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Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation
R Marais1, Y Light, H F Paterson
1CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, London, UK.
Abstract:
A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps.
Insights
Ras GTPase is crucial for activating Raf-1 kinase by recruiting it to the plasma membrane. This recruitment enables essential tyrosine phosphorylation and further activation steps, highlighting Ras as a key regulator in this signaling pathway.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Cancer research
Background:
- Signal transduction involves protein kinase cascades initiated by tyrosine kinases.
- Ras-dependent activation of Raf-1, Mek, and ERKs is central to these pathways.
Purpose of the Study:
- To investigate the role of tyrosine kinase activity in Raf-1 activation.
- To determine the properties of p74Raf-1 and oncogenic Src required for Raf-1 activation.
Main Methods:
- Utilized mammalian cell culture systems.
- Employed oncogenic Src and Ras mutants.
- Examined p74Raf-1 interaction with p21Ras-GTP.
- Analyzed tyrosine phosphorylation at specific Raf-1 sites (Tyr340/341).
Main Results:
- Activation of p74Raf-1 by oncogenic Src requires myristoylated pp60Src and interaction with p21Ras-GTP.
- Ras/Raf interaction facilitates p74Raf-1 translocation to the plasma membrane for tyrosine phosphorylation by Src.
- Co-expression with oncogenic Ras and Src resulted in a 25-fold increase in Raf-1 activation, with Ras being the limiting factor for membrane recruitment.
Conclusions:
- Ras binding to Raf-1 is essential for its recruitment to the plasma membrane.
- This recruitment enables at least two distinct activation steps, including tyrosine phosphorylation.
- Ras plays a critical role in mediating Raf-1 activation through plasma membrane localization.