Isotype-specific analysis of Ras-subfamily member signalling using xanthine nucleotide selective mutants

• Applicant: Privatdozent Dr. Ignacio Rubio
• Subject Area: Cell Biology
• Term: from 2001 to 2005
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5324397

Ras guanine nucleotide-binding proteins (GTPases) are key regulators in the intracellular signal transduction network. Amongst other functions, they transduce signals from activated cell surface receptors, eventually leading to the modulation of biological responses including proliferation, differentiation, apoptosis, senescence and cell motility. To date, the study of Ras function has mostly relied on the heterologous expression of constitutively active or blocking Ras variants. Similarly, functional investigation of downstream Ras-effector pathways has widely involved use of mutant versions of intermediary proteins. Although this approach has undoubtedly helped unravel general principles of Ras biology, the physiological relevance of such studies is a concern to many investigators. Moreover, such experiments provide little information on the specificity of individual Ras species in the physiological activation of distinct downstream effectors. Our aim is to create and exploit a system allowing the study of signal relay by wild type, "normal" Ras. The strategy is based on the generation of cell lines stably expressing. Ras proteins with altered nucleotide specificity towards xanthine nucleotids (XNPs) but with otherwise unaffected funtional properties. Perfusion of permeabilised cells with XNPs will allow us to manipulate the activation status of individual Ras-subfamily members and study their downstream cellular effects. Using this approach we intend to decipher the potency of individual Ras-subfamily members in the activation of the three established Ras-effectors Raf, PI3K and Ral-GDS.

• DFG Programme: Research Grants