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Pathways for activation of the ras-oncogene-encoded p21 protein
M R Pincus1, D Chung, D C Dykes
1Department of Pathology, SUNY Health Science Center, Syracuse 13210.
Abstract:
The ras-oncogene-encoded p21 protein is known to cause a large number of human tumors. This protein differs from its normal counterpart protein, which is present in all eukaryotic cells, in that it contains a single amino acid substitution at critical positions in the polypeptide chain, such as at Gly 12, Gly 13, Ala 59, and Gln 61. Using computer-based molecular modeling, it has been found that one region of this protein that is a candidate for interacting with other intracellular proteins is the region from residues 35 to 47. In oocyte microinjection experiments, it was found that this peptide strongly inhibits the mitogenic effects of oncogenic (Val 12-containing)p21 but does not inhibit the cellular effects of activation of normal p21 protein. Furthermore, it has been shown that the cellular effects of oncogenic p21 protein can be completely inhibited by selectively blocking protein kinase C (PKC) with a highly specific inhibitor of this protein, CGP 41 251, a staurosporine derivative. This inhibitor, however, only weakly inhibits the effects of normal cellular ras-p21 protein. In addition, a photoaffinity-labeled p21 protein has been microinjected into NIH 3T3 fibroblasts and have isolated intracellular proteins of MW 35, 43 and 61 kda covalently bound to it. The 43 kda protein is the major one and appears to be critical to the functioning of the p21 protein. Our results suggest that oncogenic and normal p21 proteins utilize overlapping but distinct pathways; the oncogenic pathway can be blocked selectively and requires the activation of PKC and the presence of the 43 kda protein.
Insights
Oncogenic ras-p21 protein, a tumor promoter, can be selectively inhibited. Blocking protein kinase C (PKC) and a 43 kDa protein disrupts oncogenic pathways, unlike normal ras-p21 protein.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- The ras-oncogene-encoded p21 protein is implicated in numerous human tumors.
- Mutations in ras-p21, such as at Gly 12, lead to oncogenic forms that differ from normal cellular p21.
- Understanding the distinct pathways of normal and oncogenic ras-p21 is crucial for targeted cancer therapies.
Purpose of the Study:
- To investigate the molecular mechanisms differentiating oncogenic ras-p21 from normal p21.
- To identify potential targets for selectively inhibiting the effects of oncogenic ras-p21.
- To elucidate the role of protein kinase C (PKC) and specific interacting proteins in ras-p21 signaling.
Main Methods:
- Computer-based molecular modeling to identify potential protein interaction regions.
- Oocyte microinjection of peptides to assess inhibition of p21 effects.
- Selective inhibition of protein kinase C (PKC) using CGP 41 251, a staurosporine derivative.
- Microinjection of photoaffinity-labeled p21 and isolation of bound intracellular proteins.
Main Results:
- A peptide corresponding to residues 35-47 of p21 selectively inhibited oncogenic ras-p21 but not normal p21.
- The specific PKC inhibitor CGP 41 251 effectively blocked oncogenic p21 effects while only weakly affecting normal p21.
- Intracellular proteins of 35, 43, and 61 kDa were found to bind to p21, with a 43 kDa protein being a major interactor.
- The 43 kDa protein appears critical for p21 protein function.
Conclusions:
- Oncogenic and normal ras-p21 proteins utilize overlapping yet distinct intracellular pathways.
- The oncogenic pathway is selectively targetable and involves the activation of PKC and interaction with a 43 kDa protein.
- These findings provide a basis for developing targeted therapies against ras-driven cancers by disrupting specific oncogenic signaling.