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Carboxyl-terminal deletion and point mutations decrease the transforming potential of the activated rat neu oncogene
Abstract:
The rat neu oncogene encodes a constitutively activated growth factor receptor/transmembrane tyrosine kinase, p185Tneu, that is structurally similar to yet distinct from the epidermal growth factor receptor. To explore the role of the carboxyl-terminal region and of putative autophosphorylation sites in regulating the activity of the rat p185Tneu (T, transforming) protein, we used site-directed mutagenesis to generate a p185Tneu mutant in which a putative tyrosine autophosphorylation site (residue 1253) at the extreme carboxyl terminus was replaced by a phenylalanine residue and a mutant in which the carboxyl-terminal 122 amino acids were deleted. These proteins were expressed in NIH 3T3 cells at comparable levels and exhibited similar autophosphorylation activity, exogenous substrate phosphorylation ability, oligomerization levels, and responsiveness to a partially purified neu-activating factor. However, the mutant p185Tneu proteins displayed a decreased transforming capacity both in vitro and in vivo. This analysis demonstrated that the carboxyl-terminal domain and at least one putative tyrosine autophosphorylation site of p185Tneu play a role in positively regulating the cell growth-regulating properties of the neu protein.
Insights
The carboxyl-terminal domain of the rat neu protein (p185Tneu) is crucial for its cell growth-regulating properties. Mutations affecting this region, including autophosphorylation sites, reduce the neu protein
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- The rat neu oncogene encodes p185Tneu, a growth factor receptor/transmembrane tyrosine kinase.
- p185Tneu is structurally similar to the epidermal growth factor receptor but distinct.
- Its activity regulation by the carboxyl-terminal region and autophosphorylation sites is not fully understood.
Purpose of the Study:
- To investigate the role of the carboxyl-terminal region and putative autophosphorylation sites in regulating p185Tneu activity.
- To determine how these regions influence the transforming capacity of p185Tneu.
Main Methods:
- Site-directed mutagenesis was used to create p185Tneu mutants.
- Mutants included replacement of a tyrosine autophosphorylation site (residue 1253) with phenylalanine and deletion of the carboxyl-terminal 122 amino acids.
- Proteins were expressed in NIH 3T3 cells and analyzed for autophosphorylation, substrate phosphorylation, oligomerization, and transforming capacity.
Main Results:
- Mutant p185Tneu proteins showed comparable autophosphorylation, substrate phosphorylation, oligomerization, and responsiveness to neu-activating factor.
- However, these mutants exhibited significantly decreased in vitro and in vivo transforming capacity.
- The carboxyl-terminal domain and at least one autophosphorylation site positively regulate neu protein's cell growth-regulating properties.
Conclusions:
- The carboxyl-terminal domain of p185Tneu is essential for its full transforming activity.
- Specific tyrosine autophosphorylation sites within this domain are critical for regulating cell growth.
- These findings highlight the importance of the carboxyl-terminal region in neu-mediated oncogenesis.