The understanding of how small molecular weight molecules transmit information from the outside of cells to the nuclei of those cells is critical for our understanding of cancer, normal cell functioning, and development.
The aim of this fellowship is to determine the molecular mechanisms by which cytokines stimulate Raf-1 kinase, thereby stimulating a pathway which can result in altered transcription in the nucleus. In order to understand the mechanism of Raf-1 activation the following will be examined: 1) the interaction of Raf-1 kinase with the JAK family of kinases (known to be activated by cytokines) 2) the change in Raf-1 phosphorylation caused by the JAK kinases and 3) the effect the phosphorylations have on the kinase activity of Raf-1. To address these questions Raf-1 (or various modified forms) will be expressed with different members of the JAK family in insect cells using the baculovirus system. Using this system it will be possible to define which domains are needed for the proteins to interact, what effects on phosphorylation result from the interaction, and what effects the phosphorylations have on Raf-1 activity. The information learned in the insect cells will be confirmed in CTLL cells stimulated with cytokines to insure that the results obtained using the baculovirus system are biologically relevant.
