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Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase
J R Fabian1, I O Daar, D K Morrison
1Molecular Mechanisms of Carcinogenesis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702-1201.
Abstract:
The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p21ras and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-1 activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that maturation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.
Insights
Tyrosine phosphorylation of Raf-1 at Tyr-340/341 activates its kinase activity. Mimicking this phosphorylation with specific mutations generates transforming Raf-1 proteins, impacting cell transformation and oocyte maturation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Raf-1 kinase activity is regulated by upstream tyrosine kinases and p21ras.
- Maximal Raf-1 activation requires coexpression of activated tyrosine kinases and p21ras.
- The precise role of tyrosine phosphorylation in Raf-1 regulation remains to be fully elucidated.
Purpose of the Study:
- To investigate the role of tyrosine kinases and tyrosine phosphorylation in Raf-1 activity regulation.
- To identify specific tyrosine phosphorylation sites on Raf-1.
- To determine the functional consequences of altering these phosphorylation sites.
Main Methods:
- Utilized the baculovirus-Sf9 expression system for coexpression studies.
- Identified tyrosine phosphorylation sites using mutagenesis.
- Assessed Raf-1 catalytic activity and transforming potential in BALB/3T3 cells and Xenopus oocytes.
Main Results:
- Identified Tyr-340 and Tyr-341 as major tyrosine phosphorylation sites on Raf-1.
- Mutations mimicking phosphorylation at these sites activated Raf-1's catalytic activity.
- These mutated proteins induced cell transformation and oocyte maturation, while non-phosphorylatable mutants blocked maturation.
Conclusions:
- Tyrosine phosphorylation at specific sites (Tyr-340/341) is a critical regulator of Raf-1 function.
- Altering these phosphorylation sites can generate constitutively active and transforming Raf-1 proteins.
- This study provides the first evidence of a single amino acid substitution generating a transforming Raf-1 protein.