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Analysis of guanine nucleotide bound to ras protein in PC12 cells
1Institute of Medical Science, University of Tokyo, Japan.
Abstract:
The ras gene product (p21) specifically binds GDP or GTP. In analogy with the reaction mechanism of other GTP-binding proteins, only the GTP-bound conformation is believed to be the biologically active one. Previously, we reported that not only oncogenic p21(Val-12) but also proto-oncogenic p21(Gly-12) could induce morphological differentiation in rat pheochromocytoma PC12 cells when microinjected in the complexed form with GTP gamma S [(1987) Mol. Cell. Biol. 7, 4553-4556]. In the present report we transformed PC12 cells with the oncogenic ras gene placed under the metallothionein I promoter. It was found that the transformed cells, when induced with Cd2+, differentiated in the absence of NGF. Then we analyzed the guanine nucleotide bound to p21 in the intact PC12 cells. It was found that conditionally induced p21(Val-12) was mostly present in the GTP-bound form, whereas the endogenous p21(Gly-12) was in the GDP-bound form. These results indicate again that p21.GTP induces the morphological differentiation of PC12 cells.
Insights
The ras gene product p21, when bound to GTP, induces cell differentiation. This study shows that the GTP-bound form of oncogenic ras p21 (Val-12) drives morphological changes in PC12 cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The ras gene product, p21, binds guanine nucleotides GDP and GTP.
- The GTP-bound conformation of p21 is considered the biologically active form.
- Previous studies suggested both oncogenic (Val-12) and proto-oncogenic (Gly-12) p21 could induce differentiation when microinjected with GTPγS.
Purpose of the Study:
- To investigate the role of ras p21-GTP in inducing morphological differentiation in PC12 cells.
- To analyze the guanine nucleotide-bound state of p21 in intact, transformed PC12 cells.
- To determine if induced differentiation occurs independently of Nerve Growth Factor (NGF).
Main Methods:
- Transformation of PC12 cells with an oncogenic ras gene under a metallothionein I promoter.
- Induction of differentiation using Cadmium (Cd2+).
- Analysis of guanine nucleotide binding to p21 in intact cells.
Main Results:
- Transformed PC12 cells differentiated upon Cd2+ induction, without NGF.
- Conditionally induced p21(Val-12) was predominantly GTP-bound.
- Endogenous p21(Gly-12) remained predominantly GDP-bound.
Conclusions:
- Ras p21 in its GTP-bound form induces morphological differentiation in PC12 cells.
- These findings reinforce the hypothesis that p21.GTP is the active signaling molecule for differentiation.
- Conditional expression of oncogenic ras can bypass the need for NGF in PC12 cell differentiation.