas ras/químicaRESUMEN
PURPOSE: From molecular modeling studies we found that two ras-p21 peptides, corresponding to p21 residues 35-47 (PNC-7) and 96-110 (PNC-2), selectively block oncogenic (Val 12-p21), but not insulin-activated wild-type, p21-induced oocyte maturation. Our purpose was to determine if these peptides block the growth of mammalian cancer cells but not their normal counterpart cells. METHODS: Since oncogenic ras has been implicated as a causative factor in over 90% of human pancreatic cancers, we have established a normal pancreatic acinar cell line (BMRPA1) and the corresponding ras-transformed pancreatic cancer cell line (TUC-3). We treated both cell lines with PNC-7 and PNC-2 and the unrelated negative control peptide, X13, attached to the penetratin sequence that allows membrane penetration and also transfected these cell lines with plasmids encoding all three peptides. RESULTS: Treatment of TUC-3 cells with each peptide resulted in their complete phenotypic reversion to the untransformed phenotype as revealed by the lack of tumor formation of these revertant cells implanted in the peritoneal cavities of nude mice. In contrast, treatment with X13-leader resulted in no inhibition of cell growth. Identical results were obtained when TUC-3 cells were transfected with plasmids expressing PNC-2, PNC-7 and X13. None of these peptides affected the normal growth of BMRPA1 cells. CONCLUSIONS: PNC-2 and PNC-7 peptides induce phenotypic reversion of ras-induced pancreatic cancer cells and have no effect on normal pancreatic cell growth. Since the plasmid encoding PNC-2 without penetratin also had the same effect on the TUC-3 cell line, we conclude that the penetratin sequence has no effect on the activity of this peptide. Since X13 attached to penetratin had no effect on TUC-3 cells, the effect is specific for PNC-2 and PNC-7 and further confirms that the effect is not caused by the penetratin sequence. PNC-2- and PNC-7-penetratin may therefore be useful in the treatment of ras-induced pancreatic carcinomas.
Asunto(s)
Modelos Moleculares , Proteína Oncogénica p21(ras) , Neoplasias Pancreáticas/genética , Péptidos/uso terapéutico , Células Tumorales Cultivadas/efectos de los fármacos , Secuencia de Bases , Humanos , Proteína Oncogénica p21(ras)/análogos & derivados , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , FenotipoRESUMEN
A Janus-2 (JAK-2) binding protein, JBP1, has been found to function as an arginine methyl transferase and is now designated PRMT5. Co-injection of plasmids encoding this protein together with oncogenic (Val 12-containing) ras-p21 protein into Xenopus leavis oocytes results in strong inhibition of oncogenic p21-induced oocyte maturation. This inhibition appears to be dependent on the methyl transferase function since a partially active R368A mutant shows diminished ability to inhibit Val 12-p21-induced oocyte maturation, and an almost totally inactive GAGRG (365-369) deletion mutant fails to inhibit Val 12-p21-induced maturation. In contrast, PRMT5 (JBP1) does not inhibit insulin-induced oocyte maturation. Since insulin-induced maturation depends on activation of cellular ras-p21, PRMT5 does not appear to inhibit the wild-type p21 protein. We also find that arginine methyl transferase inhibitors strongly block oncogenic ras-p21-activated, but not insulin-activated, wild-type ras-p21-induced oocyte maturation. Thus signaling by oncogenic p21 appears to involve methyltransferases uniquely. Surprisingly, the active site peptide, Gly-Arg-Gly, strongly suppresses insulin-induced maturation but has no effect on Val 12-p21-induced maturation. This peptide may therefore be useful in defining steps in the wild-type ras pathway.
Asunto(s)
Proteína Metiltransferasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Femenino , Insulina/farmacología , Mitógenos/farmacología , Oocitos/efectos de los fármacos , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Proteína-Arginina N-Metiltransferasas , Proteínas Proto-Oncogénicas p21(ras)/farmacología , Xenopus laevisRESUMEN
We have previously found that the protein kinase C (PKC) inhibitor, CGP 41 251, blocks oncogenic ras-p21 protein- and beta-PKC-induced oocyte maturation, but only weakly inhibits insulin-induced oocyte maturation (which requires activation of wild-type endogenous ras-p21). Because the dose-response curves for inhibition of oncogenic p21- and beta-PKC-induced oocyte maturation by CGP 41 251 superimpose and because the ras-p21-inactivating antibody, Y13-259, does not inhibit beta-PKC-induced oocyte maturation, we concluded that the oncogenic, but not wild-type, protein requires beta-PKC as a downstream target. Because multiple isoforms of PKC exist and several of these, such as epsilon-PKC, have been found to be important on ras signal transduction pathways, we have investigated which PKC isoforms are critical to each ras protein. For this purpose, we used PKC-isoform-specific inhibitors, which have been shown to inhibit selectively the function and translocation of PKC isoforms in vitro and in vivo. Specifically, the peptides KLFIMN, QEVIRN, and EAVSLKPT each inhibit beta-1, beta-2, and epsilon-PKC, respectively, but do not cross-inhibit other PKC isoforms. We find that the epsilon-PKC inhibitory peptide strongly blocks insulin- but not oncogenic ras-p21-induced oocyte maturation whereas the beta-2 inhibitory peptide more strongly inhibits oncogenic ras-p21-induced oocyte maturation, corroborating our previous studies. The beta-1 inhibitory peptide has little effect on either protein. We conclude that selective inhibition of individual PKC isoforms permits the distinction between signal transduction initiated by oncogenic and activated wild-type p21 proteins and implicate different specific PKC isoforms in mitogenic signal transduction by each of these proteins. The ability to dissect the role of individual PKC isozymes in this regulation is of therapeutic significance.
Asunto(s)
Proteína Oncogénica p21(ras)/farmacología , Oocitos/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteínas ras/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Insulina/farmacología , Isoenzimas/biosíntesis , Mitosis , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Proteína Oncogénica p21(ras)/fisiología , Oocitos/metabolismo , Péptidos/química , Péptidos/farmacología , Proteína Quinasa C/biosíntesis , Transducción de Señal , Transfección , Xenopus laevis , Proteínas ras/antagonistas & inhibidores , Proteínas ras/fisiologíaRESUMEN
GTPase activating protein (GAP) is a known regulator of ras-p21 activity and is a likely target of ras-induced mitogenic signaling. The domains of GAP that may be involved in this signaling are unknown. In order to infer which domains of GAP may be involved, we have performed molecular dynamics calculations of GAP complexed to wild-type and oncogenic (Val 12-containing) ras-p21, both bound to GTP. We have computed and superimposed the average structures for both complexes and find that there are four domains of GAP that undergo major changes in conformation: residues 821-851, 917-924, 943-953, and 1003-1020. With the exception of the 943-953 domain, none of these domains is involved in making contacts with ras-p21, and all of them occur on the surface of the protein, making them good candidates for effector domains. In addition, three ras-p21 domains undergo major structural changes in the oncogenic p21-GAP complex: 71-76 from the switch 2 domain; 100-108, which interacts with SOS, jun and jun kinase (JNK); and residues 122-138. The change in conformation of the 71-76 domain appears to be induced by changes in conformation in the switch 1 domain (residues 32-40) and in the adjacent domain involving residues 21-31. In an accompanying paper, we present results from microinjection of peptides corresponding to each of these domains into oocytes induced to undergo maturation by oncogenic ras-p21 and by insulin-activated wild-type cellular p21 to determine whether these domain peptides may be involved in ras signaling through GAP.
Asunto(s)
Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Proteína Oncogénica p21(ras)/química , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Sustitución de Aminoácidos/genética , Sitios de Unión , Cristalografía por Rayos X , Guanosina Trifosfato/metabolismo , Proteína Oncogénica p21(ras)/genética , Fosfatos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/genética , TermodinámicaRESUMEN
In the accompanying article, using molecular dynamics calculations, we found that the 66-77 and 122-138 domains in ras-p21 and the 821-827, 832-845, 917-924, 943-953, and 1003-1020 domains in GAP have different conformations in complexes of GAP with wild-type and oncogenic ras-p21. We have now synthesized peptides corresponding to each of these domains and coinjected them into oocytes with oncogenic p21, which induces oocyte maturation, or injected them into oocytes incubated with insulin that induces maturation by activating wild-type cellular ras-p21. We find that all of these peptides inhibit both agents but do not inhibit progesterone-induced maturation that occurs by a ras-independent pathway. The p21 66-77 and 122-138 peptides cause greater inhibition of oncogenic p21. On the other hand, the GAP 832-845 and 1003-1021 peptides inhibit insulin-induced maturation to a significantly greater extent. Since we have found that activated wild-type and oncogenic p21 activate downstream targets like raf differently, these GAP peptides may be useful probes for identifying elements unique to the wild-type ras-p21 pathway.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Activadoras de GTPasa/farmacología , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Proteína Oncogénica p21(ras)/farmacología , Oocitos/efectos de los fármacos , Oocitos/crecimiento & desarrollo , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Escherichia coli , Proteínas Activadoras de GTPasa/química , Insulina/farmacología , Datos de Secuencia Molecular , Proteína Oncogénica p21(ras)/química , Proteína Oncogénica p21(ras)/metabolismo , Fragmentos de Péptidos/química , Progesterona/farmacología , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Xenopus laevisRESUMEN
We have previously found that a ras switch 1 domain peptide (PNC-7, residues 35-47) selectively blocks oocyte maturation induced by oncogenic (Val 12-containing) ras-p21 protein and also blocks c-raf-induced oocyte maturation. We now find that oncogenic ras-p21 does not inhibit oocyte maturation of a constitutively activated raf protein (raf BXB) that is lacking most of the first 301 amino terminal amino acids, including the major ras binding domain and accessory ras-binding regions. We also find that a dominant negative raf that completely blocks c-raf-induced maturation likewise does not block raf-BXB-induced maturation. We conclude that PNC-7 blocks ras by binding to the amino-terminal domain of raf and that raf BXB must initiate signal transduction in the cytosol.
Asunto(s)
Mitógenos/farmacología , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Proteína Oncogénica p21(ras)/farmacología , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Sitios de Unión , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Mitógenos/química , Mitógenos/metabolismo , Proteína Oncogénica p21(ras)/química , Proteína Oncogénica p21(ras)/metabolismo , Oocitos/efectos de los fármacos , Oocitos/crecimiento & desarrollo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/genética , Xenopus laevisRESUMEN
PURPOSE: We have previously found that the staurosporine derivative, CGP 41 251, that has a high specificity for inhibiting protein kinase C (PKC), selectively blocks oncogenic ras-p21-induced oocyte maturation and that PKC and jun-N-terminal kinase (JNK), with which oncogenic ras-p21 directly interacts, reciprocally require each other's activation. We sought to determine whether CGP 41 251 blocks proliferation of ras-transformed mammalian cells and whether it synergistically exerts this effect with a ras-p21 peptide (residues 96-110) that interferes with the interaction of ras-p21 with JNK. METHODS: We incubated ras-transformed rat pancreatic cancer TUC-3 cells and their normal counterpart pancreatic acinar BMRPA1 cells with CGP 42 251 alone and in the presence of the ras-p21 96-110 peptide, both in pre- and post-monolayer phases and determined cell counts and morphology and, for TUC-3 cells, their ability to grow on soft agar. In the post-monolayer experiments, we also evaluated these parameters after withdrawal of these agents. RESULTS: CGP 41 251, but not its inactive analogue, CGP 42 700, blocked pre-monolayer growth and reduced post-monolayer cell counts of both TUC-3 and BMRPA1 cells (IC(50) 0.28 and 0.35 micro M, respectively). After 2 weeks of treatment, all the remaining TUC-3 cells exhibited the untransformed phenotype. Withdrawal of CGP 41 251 resulted in almost complete regrowth of the normal BMRPA1 cells while the reverted TUC-3 cells grew much more slowly. These effects were greatly enhanced by the presence of the ras-p21 96-110 peptide. CONCLUSIONS: CGP 41 251 strongly blocks growth of ras-transformed pancreatic cancer cells by causing cell death and by induction of phenotypic reversion. The enhancement of this effect by the ras-p21 96-110 peptide indicated synergy between it and CGP 41 251, allowing it to block proliferation of the transformed cells selectively. These findings suggest the possibility of using these two agents in anticancer therapy.