Plasmid expression of a peptide that selectively blocks oncogenic ras-p21-induced oocyte maturation.

PURPOSE: We have previously found that a synthetic peptide corresponding to ras-p21 residues 96 110 (PNC2) selectively blocks oncogenic (Val 12-containing) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferation, we synthesized an inducible plasmid that expressed this peptide sequence. Our purpose was to test this expression system in oocytes to determine if it was capable of causing selective inhibition of oncogenic ras-p21. METHODS: We injected this plasmid and a plasmid expressing a control peptide into oocytes either together with oncogenic p21 or in the presence of insulin (that induces maturation that is dependent on normal cellular ras-p21) in the presence and absence of the inducer isopropylthioglucose (IPTG). RESULTS: Microinjection of this plasmid into oocytes together with Val 12-p21 resulted in complete inhibition of maturation in the presence of inducer. Another plasmid encoding the sequence for the unrelated control peptide, X13, was unable to inhibit Val 12-p21-induced maturation. In contrast, PNC2 plasmid had no effect on the ability of insulin-activated normal cellular or wild-type ras-p21 to induce oocyte maturation, suggesting that it is selective for blocking the mitogenic effects of oncogenic (Val 12) ras p21. CONCLUSION: We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p21 and may therefore be highly effective in blocking proliferation of ras-induced cancer cells. Also, from the patterns of inhibition, by PNC2 and other ras- and raf-related peptides, of raf- and constitutively activated MEK-induced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.

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.

PURPOSE: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. METHODS: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MEK-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. RESULTS: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. CONCLUSION: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MEK inhibitors. The results imply that blockade of both MEK and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras-induced tumors.

Glutathione-S-Transferase as a selective inhibitor of oncogenic ras-p21-induced mitogenic signaling through blockade of activation of jun by jun-N-terminal kinase.

We have identified the intracellular detoxification enzyme, glutathione-S-transferase (GST), as a potent inhibitor of the activation of jun by its kinase, jun-N-terminal kinase (JNK), in vitro. All three major isozymes (alpha, mu, and pi) bind to JNK-jun complexes and inhibit activation of jun by JNK. We now find that GST inhibits JNK-induced oocyte maturation in vivo and strongly inhibits oocyte maturation induced by oncogenic ras-p21 protein, but not by insulin-activated normal cellular p21 protein. These results correlate with the finding that oncogenic, but not insulin-activated normal, p21 induces high levels of activated JNK. GST also strongly blocks induction of oocyte maturation by protein kinase C (PKC) which is a critical downstream target of oncogenic but not normal ras-p21. Thus, we have established a new function for GST as a potent physiological inhibitor of the ras-JNK-jun pathway.

Inhibition of oncogenic and activated wild-type ras-p21 protein-induced oocyte maturation by peptides from the guanine-nucleotide exchange protein, SOS, identified from molecular dynamics calculations. Selective inhibition of oncogenic ras-p21.

In the preceding paper we performed molecular dynamics calculations of the average structures of the SOS protein bound to wild-type and oncogenic ras-p21. Based on these calculations, we have identified four major domains of the SOS protein, consisting of residues 631-641, 676-691, 718-729, and 994-1004, which differ in structure between the two complexes. We have now microinjected synthetic peptides corresponding to each of these domains into Xenopus laevis oocytes either together with oncogenic (Val 12)-p21 or into oocytes subsequently incubated with insulin. We find that the first three peptides inhibit both oncogenic and wild-type p21-induced oocyte maturation, while the last peptide much more strongly inhibits oncogenic p21 protein-induced oocyte maturation. These results suggest that each identified SOS region is involved in ras-stimulated signal transduction and that the 994-1004 domain is involved uniquely with oncogenic ras-p21 signaling.

Identification of the site of inhibition of oncogenic ras-p21-induced signal transduction by a peptide from a ras effector domain.

We have previously found that a peptide corresponding to residues 35-47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras-p21 peptide corresponding to residues 96-110 that blocks ras-jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation. We now find that the p21 35-47, but not the 96-110, peptide completely blocks c-raf-induced maturation. This finding suggests that the 35-47 peptide blocks oncogenic ras at the level of raf; that activated normal and oncogenic ras-p21 have differing requirements for raf-dependent signaling; and that the two oncogenic-ras-selective inhibitory peptides, 35-47 and 96-110, act at two different critical downstream sites, the former at raf the latter at JNK/jun, both of which are required for oncogenic ras-p21 signaling.

Evidence that oocyte maturation induced by an oncogenic ras-p21 protein and insulin is mediated by overlapping yet distinct mechanisms.

We have recently shown that a peptide (residues 35-47) from a functional region of the ras p21 protein, thought to be involved in the binding of p21 to GTPase activating protein, the antibiotic azatyrosine, known to induce the ras-recision gene, and the selective protein kinase C inhibitor, CGP 41,251, all inhibit oncogenic p21 protein-induced maturation of oocytes in a dose-dependent manner. We now show that these three agents only partially inhibit insulin-induced oocyte maturation, known to be dependent on activation of cellular p21 protein. On the other hand, the anti-p21 protein antibody Y13-259 completely inhibits both insulin- and oncogenic p21 protein-induced maturation as does a tetrapeptide, CVIM, known to block the enzyme farnesyl transferase which covalently attaches the farnesyl moiety to the p21 protein allowing it to attach to the cell membrane. Our results suggest that while the oncogenic and insulin-activated normal p21 proteins share certain elements of their signal transduction pathways in common, these pathways diverge and allow for selective inhibition of the oncogenic pathway.

Evidence that the ras oncogene-encoded p21 protein induces oocyte maturation via activation of protein kinase C.

The ras oncogene-encoded p21 protein is known to induce cell maturation of Xenopus laevis oocytes and malignant transformation of NIH 3T3 mouse fibroblasts. The pathways involved in oocytes and NIH 3T3 cells appear to be similar to one another. For example, in both cases, the ras p21-induced cellular events involve increased intracellular levels of the second messengers diacylglycerol and inositol phosphates, the former of which activates protein kinase C (PKC). To investigate the pathway of ras-induced oocyte maturation, we have explored the relationship between p21 protein and PKC. We show that the maturation signal from oncogenic p21 microinjected into Xenopus oocytes is completely blocked by the relatively specific PKC inhibitor CGP 41251, a staurosporine analogue that selectively inhibits PKC, but not by an inactive analogue of staurosporine, CGP 42700. Microinjection of purified PKC or of phorbol ester induces maturation of oocytes. PKC-induced maturation is inhibited by CGP 41251 but not by CGP 42700. Maturation induced by microinjected PKC is also not inhibited by two specific anti-p21 agents, the inactivating anti-p21 monoclonal antibody Y13-259 and the amino acid derivative azatyrosine. Both of these agents block p21-induced cell maturation. These results suggest that ras effects depend upon the action of PKC, whose activation is an event that occurs downstream of p21 in the maturation signal pathway.

A peptide from the GAP-binding domain of the ras-p21 protein as well as azatyrosine block ras-induced maturation of Xenopus oocytes.

The ras-oncogene-encoded p21 protein is known to produce malignant transformation of NIH 3T3 cells as well as maturation of Xenopus oocytes when microinjected into these cells. p21 protein is known to bind a GTPase activating protein (GAP) intracellularly; residues 32-45 have been implicated in interacting with GAP. We demonstrate here that a peptide corresponding to residues 35-47 of p21 as well as the antibiotic azatyrosine inhibit the ras-induced maturation of Xenopus oocytes in a dose-related manner upon microinjection. We have previously shown that this p21 peptide and azatyrosine could inhibit the effects of p21 protein on cell transformation and pinocytosis in NIH 3T3 cells. In the present study, in which we have extended these results to the oocyte system, we also demonstrate that both partially inhibit insulin-induced oocyte maturation, a process which is thought to involve activation of endogenous p21 protein; on the other hand, both agents fail to inhibit oocyte maturation induced by progesterone, which is known not to act through p21 protein activation. Control studies with other peptides and tyrosine analogues support the selective nature of these events. These results suggest that both the p21-related peptide and azatyrosine have potent anti-ras effects intracellularly.

A peptide from the GAP-binding domain of the ras-p21 protein and azatyrosine block ras-induced maturation of Xenopus oocytes.

The ras-oncogene-encoded p21 protein causes malignant transformation of NIH 3T3 cells and maturation of Xenopus oocytes when microinjected into these cells. P21 is known to interact with GTPase activating protein (GAP) intracellularly. Residues 32-45 of p21 have been implicated in interacting with GAP. In a previous study, we demonstrated that a synthetic peptide containing residues 35-47 from the GAP-binding region of p21 could block in vivo the effects of oncogenic p21 protein. It has also been found that an antibiotic, azatyrosine, blocks ras-initiated cell transformation. We now demonstrate that both of these agents inhibit the ras-p21 protein-induced maturation of Xenopus oocytes in a dose-related manner when microinjected into oocytes. The effects of each of these agents is specific. Both agents block insulin-induced maturation of oocytes, a process which is known to involve activation of endogenous normal p21 protein. On the other hand, neither agent inhibited oocyte maturation induced by progesterone, which is known to initiate oocyte maturation by ras-independent pathways. The inhibitory effects of the peptide were not mimicked by a control peptide from the CD4 receptor protein. Furthermore, the effect of azatyrosine was not mimicked by L-tyrosine. These results suggest that both the peptide and azatyrosine have potent anti-ras effects intracellularly.